Best Practice & Research Clinical Haematology Volume 24, Issue 1 March 2011

CLINICAL HAEMATOLOGY The Best Practice & Research series aims to provide a topical serial publication describing and i...

Author: W. Fibbe

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CLINICAL HAEMATOLOGY The Best Practice & Research series aims to provide a topical serial publication describing and integrating the results from the latest original research articles into practical, evidence-based review articles. These articles seek to address the key clinical issues of diagnosis, treatment and patient management. Each issue follows a problem-orientated approach which focuses on the key questions to be addressed, clearly defining what is known and not known. Management is described in practical terms so that it can be applied to the individual patient. The serial is aimed at the physician in both practice and training. Best Practice & Research Clinical Haematology is abstracted and indexed in the following sources: Index Medicus, Medline, PubMed, Current Contents/Clinical Medicine, Current Contents/Life Sciences, Science Citation Index, SciSearch, Research Alert and EMBASE/Excerpta Medica.

Editor-in-Chief Jacob M. Rowe MD Dresner Professor of Hemato-oncology; Technion Israel Institute of Technology Chief, Department of Hematology and Bone Marrow Transplant Rambam Medical Center Haifa 31096, Israel EDITORIAL BOARD T. Barbui, Italy I. M. Franklin, UK D. G. Gilliland, USA A. Gratwohl, Switzerland C. Hershko, Israel

V. Hoffbrand, UK V. J. Marder, USA M. S. Tallman, USA J. C. Wade, USA

Best Practice & Research Clinical Haematology 24 (2011) 1–2

Contents lists available at ScienceDirect

Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha

Preface

Introduction

Mesenchymal stromal cells (MSCs) were recognized more than 40 years ago by Alexander Friedenstein, who described a population of adherent cells from the bone marrow which were non phagocytic, had a ﬁbroblastic appearance and that exhibited in-vitro multi-lineage differentiation into bone, cartilage, adipose tissue, tendon and muscle. Following transplantation under the kidney capsule, these cells gave rise to ossicles that contained multiple mesenchymal lineages. In recent years, there has been a renewed interest in MSCs that is mainly driven by their potential for clinical application in a variety of disorders. A vast amount of data suggest that MSCs can be applied for tissue regeneration, for instance to repair bone, cartilage or muscle defects. Perhaps more importantly, MSCs have been demonstrated to regulate immune responses in the context of tissue injury, autoimmunity and alloimmunity in particular transplantation. MSCs have been used therapeutically in clinical trials in stem cell transplantation, with the aim to promote engraftment or to prevent or treat acute graft-versus-host disease. Following encouraging experiments in experimental animals suggesting that MSC treatment may alleviate the symptoms in several models of autoimmune conditions, such as Crohn’s disease and Systemic Lupus Erythematosus, clinical trials have now began to explore their potential efﬁcacy in the setting of human autoimmune disorders. The possibility to expand the cells ex-vivo under appropriate culture conditions has greatly facilitated their clinical use. At present, most MSC products are expanded in media containing fetal calf serum or platelet lysate. Under these conditions, cells can be culture expanded to numbers required for clinical use within a period of several weeks. However, the effects of culture conditions or donor selection on the functional and therapeutic properties of clinical grade MSCs is still unclear. Very few markers are available for the prospective isolation of MSCs and in most instances, isolation still relies on their ability to adhere to plastic surfaces. An enrichment of CFU-F activity has been reported for markers including STRO1 and the low afﬁnity Nerve Growth Factor Receptor CD271. However, these markers are rarely used for prospective isolation. The clinical potential of MSCs is also determined by host factors and it has been suggested that MSCs need to become activated in the host environment in order to mediate their immune modulatory effects. Heterogeneity in host factors, in particular the availability of an appropriate pro-inﬂammatory environment, may explain the variability that is observed in clinical trials. It is therefore attempted to mimic the pro-inﬂammatory environment by the addition of cytokines, including interferon gamma. I am happy that leading experts in the ﬁeld of MSC biology, translation and clinical application have accepted the invitation to contribute to this special issue. It covers not only the biology of murine and human MSCs, their ex-vivo expansion and their role in the hematopoietic microenvironment, but also their potential for clinical application in autoimmune disorders, neuroregenerative disorders and allogeneic stem cell and cord blood transplantation.

1521-6926/$ – see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.beha.2011.02.001

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Preface / Best Practice & Research Clinical Haematology 24 (2011) 1–2

Willem E. Fibbe, Prof.* Leiden University Medical Centre, Department of Immunohematology and Blood Transfusion, Albinusdreef 2, Building 1, E3-Q P.O. Box 9600, 2300 RC Leiden, Netherlands Tel.: þ31 71 5263827; Fax: þ31 71 5265267. E-mail address: [email protected]

Best Practice & Research Clinical Haematology 24 (2011) 3–11

Contents lists available at ScienceDirect

Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha

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Multipotent adult progenitor cells Abhishek Sohni, MSc, Doctoral fellow *, Catherine M. Verfaillie, MD, Director, Stem cell institute Leuven * Interdepartementaal Stamcelinstituut, Katholieke Universiteit Leuven, O&N1, bus 804, Herestraat 49, 3000 Leuven, Belgium

Keywords: MAPC pluripotency reprogramming

We here discuss the potency and characteristics of various adult derived adherent stem cells with special focus on multipotent adult progenitor cells (MAPC) isolated ﬁrst in 2002 in our lab. We describe the potency of MAPC, our current understanding in relationship with novel insights gained in epigenetic modiﬁcations that increase cellular potency, and their possible clinical applications. Ó 2011 Published by Elsevier Ltd.

Introduction A stem cell is characterized by its unique ability to self-renew, some without senescence. When selfrenewal divisions are symmetrical, the two daughter cells become stem cells, leading to expansion of the stem cell pool. In postnatal life, most stem cell divisions are asymmetrical, yielding one stem cell and a more differentiated cell, or progenitor cell, which has limited self-renewal ability. The potency of a stem cell is deﬁned by its ability to divide and produce one to many different cell types. The zygote is totipotent as it can contribute to the cells of embryonic as well as extraembryonic tissue. An embryonic stem cell (ESC), derived from the inner cell mass (ICM) of the embryo, is pluripotent as it differentiates to all the cell types of embryo proper, but no longer the trophoblast. Stem cells derived from different tissues are multipotent as they can give rise to all cells of the tissue they are derived from, but not cells of other tissues. The third part of the deﬁnition of stem cells is that they can functionally repopulate a tissue (even undamaged) in vivo. The most extensively studied adult stem cell is the hematopoietic stem cell (HSC) – isolated from bone marrow (BM) [1], from peripheral blood following mobilization from the BM or from umbilical cord blood (UCB) – which can replenish the entire blood lineage of lethally irradiated organisms upon transplantation. Since the characterization of HSC, many other adult stem cells have been deﬁned,

* Corresponding authors. Tel.: þ32 16 33 02 95; Fax: þ32 16 33 02 94. E-mail addresses: [email protected] (A. Sohni), [email protected] (C.M. Verfaillie). 1521-6926/$ – see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.beha.2011.01.006

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including neural stem cells (NSC) in the hippocampus and subventricular zone of adult brain [2–5], gastrointestinal stem cells [6], epidermal stem cells [7,8], among others. Friedenstein and colleagues demonstrated in 1970 that a population of ﬁbroblast like cells can be isolated from BM, that can form bone and cartilage and reconstitute the bone marrow microenvironment [9], which later were called mesenchymal stem cells or marrow stromal cells (MSC) [10,11]. In a recent study by the Bianco group, it was shown that MSC isolated freshly from BM can recreate bone and the bone marrow microenvironment, from which MSC can again be isolated, demonstrating for the ﬁrst time that MSC indeed have stem cell properties [12]. Although it is generally accepted that the adult stem cells named above have a restricted differentiation ability; i.e. they generate cells from the tissue from which they were derived, since the late 1990s, many studies have suggested that under some conditions, adult tissue derived stem cells may have broader differentiation potency, also termed stem cell plasticity. Jiang et al. in 2002 cultureisolated such an adult cell from bone marrow, called Multipotent Adult Progenitor Cells [13]. An update on our understanding of the characteristics and origin of MAPC will be given, speciﬁcally describing rodent MAPC, and MAPC will be placed in relationship to current knowledge on different stem cells and ability of cells to acquire novel fates. Embryo and non-embryo-derived stem cells Embryonic tissue derived stem cell The most extensively studied and characterized embryo derived stem cells are the ESC. ESC are classically derived from the ICM of the blastocyst at E 3.5 and E 4–5 in mouse and man, respectively. Murine (m) and human (h) ESC are pluripotent [14,15]. This can be demonstrated by tetraploid complementation studies in mouse, where mESC give rise to all cells of the somatic and germline lineages [16,17]. Another method to demonstrate pluripotency is by formation of teratomas from either hESC or mESC, containing cells of all three somatic germ layers. Stem cell populations have also been isolated from other stages of early murine conspectuses, including from the 4–8 morula [18] stage and from the pre-gastrula stage from mouse embryos [19].The former are like mESC – pluripotent, as they contribute to all the somatic and germline cell types, and are maintained in vitro like ICM derived mESC. Cells isolated from pre-gastrula embryos, also termed epiblast-stem cells (EpiSC) express like mESC, Oct4, Nanog and Ssea-1, and form teratomas. However unlike mESC, EpiSC do not require feeders or leukemia inhibitory factor (Lif) for culture but can be expanded in the presence of Activin/nodal signaling [19]. In contrast to preblastocyst derived stem cells, EpiSC were unable to contribute to somatic cells and the germline following injection into blastocyst or following morula aggregation [18,19]. It is believed that hESC may be more akin to mEpiSC, based on gene expression proﬁling data and similar culture conditions required to maintain hESC in vitro [19]. Jannet Rossant’s group isolated stem cells from extraembryonic endoderm called XEN [20]. These cells express Sox7, Hnf4, Gata4 and Foxa2 but lack expression of Oct4 and Nanog [20]. In a chimera assay, XEN cells contributed to the parietal endoderm and to the parietal yolk sac at later stages during embryo development [20]. More recently, Debeb et al. isolated an other population of Oct4 expressing cells from rat blastocysts, termed extraembryonic endodermal precursor cells or XEN-P cells. Rat XENP cells, like rat or mouse ESC, express Oct4 and Ssea-1 and require leukemia inhibitory factor (Lif) for ex vivo maintenance. However, XEN-P cells do not express Nanog or Sox2, but express Sox7, Sox17, Gata4 and Gata6, similar to the hypoblast fraction of cells within the ICM that also do not express Nanog, but express Gata6 [21]. Upon morula aggregation or injection in the blastocyst, XEN-P cells contribute to primitive/visceral and parietal extraembryonic endodermal lineages but not the embryo proper [21], and even though they form tumors when injected postnataly, the tumors are akin to yolk sac tumors, not teratoma [22]. Yet another cell type of the embryonic lineage is the trophoblast stem cell. As the name suggests, they are derivative of the trophectoderm of the blastocyst, and require ﬁbroblast growth factor-4 (FGF4) for ex vivo maintenance. These cells contribute to the embryo part of the placenta, an important cell lineage for the embryo survival in utero [23]. Matsui et al. isolated embryonic germinal stem cells (EGC) with potency similar to ESC [24]. These cells were derived from primodial germ cells of 8.5 days post

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coitum embryos and exhibit properties similar to ESC including germ line transmission, teratoma formation and chimeric contribution to embryo proper [24]. EGC have also been isolated from human abortuses, and like their mouse counterparts exhibit many characteristics of ESC [25]. Stem cells derived from non-embryonic tissues As many of the stem cells discussed below will be discussed at length in other sections of this special issue, the description of stem cells from non-embryonic tissue, except for MSC, will be very brief. HSC, isolated from BM, blood or UCB, have been the best characterized. HSC can be puriﬁed to homogeneity in mouse and highly enriched in human, based on a complement of cell surface antigens, and many signals that govern their quiescence and differentiation have been identiﬁed. Likewise, stem cells that generate skin and its appendages and gastrointestinal stem cells can be puriﬁed to near homogeneity based on cell surface antigens [6–8]. Based on these cell surface determinants as well as based on the fact that they proliferate rarely, and hence retain bromodeoxyuridine (BrdU) long term has allowed investigators to identify their position in the different tissues [26–28]. This has lead to characterization of the niche cells surrounding the stem cells. These studies have indicated that signals that govern selfrenewal and differentiation of HSC (Wnt’s, TGFb-family members, Notch-signaling) are highly similar for epidermal and gastrointestinal stem cells. Similarly, NSC have been isolated from postnatal brain where they are located in the subventricular zone (SVZ) and the hippocampus. Even though prospective isolation as for HSC, and epidermal and gastrointestinal stem cells is less completely deﬁned, the location of NSC in the SVZ has been deﬁned. Morphogens such as Wnt’s, TGFb-family members and signals via the Notch system also play a role in NSC homeostasis and differentiation. In the 1970s Friedenstein et al. demonstrated that aside from HSC another population of cells could be cultivated from bone marrow, that had the appearance of ﬁbroblasts, but can generate aside from ﬁbroblasts, also adipocytes, chondrocytes and osteocytes [9,29]. These cells were initially termed ﬁbroblast colony forming cells (CFU-F), and were later renamed by Caplan and colleagues and Prockop and colleagues as mesenchymal stem cells or MSC [10,11]. MSC are commonly isolated from BM, and since the initial description by Friedenstein, also from many other tissues, such as adipose tissue, fetal lung, placenta, Wharton’s jelly and UCB, among others [30–33], by culturing cells from these tissues on plastic dishes, to which they attach. MSC give rise to ﬁbroblasts, adipocytes, chondrocytes, osteocytes, smooth muscle cells, and hematopoietic supportive “stromal” cells [9–11,34]. Some have also suggested that MSC may give rise to endothelial cells and cardiac muscle cells, two other mesodermal cell types, although this notion remains contested [35–38]. During the past 3–4 years, more insights have been gained related to the origin of MSC, as well as their stem cell capacities. Studies from the Bianco and the Peault groups have demonstrated that MSC in different tissues are located surrounding blood vessels and are a subpopulation of pericytes [12,39]. They can be prospectively isolated based on expression of the cell surface antigen CD146 [12], and as alluded to earlier, can be grafted in vivo where they reconstitute bone tissue from which they can be re-isolated, demonstrating that they represent true stem cells. Although the location of MSC in vivo has been deﬁned, what the signals are that regulate the homeostasis of MSC in vivo has yet to be deﬁned. MSC, isolated by culturing in the presence of serum on plastic surfaces, are thought to contain only a small fraction of the cells that can be prospectively isolated, as transcriptome studies have shown that the expressed gene proﬁle changes very fast upon culturing MSC in vitro (PJ Simons, personal communication). Whether these cultureinduced changes may also be responsible for changing/broadening the fate of MSC, will be discussed below. Stem cells derived from non-embryonic tissues with broader potential During the last 10 years, a number of groups have isolated cells with broader differentiation ability than MSC from BM and other tissues, such amniotic ﬂuid and UCB, cells by plastic adherence, as is done for MSC. These include MAPC (to be discussed further below), very small embryonic stem cell like (VSEL) stem cells, marrow isolated adult multilineage inducible (MIAMI), multipotent adult stem cells (MASC), amnitotic ﬂuid stem cells (AFS), and unrestricted somatic stem cells (USSC), among others [40–43]. Cell surface phenotype and differentiation potential has been summarized in Table 1. VSEL, are

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Table 1 Adherent adult stem cells with cell surface phenotype and differentiation capacity. Cell type

Derived from

Potency

Surface phenotype

Reference

Human mesenchymal stem cell (MSC)a

Bone marrow, cord blood, tissue resident

Mesenchymal lineage

[10–12]

Human marrow isolated adult multilineage inducible cells (MIAMI) Human Unrestricted somatic stem cells (USSC) Human very small embryonic like (VSEL)

Bone marrow

Mesoderm, endoderm and ectoderm

CD105þ, CD90þ, CD73þ, CD79a, CD45, CD34, CD19, CD14, CD11b, HLA-DRCD164þ, CD122þ, CD81þ, SSEA4þ, CD45, CD34, c-Kit-,

Cord blood

Mesoderm, endoderm and ectoderm Neuroectoder, pancreatic cells, cardiomyocytes

CD10 low, Flk1 low, CD45, CD34, c-kitCXCR4þ, AC133þ, Sca-1þ, CD45-, SSEA1þ(mouse), SSEA4þ(Human), Lin-, MHC-I-

Pancreatic cells, neuronal, mesenchymal

CD49bþ/CD90þ, CD13þ, CD105 low, CD73 low, CD44 low, HLA-ABC low, CD133, CD45, CD34, CD14, HLA-DRCD105þ, CD90þ, CD73þ, CD44þ, CD29þ, MHC-Iþ, c-Kitþ, MHC-II low, CD133, CD45, CD34 c-Kitþ, CD9þ, CD13þ, CD31þ, CD44, MHC-I-, CD45, Thy1-

Human Multipotent adult stem cells (MASC)

Umbilical cord blood, bone marrow mobilized Bone marrow, heart, liver

Human amniotic ﬂuid stem cell (AFS)

Amniotic ﬂuid

Mesenchymal, neuronal, endothelial, hepatic

Rodent multipotent adult progenitor cells (MAPC)

Bone marrow, muscle, brain

Mesoderm, endoderm and ectoderm

a

[41]

[46] [42,43]

[40]

[45]

[47,49, 61,79]

Phenotype based on the International society for Cellular therapy criteria.

cells derived from bone marrow (mouse) and cord blood (Human) [42,43]. While human cord blood derived VSEL cells cannot be expanded ex vivo [42] their mouse counter parts can be grown as spheres in co-culture on C2C12 cells. These cells have been shown to differentiate to cardiomyocytes, neuroectoderm and pancreas [43,44]. They reportedly express Oct4 and Nanog, two transcription factors that govern pluripotency of ESC. MASC are isolated from BM, heart and liver [40]. MASC can divide in vitro for more than 40 population doublings without shortening of telomere length, and can differentiate into neuroectodermal, hepatic and mesodermal lineages in vitro. MASC cells were also reported to express Sox2, Oct4 and Nanog. AFS are isolated from amniotic ﬂuid, express Oct4 and Nanog, proliferate well beyond 100 population doublings and generate mesenchymal, endothelial, hepatic and neuronal lineages [45]. The other cell populations, MIAMI and USSC, by contrast do not express markers associated with pluripotency such as Oct4, Nanog and Sox2. MIAMI cells, isolated from BM, can proliferate extensively, and give rise to pancreatic and neuronal lineages apart from mesenchymal cells [41]. USSC in vitro have the capacity to form fat, bone and cartilage tissue and can generate hepatocyte and neuroectodermal cell types [46]. When implanted in pre immune sheep USSC can contribute to chondrocytes, cardiomyocytes, hematopoietic cells and certain neuron-like cells [46]. Culture conditions for all these cell populations are relatively similar, even though differences exist in cytokines and serum used and ambient oxygen levels. Whether these differences in culture conditions are responsible for the differences between MASC, MIAMI, AFS, USSC and VSEL populations, and between these cell populations and “classical” MSC is unknown. Isolation and characteristics of rodent MAPC We will here discuss the isolation of MAPC from the bone marrow of rat and mouse. An extensive protocol for the isolation of rodent MAPC has recently been published Subramanian et al. [47]; hence we will only brieﬂy highlight the isolation procedure here. Rodent bone marrow cells are ﬂushed from the long bones of young rodents, and plated with EGF, PDGF-BB, LIF in 2% serum containing medium on plastic plates for 4 weeks. Subsequently the cells are depleted of CD45 cells and subcloned at very low

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densities (5 cells/well of 96 well plate) [13,47,48] After 6–8 weeks, clones of cells with MAPC morphology and typical MAPC gene expression (Oct4) and cell surface characteristics (SSEA1 and CD31 for rat MAPC, for example) can be detected. To generate clonal populations, such cell populations can be re-cloned at 0.8 cells/well. Compared with the isolations described by Jiang et al. in 2002, the current protocol differs in that isolations and maintenance of rodent MAPC are done using fetal calf serum under hypoxic conditions. Murine and rat MAPC do not express CD45, a hematopoietic marker nor MHC class II and low to negligible levels of MHC class I. Mouse MAPC express c-Kit, EpCam and CD9 and lack CD34 and CD44, whereas rat MAPC express CD31, SSEA1, and lack CD44. Clones of rat MAPC express Oct4 at levels nearly equivalent to that of mouse embryonic stem cells, and mouse MAPC at levels 10–20% of that in ESC [49]. Of note rodent MAPC do not express Nanog or Sox2. Rodent MAPC differentiate into cells of all the three germ layers [13,49]. Some of these aspects will be highlighted here. Cardiovascular system The cardiovascular system is made up of tissue of the heart and blood vessels. MAPC differentiate robustly to endothelium and smooth muscle making up the components of blood vessel. Aranguren et al. demonstrated that MAPC can be differentiated to both venous and arterial endothelial cells [50] which was conﬁrmed further by Liu et al. and Xu et al. [51, 52]. In a functional analysis study, Aranguren et al. also demonstrated that undifferentiated MAPC could restore muscle function as well as blood ﬂow in a limb ischemia model in part by direct contribution to smooth muscle and endothelium, as well as skeletal muscle, and in part by elaborating trophic factors that enhance endogenous angiogenesis and myogenesis [53]. Ross et al. demonstrated that MAPC when treated with TGF-b and PDGFbb under serum free conditions can form smooth muscle cells (SMC) [54]. These MAPC-SMC express genes such as Transgelin (Tagln or Sm22), Calponin h-1 (CNN1) and more mature markers of smooth muscle such as alpha smooth muscle actin (aSMA) and smooth muscle myosin heavy chain (SM-MHC). MAPC-SMC function like primary mature smooth muscle cells under mechanical stress and chemical stimulation, and express calcium channels similar to those found on mature smooth muscle cells [54]. Tolar et al. and Pelacho et al. demonstrated that when mouse MAPC were grafted in an acute myocardial infract in mice, functional improvement occurred, even though no direct contribution of the MAPC to cardiac muscle was observed, but because of trophic factors generated by MAPC that decreased the infarct size and enhanced angiogenesis [55,56]. Hematopoietic system Seraﬁni et al. demonstrated that when mouse MAPC were injected into lethally irradiated mice, reconstitution of the hematopoietic system could be seen, with generation of HSC that could repopulate secondary recipients. However, 1000 fold more MAPC were required compared to control (KTLS) HSC for reconstitution [57]. Osteoblast differentiation Maes et al. demonstrated that like MSC, MAPC can generate osteoblasts, which is enhanced by placenta-like growth factor [58]. Unpublished studies have also shown that rodent MAPC can differentiate into adipocytes, again akin to MSC. Hepatic differentiation MAPC have been instrumental in development of robust protocol [59] for differentiation to functional hepatocyte-like cells. This protocol is not only applicable for differentiation of MAPC to liver cells, but also can be adapted for mouse and human ESC [60]. Roelandt et al. have demonstrated that by mimicking early embryonic developmental cues that MAPC and ESC can be differentiated to functional hepatocyte-like cells. These hepatocyte-like cells express early hepatic markers such as Afp, Ttr, Alb and mature genes such as G6P, Cyp1a2, and the coagulation Factor-V. Roelandt et al. also demonstrated that

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these MAPC derived hepatocyte-like cells can secrete albumin, produce urea, store glycogen, and express functionally active cytchrome p450 enzymes [60]. Neural differentiation Jiang et al. in 2002 demonstrated that rat and mouse MAPC can differentiate to the neuroectodermal lineage, generating neurons with electrophysiological properties similar to CNS neurons [61]. More recently, differentiation toward neural stem cell-like cells has been achieved using a modiﬁed Conti et al. protocol [62], although maturation to neurons and astrocytes has not yet been achieved [49]. Immunomodulatory role of rodent MAPC Immunomodulation has been associated with certain adult stem cells and has been extensively studied in MSC. MSC derived from BM as well as UCB lack MHC-II and co-stimulatory molecules CD40, CD40L (ligand), CD80 & CD86 on their cell surface [63–65]. MSC inhibit the function of T-cells, B-cells and dendritic cells, and are therefore being tested clinically in immune disorders such as graft versus host disease, and Chrohn’s disease, among others [63,66]. Clones of murine and rat MAPC also demonstrate such immunomodulatory effect. Kovacsovics-Bankowski et al. have demonstrated that clinical scale expanded rat MAPC inhibited GVHD (graft versus host disease) response [67]. These rat MAPC were not sensitive to NK-cell lysis and could in a dose dependent manner suppress T-cell proliferation in mixed lymphocyte response (MLR) [67]. Similarly, mouse MAPC can inhibit in vivo Tcell alloresponse and GVHD, however, the GVHD suppression was only found upon intrasplenic delivery of the cells [68], or a popliteal lymph node assay [69] and not systemic delivery [68]. What is the developmental analog of rodent MAPC? Although rodent MAPC share certain characteristics with MSC, a transcriptome study comparing MAPC to MSC and ESC[49], demonstrated that the expressed gene proﬁle of MSC and rat and mouse MAPC differs signiﬁcantly. Importantly, mouse and rat MAPC express pluripotency markers such as Oct4, c-Myc, Klf4, Sall4, and a number of ESC-associated transcripts (Ecats) [49] similar to those found in ESC, but not expressed in mouse MSC. However, unlike ESC, MAPC do not express Nanog and some Ecats. Interestingly, MAPC express a number of endodermal genes such as Sox17, Foxa2, Hnf1b, Gata4 and Gata6, as well as Sox7 [49]. We concluded from this study that MAPC were most equivalent to extraembryonic endoderm cells (XEN) cells, described by the Rossant group. In 2009 Debeb et al. described the direct isolation from rat blastocysts of extraembryonic progenitor cells, termed XEN-P cells, to which MAPC likely most closely compare. Like XEN-P cells, but unlike XEN cells, MAPC express Oct4, Hnf1b and not Hnf4a, and Ssea-1, and require LIF for their in vitro maintenance [21,49]. Like XEN-P cells and XEN cells, MAPC express Gata4, Gata6, Sox7 and Sox17, but not Nanog. Rodent MAPC express high levels of the pluripotency gene Oct4 whose expression is limited to ESC and cells of the gonads in adult [70]. Oct4 is not required in postnatal life and is not expressed outside of the gonads in adult tissues [70]. We have evidence that Oct4 is not expressed in fresh rat or mouse BM, but that the expression is acquired during in vitro culture. Such a “reprogramming event” is in line with a number of recent observations. Takahashi et al., demonstrated that it is possible to reprogram terminally differentiated adult somatic cell such as ﬁbroblasts to ESC-like called induced pluripotent (iPS) cells by introduction of four transcription factors [71]. Since then, reprogramming has also been achieved by transfection of the factors as proteins, and by using fewer transcription factors in combination with small molecules that affect the epigenetic state of cells. In addition, several groups have demonstrated that culture of spermatogonial stem cells, that express Oct4 but not Nanog and Sox2, under ESC culture conditions can reprogram these single lineage stem cells to cell with most if not all features of ESC [24,72–75], We therefore hypothesize that the rodent MAPC is induced by the longterm culture leading to de-differentiation of rare cells to cells that express Oct4 as well as primitive endoderm genes. How closely related are MAPC and XEN-P cells, i.e. whether MAPC are a cultureinduced reprogramming of BM cells to hypoblast-like cells, is currently being evaluated.

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Isolation, characteristics of human MAPC We and others have also isolated MAPC from human BM. A full description of the isolation and in vitro differentiation ability of human MAPC, in comparison with MSC and Mesoangioblasts, including a transcriptome comparison is forthcoming. Human MAPC can be expanded long-term (more than 70 population doublings), and unlike MSC can robustly generate endothelial cells [50,76]. Compared with rodent MAPC human MAPC are cultured without LIF, but like rodent MAPC with FCS, EGF and PDGF-BB. Like MSC they have extensive immunomodulatory properties in vitro and in vivo [67,77] [and manuscript in preparation]. When grafted in vivo in ischemia models, human MAPC like rodent MAPC, signiﬁcantly increase angiogenesis and endogenous stem cell proliferation, leading to improved CNS [78], cardiac [77] and skeletal muscle function [53]. This has lead to the creation of cGMP-grade human MAPC, termed MultiStemÒ, which is currently being evaluated in phase I clinical trials. Future prospective The premise of studying stem cells is their potential for future cell based therapies for various pathologies. Rodent and murine models and stem cells derived from them have been used widely as an excellent tool to understand the development and pathogenesis of a disease. Their role in human therapies is limited to initial screens. Nevertheless, the studies relating to tissue speciﬁc differentiation of rodent and murine stem cells has been instrumental in understanding and devising better protocols for human stem cell differentiation. Rodent and murine MAPC are being used to study the molecular mechanisms underlying tissue development. The clinical use of human embryonic stem cells is limited not only due to their tumorigenicity in their undifferentiated state but also have ethical limiations. While on the other hand, adult stem cells have more limited differentiation capacity and therefore also less tumorigenic potential, are being considered for clinical use. MAPC of human origin (MultiStemÒ) are currently being evaluated in a number of preclinical vascular disease models, as well as in a phase I clinical trial in patients with acute myocardial infarct, and stroke. It has been demonstrated that MultiStemÒ are tolerated well and positively affect cardiac function [77], even though the functional effects will need to be further established by phase II and III clinical trials. The immunomodulatory properties of MultiStemÒ are currently being tested clinically in a phase I trial to prevent GVHD and in a phase I trial for Crohn’s disease, as well as in preclinical models of solid organ transplantation, among others. The very extensive expansion potential of MultiStemÒ, and their immunomodulatory properties, should allow for clinical trials to be done in an allogeneic setting and using a single donor derived bank of cells, limiting the variability of the graft when multiple donors are required or when autologous cells are used. Their outspoken proangiogenic phenotype also suggests that MultiStemÒ are a good cell source for therapy of ischemic disorders. Conﬂict of interest statement Catherine M. Verfaillie is a consultant to Athersys Inc. References [1] Spangrude GJ, Scollay R. A simpliﬁed method for enrichment of mouse hematopoietic stem cells. Exp Hematol 1990;18: 920–6. [2] Burns AJ, Pasricha PJ, Young HM. Enteric neural crest-derived cells and neural stem cells: biology and therapeutic potential. Neurogastroenterol Motil 2004;16(Suppl. 1):3–7. [3] Joseph NM, Mukouyama YS, Mosher JT, et al. Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial ﬁbroblasts in addition to schwann cells. Development 2004;131:5599–612. [4] Zhang J, Duan X, Zhang H, et al. Isolation of neural crest-derived stem cells from rat embryonic mandibular processes. Biol Cell 2006;98:567–75. [5] Brandl C, Florian C, Driemel O, et al. Identiﬁcation of neural crest-derived stem cell-like cells from the corneal limbus of juvenile mice. Exp Eye Res 2009;89:209–17. [6] Barker N, Huch M, Kujala P, et al. Lgr5(þve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 2010;6:25–36.

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[45] De Coppi P, Bartsch Jr G, Siddiqui MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100–6. [46] Kogler G, Sensken S, Airey JA, et al. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 2004;200:123–35. [47] Subramanian K, Geraerts M, Pauwelyn KA, et al. Isolation procedure and characterization of multipotent adult progenitor cells from rat bone marrow. Methods Mol Biol 2010;636:55–78. [48] Breyer A, Estharabadi N, Oki M, et al. Multipotent adult progenitor cell isolation and culture procedures. Exp Hematol 2006;34:1596–601. *[49] Ulloa-Montoya F, Kidder BL, Pauwelyn KA, et al. Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity. Genome Biol 2007;8:R163. [50] Aranguren XL, Luttun A, Clavel C, et al. In vitro and in vivo arterial differentiation of human multipotent adult progenitor cells. 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Contents lists available at ScienceDirect

Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha

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Prospective identiﬁcation and isolation of murine bone marrow derived multipotent mesenchymal progenitor cells Fernando Anjos-Afonso, Ph.D., Senior Research Fellow *, Dominique Bonnet, Ph.D., Senior Group Leader * Haematopoietic Stem Cell Lab, Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom

Keywords: mesenchymal stroma cell (MSC) mesenchymal progenitor cell (MPC) sca-1 multipotent differentiation pericyte adventitial reticular cell vascular smooth muscle cell (VSMC)

Enormous confusion still exists in the scientiﬁc community regarding the in vivo identity of putative bone marrow (BM) multipotent mesenchymal progenitor cells (MPCs). There is still lack of consensus between laboratories on this issue but recent advancements in this ﬁeld have shed light on the identity of these cells in humans. However, in mice there are limited and reproducible data available that convincingly deﬁne prospectively these cells in vivo. In this review we will critically address: 1) important considerations on how to interpret MPC nomenclature, heterogeneity and differentiation abilities; 2) potential surface antigens that could aid in the isolation of MPC from mouse BM; 3) and their topography and prospective cellular relationship with pericytes, adventitial reticulocytes (ARCs) and vascular smooth muscle cells (VSMCs). Ó 2010 Elsevier Ltd. All rights reserved.

A brief historical and nomenclatural background Detailed introduction of the history of MSCs and their deﬁnition, differentiation, immuno-modulatory properties and potential use of these cells in different disease models are covered by other contributors of these series of reviews. We will brieﬂy introduce these cells and the nomenclature used here. Mesenchymal cells are fundamental cells that form the connective tissue throughout the body. In the bone marrow (BM) they are thought to be mainly mesodermally but also neuro-epithelially derived [1–3]. Many scientists believe that putative mesenchymal stem or progenitor cells exist in adult organisms and they are the founders of ﬁbroblasts, osteoblasts, chondrocytes, adipocytes and smooth

* Corresponding authors. Tel.: þ44 (0) 20 7269 3281; Fax: þ44 (0) 20 7269 3581. E-mail addresses: [email protected] (F. Anjos-Afonso), [email protected] (D. Bonnet). 1521-6926/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2010.11.003

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muscle cells in vivo. The deﬁnitive evidence that bone marrow includes cells that can generate connective tissue-forming cells was originally provided by the pivotal work of Friedenstein and his coworkers [4,5]. First, they demonstrated by heterotopic transplantation the existence of a minor population of cells in human BM that are precursors of osteoblasts [4,5]. These cells were distinguishable from the majority of haematopoietic cells by their rapid adherence to plastic and by the elongated ﬁbroblast-like appearance in culture [4,5]. Then, they were able to show that seeding BM cells at clonal level resulted in the formation of colonies initiated by single cells, named the colony-forming unit ﬁbroblastic, CFU-Fs. CFU-Fs have since been used as the hallmark for the quality and growth potential of human MSC isolates in vitro. However, in the murine system CFU-Fs are highly contaminated with haematopoietic cells, at least in early cultures initiated with unfractionated BM [6,7] making this assay inappropriate as a predictive factor for the quality and growth potential of murine stromal cells. Since then, others have extended these observations supporting the ﬁnding that the cells identiﬁed by Friedenstein were multipotent. In particular work done by Pittenger et al., showed that tri-lineage potential (osteoblast, chondrocyte and adipocyte lineages) clones were present in human BM and provided a substantial description of the cell surface phenotype of these cells [8]. However, the nomenclature used to describe multipotent mesenchymal stromal cells has varied throughout the years and until today there is still no universal consensus to name them. These cells originally termed as “osteogenic stem cells” by Friedenstein [4], were then being introduced as “Mesenchymal Stem Cells” by Arnold Caplan [9]. The latter, although not the most appropriate designation (discussed below), became more widely used by many scientists. In this review we followed the recommendations of the International Society for Cellular Therapy, which proposed the use of “Multipotent Mesenchymal Stromal Cell” (MSC) [10]. This is due to the fact that the current known markers used to isolate these cells are shared by a variety of different mesenchymal cell-types. Consequently, the resulting cultures are stromal cultures that might contain a proportion of cells with immature features, which could be the “Mesenchymal Progenitor Cells (MPCs)” as initially suggested by Dennis et al. [11]. Some important considerations Before starting a detailed examination on the main headlines of this review, we would like to bring up some important issues that could help new investigators in the ﬁeld to better understand the biology of MSCs and how to interpret the available data with a cautious and critical view. From man to mice? MSC biology is one of those unorthodox ﬁelds where we have a better understanding of the human than the murine system. Mouse strain variations and more difﬁcult methods to culture murine MSCs (as they depend on the haematopoietic contaminants to thrive in early cultures) [6,7,12,13]; have hindered the understanding of these cells in mice. Consequently, some examples given in this review will be based on human studies. However, we would like to stress that ﬁndings from the human studies are not necessarily the same in mice. There are numerous examples of this, for instance: Stro-1, one of the best-known human MSC markers has no mouse counterpart; the cell surface epitopes and in vitro differentiation capacities vary between mouse strains, especially the (reduced) capacity of murine MSCs (mMSCs) to form cartilage-like tissue [12,13]; and mMSCs are karyotypically very unstable in culture right from early cultures whereas human MSC are not [14–16]. Therefore, when analysing data from human studies, a cautious inspection is required to apply the biology to mMSCs and vice-versa. Can we call MSCs “Stem Cells”? If we use the very stringent deﬁnition of a “Stem Cell”, which is deﬁned as “at single cell level, a cell capable not only to give rise to different type of progenies but also of self-renewal capacity in vivo assayed by serial transplantation” then presently, MSCs are not fulﬁlling these rigorous criteria (routinely used to evaluate Haematopoietic Stem Cells (HSCs) function). Indeed, the self-renewal capacity of MSCs has been merely associated with their continuous growth in culture with the preservation of in vitro differentiation after multiple cell passages. However, these in vitro features correlate

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poorly with results of in vivo differentiation assays as exempliﬁed by a recent work by Bianco and coworkers showing that the generation of bone tissue in vitro does not appear to reﬂect the capacity of forming bone (also of forming stroma and adipocytes) in vivo, and only 10% of the clones were able to do so [17]. Unlike HSCs, which can be systemically transplanted in vivo with high engraftment efﬁciency, MSCs have to be expanded in sufﬁcient numbers and must be transplanted locally to exert their in vivo functions. One could argue that the feeble knowledge on their identity has hampered their use at single cell level. We still don’t know if all cultured cells have similar regenerative capacity for different lineages in vivo even when prospectively isolated using the current known surface markers. Furthermore, we have to consider that the turnover of cells in connective tissue in an adult organism is relatively low and consequently the rate of self-renewal divisions for the putative MPC in vivo under homeostasis (assuming that really takes place) should also be very low. Moreover, there is no data supporting that in vivo serial transplantation is feasible and therefore assessing self-renewal and multipotency in vivo of MPCs or other stromal cells might require different assays from those applied for HSCs. That being said, evidence for self-renewal of MSCs had recently emerged. Paolo Bianco’s team has shown that clonogenic human CD146þ BM derived cells were able to self-renew. When clonally derived cells were implanted subcutaneously into immunodeﬁcient mice they were able to retrieve a minor population of cells weeks later with a similar phenotype as the initial inoculum and re-grow these cells at a clonal level [18]. Overall, there is a lack of deﬁnite data supporting the idea that MSCs isolated from tissues other than BM contain “Stem Cells”. We would also like to call the attention of the readers to the claims of MSC multipotency in numerous publications based solely on in vitro assays. As mentioned above, the outcome of these assays does not necessarily correlate with in vivo differentiation potential of MSCs. Furthermore, most of the data published relied heavily on representative ﬁgures of speciﬁc lineage staining without any accompanied quantiﬁcation. As an example, it is often found ﬁgures showing just very few Oil Red O positive cells (that depicts adipocytes) claiming that the MSCs in question have adipogenic differentiation. Such data can be misleading and create a barrier for the readers to compare data from different laboratories while also being biologically meaningless. Assessment of the degree of differentiation from MSCs to each speciﬁc lineage by quantiﬁcative methods should be implemented to be able to evaluate the potential of the cells isolated using different methods and from different tissues.

Expansion/Differentiation and heterogeneity Currently, most of the in vitro culture and expansion protocols are based on a basic medium supplemented with 10–20% of selected batches of Foetal Bovine Serum, which certainly does not constitute the most suitable way to maintain the undifferentiated state of MSCs. This is crucial, as the interpretation of the immense data available on their heterogeneity and differentiation potential depend on how MSCs are grown. BM MSCs are widely perceived as a heterogeneous population of cells in vitro, despite the homogeneous expression of most surface antigens used to describe them. This view has been supported by many reports showing that at clonal level not all the cells have the same in vitro differentiation potential [11,19–21]. One of the earliest works reporting this observation was from Muraglia et al., which demonstrated that approximately 30% of all human MSC clones exhibit a tri-lineage differentiation potential [19]. A more recent study using mouse cells from O’Connor’s group reported that tri-lineage MSCs accounted for nearly 50% of the CFU-Fs [20]. The most prominent study on the heterogeneity of cultured MSCs comes from Prockop’s group. They showed that at steady state, MSC cultures contain a minor population of small, agranular and quiescent cells (named RS-1 cells). These RS-1 cells express an antigenic proﬁle that is different from the most abundant fast growing and committed precursors (mature-MSCs) and for example, express the high-afﬁnity nerve growth factor receptor, Trk [22]. When studying a precursor-progeny relationship between RS-1 and other cell-types, it was concluded that the high proliferative capacity of mature-MSCs depends on the presence of RS-1 cells [22]. Moreover, RS-1 cells display a more robust differentiation capacity than mature-MSCs. It seems that RS-1 cells may

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represent a subset of uncommitted cells [22] and therefore RS-1 sub-population could be the putative MPC enriched fraction. The assumption that cultured MSCs are heterogeneous is well supported but whether it is the same for cells in vivo is debatable. Most current methodologies applied for in vitro differentiation assays require a certain cell number and density to drive the cells to a particular lineage [8,12,13,21–23]. It is thus necessary to expand these cells beforehand in order to accomplish in vitro assays. One must consider that in vitro expansion with commonly used medium might cause a substantial number of MSCs to lose their differentiation capacity, even at early passage. This has been supported by many studies which show not only that the differentiation capacity of cultured MSCs is gradually reduced upon cell passaging [24–26], but also that, as MSC culture are passaged, they become heterogeneous as they expand and contain at least 2 subpopulations of cells: small, rapidly self-renewing MSCs (RS-1 MSCs) and larger, slowly renewing MSCs (mature-MSC) [22]. Without a more in-depth knowledge on how to maintain the primitive features of MSCs we have to consider the possibility that MSCs might not be as heterogeneous in vivo. Indeed, some authors have questioned the concept of the heterogeneity of MSC. Jones et al., showed that by using a combinatorial analysis of surface antigens by ﬂow-cytometry that most cells that have CFU-F capacity express many of the commonly known MSC markers uniformly [27,28]. They showed this by ﬁrst positively selecting bone marrow cells using an antibody that recognises ﬁbroblasts (anti-D7-FIB antibody) and then eliminating haematopoietic cells from this analysis. As a result, the remaining cell fraction expresses CD73 (ecto-50 -nucleotidase), CD105 (endoglin), CD90 (Thy-1) and very interestingly, another nerve growth factor receptor, CD271 almost homogeneously [27,28]. Similarly, another paper reported that most of the human BM CFU-F activity comes from the non-haematopoietic CD271þ fraction [29]. Nevertheless, it is likely that some of these markers are shared in vivo with other lineage speciﬁc mesenchymal precursors (or even their subsequent progenies), like adipocyte-precursors whose phenotypes are not well deﬁned for human cells and ﬁbroblasts that have limited differentiation capacities [18,30]. It is possible that these lineage speciﬁc precursors and other cell-types can still form CFU-Fs. Until we can distinguish all the differentiation stages phenotypically for the different mesenchymal cell lineages using surface markers or other means, then apply these measures to further dissect the non-haematopoietic/endothelial CD271þ or CD73þ or other similar BM populations, we can not be sure that these populations are homogeneous. What is clear at this stage is that the heterogeneity observed from MSC cultures is likely a consequence of inefﬁcient in vitro culture systems that are unable to maintain the original features of these cells. Potential surface antigens that could aid the isolation of mouse BM derived MSCs In this section we will summarise what is currently known about the phenotype of mMSCs mostly from data generated by analysis of cultured cells. Before going into more details, a few important issues should be highlighted. First, most of current surface antigens known to be expressed on MSCs are not speciﬁc to MSCs; there is a myriad of positive markers that are also shared by different cell types. Secondly, the expression of some of the positive markers changes during in vitro culture. In addition to the examples mentioned above, the expression of Stro-1 and CD271 are down regulated in culture [27,28,31,32]. In the murine system, the reverse also happens: some antigens that are highly expressed on cultured cells are difﬁcult to detect directly from BM cells (see below). Thirdly, some of the antigens are not universal for all the mouse strains. Altogether these impede the establishment of a panel of surface antigens that can be used consistently to distinguish the most primitive sub-fraction from the rest of the cultured cells and the prospective isolation of these cells from BM of different mouse strains. MPC: how to ﬁnd the needle in a haystack? There is a general consensus that, like in human cells, mMSCs do not express most common surface antigens that are found on haematopoietic and endothelial cells. These include: CD11b (Mac-1), Ter119 (an erythroid lineage marker), CD45 (protein tyrosine phosphatase, receptor type C; a pan haematopoietic marker) and CD31 (PECAM-1; endothelial and haematopoietic marker) [6,7,12,13]. Therefore, the common view is that mMSCs are conﬁned in the CD45CD11bTer119CD31 fraction. Other

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surface antigens that are commonly found in different stem/progenitor populations, such as CD117 (c-Kit; receptor for Stem Cell Factor, SCF), are not found on cultured MSCs [6,7,12,13,33]. Although CD90 (Thy-1) is expressed in human MSCs, the expression of this antigen has been repeatedly reported by many groups, including ours, to be absent on mMSCs derived from most commonly used mouse strains including C57BL/6 mice [6,7,12,13,33]. However, two recent reports have reported CD90 expression in mMSCs from C57BL/6 mice [34,35]. The reason for this discrepancy is unclear and it deserves further analysis. The CD34 (a protein in the sialomucin family) antigen is known to be expressed on cultured MSCs derived from some mouse strains like C57BL/6, FVB/N and NOD/SCID, but not in DAB1 and BALB/c mice [7,12]. Thus, CD34 cannot be used as a universal cross-strain positive surface antigen for identifying mMSCs in BM. The other two highly expressed antigens that are commonly found on both cultured and freshly isolated human MSCs, CD105 and CD73, can also be found in cultured mMSCs derived from different strains [7,12,33] albeit at different levels of expression (Anjos-Afonso, unpublished data). Unfortunately, none of these antigens are very useful in prospectively isolating an enriched population of mMPCs. As an example, the CD73 antigen is difﬁcult to detect in fresh cells from NOD/SCID and ROSA26 mice (C57BL/6 background) (Anjos-Afonso, unpublished data). Interestingly, CD73 is highly expressed on cultured cells derived from the same mouse strains, highlighting the upregulation of CD73 upon culture as mentioned previously (Anjos-Afonso, unpublished data). Our group has found that CD105 is expressed at low levels in freshly isolated BM CD45CD11bTer119CD31 fraction from the aforementioned mouse strains. However, CD105 has been reported to be undetectable in mouse calvaria in vivo or calvaria-derived cells in vitro [36]. Sca-1, the best marker in the mouse system? Sca-1 (stem cell antigen) is a well-known marker used to enrich adult murine HSCs and can be used to isolate a nearly pure HSC population when used in conjunction with additional markers. Sca-1 is an 18-kDa mouse GPI-AP (glycosyl phosphatidylinositol-anchored cell surface protein) of the Ly-6 gene family [37]. Despite our lack of knowledge regarding its physiological role, Sca-1 is used regularly in conjunction with negative selection against mature markers to enrich stem and progenitor haematopoietic cells. There is some solid evidence supporting the idea that Sca-1 could be also a potential positive marker for the identiﬁcation of mMPCs. First, Sca-1/ mice exhibit age-related osteoporosis characterised by weakening in bone material, microarchitectural, and mechanical properties [38]. Decreased osteoprogenitors, osteoblasts and bone formation are apparent in these mutant mice and are the result of reduced numbers of MSCs. Also, Sca-1/ mice display reduced adipogenesis in vitro [38]. In addition to these ﬁndings, some reports have shown that the tri-lineage potential of cells isolated based on Sca1 expression alone [34–36,39] or in combination with other antigens is conﬁned to the Sca-1þ fraction whereas the Sca-1 fraction displays mainly osteo- and chrondrogenic potential [34–36,39]. Unfortunately, Sca-1 expression varies between mouse strains. Strains with the Ly6.2 variants, such as C57BL/6, FVB/N and 129 have a higher percentage of Sca-1 expressing cells in fresh BM cells [40,41], whereas in Ly6.1 strains such as BALB/c and DAB1 display lower percentage of Sca-1þ cells in vivo and an absent-low Sca-1 expression in cultured MSCs [12,41]. The NOD/SCID mouse strain and its variants are widely used mouse models in the study of human haematopoiesis in vivo and there is an increased interest in dissecting how human HSCs interact with cells from the BM niche. Although Sca-1 is highly expressed on cultured MSCs isolated from these immunodeﬁcient mice [7,13], the percentage of Sca-1þ cells in the BM CD45CD11bTer119CD31 is much lower than in C57BL/6 mice (Anjos-Afonso, unpublished data). Even though Sca-1 is one of the best surface antigens known to date, this limits its use as a cross-strain marker to enriched mMPCs. Putting aside this limitation, Sca-1 has been used in combination with other markers such as CD166 (Alcam), CD51 (integrin alpha V) and PDGFRa (plateletderived growth factor receptor alpha) to further enrich mMPCs in vivo [34,35,39,42]. What other markers could be used? An early study from Suda’s group showed that most of the multilineage differentiation capacity of foetal limb perichondrium MSCs resides mainly in the CD166þ fraction [42]. However, our group

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has been unable to identify co-expression of CD166 with Sca-1 in the adult C57BL/6 BM CD45CD11bTer119CD31 fraction. This lack of co-expression has recently been conﬁrmed by the aforementioned authors, demonstrating that adult BM CD45Ter119CD166þ cells are an osteoblastenriched population instead [35]. CD51 is also known be expressed in osteoblasts and has been used to enrich osteoblasts from the BM [39,43], and when combined with Sca-1, a double positive subpopulation can be detected [43]. However, there is no functional data available demonstrating that this double positive subpopulation is enriched for mMPCs. Both PDGFRa and PDGFRb are expressed by different mesenchymal cells. Not only is the PDGF signalling pathway important in promoting survival and proliferation in mesenchymal cells [44,45], but these receptors have also been used in combination with VEGFR2 (vascular endothelial growth factor receptor 2) to identify the point of lineage divergence between lateral and paraxial mesoderm [1]. This makes them ideal as prospective positive markers. When combined with Sca-1 staining, Matsuzaki’s group have recently demonstrated that the CD45Ter119þSca-1þPDGFRaþ BM subpopulation, derived from C57BL/6, is enriched with MPCs with tri-lineage capacity whereas other cellular fractions have a more restricted differentiation potential, mainly lacking fat-forming capacity [34]. Moreover, this population can be found in different mouse strains like DBA-1 and BALB/c mice. In this study the level of Sca-1 expression was similar between all strains tested [34]. The reason for the observed differences from other published studies is currently not clear. Then, the authors went on showing that this population isolated from green ﬂuorescent protein (GFP) mice when injected intravenously into non-GFP recipients, the inoculated cells were able to give rise to some GFPþ osteoblasts and adipocytes in vivo. Although this study provides the ﬁrst plausible enrichment of mMPC using Sca-1 and PDGFRa expression, there are some caveats. First, the subpopulation in question, Sca1þPDGFRaþ, is almost undetectable in the BM and most of the study was performed using cells isolated from bone fragments after collagenase digestion. As such, the isolated cells are mainly bone-derived and not BM derived. It was assumed that the BM has a similar subpopulation based on a histological staining locating two Sca-1þPDGFRaþ cells near a BM vasculature [34]. This assumption is only valid if similar in vitro and in vivo functions are demonstrated and compared between the two populations. Secondly, the seeding efﬁciency of these cells was very low, reaching w25 CFU-Fs/1000 puriﬁed cells. While this frequency is comparable from human studies using non-haematopoietic/endothelial CD164þ or CD73þ BM cells [18,46], one must bear in mind that the Sca-1þPDGFRaþ population was extracted from bone fragments and as the frequency of this population is w40x lower in the BM, this suggests that the seeding efﬁciency should be lower than 1 CFU-Fs/1000 puriﬁed cells. Most importantly, it is known that murine MSCs depend on the haematopoietic contaminants to thrive in early cultures. Consequently, we cannot exclude that non-Sca-1þPDGFRaþ fractions could also contain some mesenchymal progenitors unable to survive in culture and therefore excluded from the analysis. Thirdly, most of the clonally derived populations showed very weak adipogenic differentiation capacity in vitro [34], implying that the Sca-1þPDGFRaþ population it is still heterogeneous. A recent paper showed that the bone-fragment derived non-hematopoietic/endothelial Sca-1þPDGFRaþ cells have similar gene expression proﬁle to Sca-1þCD166 population despite the lack of chondrogenic differentiation potential by the latter [35]. Altogether it highlights the possibility for the Sca1þPDGFRaþ subpopulation to be the fraction where most mMPCs might reside. That said, the literature available is too limited at this stage to make any absolute claims. Moreover there is a necessity to ﬁnd more stable, universal (cross-strain) and highly expressed surface antigen (specially in fresh BM cells) that could substitute or complement the currently known murine markers. MSC topography and the prospective cellular relationship with adventitial reticulocytes, pericytes and vascular smooth muscle cells All the same or different members of the same family with the same outﬁt? Recently, many investigators have used immunohistology to revisit the topography of MSCs in vivo [18,28] and their ﬁndings suggest that MSCs may be identical to BM adventitial reticulocytes (ARCs) [47–49]. The morphological and phenotypical similarities between MSCs and ARCs are well supported (e.g. both express CD10, CD13, CD271, CD146, tissue-non speciﬁc Alkaline Phosphatase (AP))

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[8,18,27,28,47,48]. Findings from these recent reports are similar to that published in the early 90’s when Colombo’s group ﬁrst used CD271 to locate potential BM stromal cells in human trephine sections [48]. They found that CD271þ cells have an oval nucleus and a scant cytoplasm with long dendrites that intermingle with the haematopoietic cells. They also found that a substantial number of CD271þ cells are lining in the abluminal side of sinus endothelial cells and provide the scaffold for the haematopoietic marrow [48]. Moreover, they showed that the number of CD271 expressing cells is correlated with the traditional reticulin, vimentin, CD13 and AP expression [48], thus providing the ﬁrst compelling evidence that CD271 expressing cells represent ARCs. Some investigators have also suggested that MSCs might be vascular pericytes [50–52]. This suggestion has a basis in that: historically, ARCs have also been also named pericytes of the venous sinusoids [47,48] as they are found, though not exclusively, in the perivascular areas; human Stro1þCD106þ or Stro-1þCD146þ express aSMA (alpha smooth muscle actin; a marker that is expressed by most, but not all, pericytes and in VSMCs) or were positive for the 3G5 antigen, which is thought to be speciﬁc for pericytes [31,53]; pericytes have been shown to be able to differentiate into adipocytes, chondrocytes and osteoblasts [54,55]; and several antigens that are expressed in MSCs are also found to be expressed in pericytes including: CD13, CD73, CD146, Stro-1, CD90, aSMA, etc [50,53,56–59]. With respect to these ﬁndings, some authors suggest that the reason why MSC populations could be isolated from different tissues other than BM is because of the presence of tissue-resident vascular pericytes as they form a subendothelial network that spans the microvasculature in most tissues [50–52]. However, VSMCs also share some of the markers of pericytes, such as CD146, PDGFRs, CD13, NG2 and CD105 and therefore it could be argued that they are also similar to MSCs [1,60–65]. Furthermore, VSMCs have also been shown to have tri-lineage differentiation capacities in vitro [66–68]. Apart from pericytes and VSMCs, progenitors with multilineage capacities have also been isolated from the mesenchyme of tunica adventitia (discussed below) [69,70]. This makes it difﬁcult to interpret and distinguish some of the results published when different mesenchymal cell-types that have overlapping features are present in the same tissue and can be co-isolated using the current protocols. As such, stromal cells obtained based on selection with one positive marker or simply by adherence to plastic, are likely to contain different mesenchymal cell-types such as pericytes, VSMCs and adventitia progenitors (if we assume that these cells are distinct from each other). One must acknowledge that there is indeed a high degree of similarity between MSCs and these other cell-types. Moreover, it seems that most MSCs appear to have perivascular topography [18,47,53], although MSC cultures could also be obtained from articular cartilages, which are avascular [71]. However, it is worth noting that some of the data available might have led to presumptive conclusions and one should thus consider the following: 1. Two simple facts that are often forgotten are that all cell-types mentioned are mesenchyme by nature and therefore, it is not surprising that they share numerous cell surface antigens. Second, most tissues, including BM, are very vascular and it is not surprising that stromal cells may have perivascular localization as their main function is to form the supporting network of other structures, including the vascular system. 2. As described previously, some of the markers used to identify MSCs can be up regulated upon cultivation. Hence, all these mesenchymal cell-types that seem to share a list of common antigens could be a consequence of culture. Currently, there is limited data available where multiparameter analyses have been conducted in situ or with freshly isolated cells and then applied these mutiantigen staining approaches for the subsequent isolation of the cells in question. This raises the possibility that some “shared” surface antigens may be actually be useful to distinguish the aforementioned cell-types prior to cultivation. 3. The in vitro differentiation assays that are used to demonstrate the “plastic” potential of the cells might not be the most accurate assays to determine MSC potential, and could be an artifact from the in vitro culture employed. It has been reported that more mature mesenchymal cell-types, such as adipocytes, chondrocytes and osteoblasts seem to be able to de-differentiate and then convert into another lineage in response to inductive extracellular cues [71–74]. In this context, VSMCs have also been shown to be prone to de-differentiation during in vitro cultivation, and consequently being able to re-direct them to adipo- or osteo- or chondrocytic lineages in vitro [66–68]. One could argue that this

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could also happen with pericytes although there is no formal data available to support this view yet. Interestingly, it has been shown that rat aortic smooth muscle cells can become pericytes during angiogenesis in vitro [75]. 4. Variation in expression of markers used to characterise these cell-types exists in other tissues. Interpreting data from experiments in tissues other than BM might not necessarily relate to the same cells derived from BM. In disguise, can we hunt them down? The question remains whether we can delineate the differences between all these cell-types. At very least we can attempt to exemplify their differences. For this, one must ﬁrst deﬁne pericytes: the cells that are embedded within the vascular basement membrane of blood microvessels where they make speciﬁc focal contacts with the endothelium [76,77]. Generally, the arteries and veins are surrounded by single or multiple layers of VSMCs (tunica media), whereas the smallest capillaries are partially covered by single pericytes. The latter are found around capillaries, pre-capillary arterioles, post-capillary venules, and collecting venules. The distinction between pericyte and VSMC morphology and location is not absolute but exists as a continuum of properties ranging from the usual VSMC to the typical pericyte, distributed along intermediate size to small vessels [76,77]. The morphological features are important because in a few studies where multipotent mesenchymal cells were isolated based on CD146 expression, the demonstration of their in situ localisation seems to have depicted VSMCs (with multinuclei layer of cells surrounding endothelial cells) instead of pericytes claimed by the authors of these reports [50,69]. Typically pericytes are positive for CD13, NG2, desmin, aSMA, PDGFRb, CD146 (human) and for the species-speciﬁc 3G5 antibody staining (human and bovine) [50,53,56,77–79]. However, none of these markers are pan-pericyte markers, as already mentioned, with CD13, NG2, desmin, aSMA, PDGFRb and CD146 also being expressed in VSMCs [60–65]. Moreover, the expression of some of these markers is dynamic and varies between tissues. The classic example is the expression of aSMA, which is not found in both bovine retina and rat mesenteric mid-capillary pericytes [80]. Skin and CNS pericytes have almost no expression of aSMA [77,81]. Similarly, NG2 expression is restricted to arteriolar and capillary perivascular cells and is absent on venous pericytes in rats [82]. Endoglin, a prominent MSC marker is also expressed at low levels in human cultured and arterial tissue VSMCs [60,61], and therefore makes its use inappropriate to differentiate between pericytes and VSMCs. The usefulness of other conventional markers like CD90 and CD73 is still unclear. In most studies CD90 has been shown to be expressed on pericytes [50,57,69] but convincing data are lacking on its in situ expression in VSMCs. However, commercially available human VSMC lines do not express CD90. Overall, the available data seem to indicate that CD90 might be useful for this lineage delineation but further studies are required to conﬁrm this idea. There is also ambiguous data concerning whether CD73 is present in pericytes. Human dermal- [79] and adipose- [69] derived pericytes appear to express low levels of CD73, while high expression has been found on cells derived from human skeletal muscle [50] but absent in mouse retinal pericytes [58]. Again, expression data of CD73 in VSMCs is lacking. What can then be used to identify putative MPCs or immature mesenchymal cells from typical pericytes? An evaluation of the literature and our experimental data indicates that desmin expression might be an appropriate candidate. Freshly isolated and cultured cells while sub-conﬂuent (desmin expression can be artiﬁcially induced if the cells are being cultured in constant conﬂuency) do not express desmin in both human and murine MSCs at the protein level [7,83,84,85]. Desmin is expressed in pericytes, albeit at different levels depending on their maturity status [76,81]. As such, reporter mice for desmin might help to address this question. If we consider that Sca-1 is the primary surface antigen for mMSCs and NG2 is expressed in all typical BM pericytes then, according to our ﬁndings, putative mMPCs are unlikely to be pericytes as we found little or no co-expression of these two antigens in freshly isolated BM cells (Anjos-Afonso, unpublished data). Validation on whether NG2 can depict all typical pericytes is necessary to conﬁrm this observation. Nevertheless, recent ﬁndings examining progenitors from muscle and adipose tissues might support our observations. Mesenchymal progenitors derived from adult skeletal muscle (deﬁned as non-haematopoietic/endothelial/muscle Sca-1þCD90þPDGFRbþ cells that reside in the interstitial

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space of muscle tissue) have been shown to have tri-lineage differentiation potential [86]. These cells of mesenchymal origin (vimentinþ) do not express any other pericytic marker including NG2 and aSMA [86]. These cells phenotypically resemble progenitors identiﬁed in mouse aortica adventitia (Sca1þPDGFRbþ cells), which do not express smooth muscle cell markers but can easy differentiate into VSMCs upon PDGF stimulation [86]. Unfortunately, the tri-lineage differentiation capacity was not demonstrated in this study. Interestingly, high adipogenic differentiation capacity can also be achieved from adventitial cells (CD34þaSMACD90þCD146) isolated from human adipose tissue, which is distinctive from what has been suggested as “pericytes” by the lack of CD146 expression [69]. Furthermore, retinal and brain pericytes have different morphology than conventional MSCs in vitro [87,88]. These pericytes have smooth muscle cell-like morphology and upon reaching conﬂuency they form multilayered areas, which retract and form nodules, the extracellular matrix of which becomes mineralised [55,87,88]. The mineralised matrix by the microvascular pericytes is associated with the expression of markers of the osteoblast lineage (AP, bone sialoprotein, osteocalcin, osteonectin and osteopontin) [55,87,88]. These features are not found in conventional MSC cultures. In addition, despite reports claiming that pericytes have adipogenic potential in vitro, stimulated pericytes are hardly stained by Oil Red O dye (which stains for neutral triglycerides and lipids) upon closer inspection [50,54,55,87,88]. Altogether, it suggests not only that typical pericytes may be distinct from putative MPCs but they also don’t seem to have the same degree of tri-lineage potential that is found in conventional BM derived MSC cultures. Conclusions In summary, we currently believe that the murine BM stromal compartment is largely unexplored compared to their human equivalent. The phenotype of the potential BM MPC is still vague but most probably conﬁne to the Sca-1þPDGFRbþ fraction. Moreover, the data available points to the idea that MSCs are related to both progenitors from adventitia and pericytes, (perhaps slightly closer to the former) but further apart from classical VSMCs. We might hypothesise that in adult BM the primitive mesenchymal compartment is composed of a myriad of cell-types with similar phenotypes and differentiation potentials and even perhaps with the capacity of lineage inter-convertibility in vivo rather than one identiﬁable mesenchymal progenitor population, thus explaining the challenges/ hurdles that this ﬁeld had faced over the years trying to identify/isolate these putative cells in vivo. Conﬂict of interest statement No conﬂicts of interest to declare. Acknowledgments We would like to thank Erin Currie and Katie Foster for their valuable comments on the manuscript. Cancer Research UK supported this work. References [1] Sakurai H, Era T, Jakt LM, et al. In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility. Stem Cells 2006;24:575–86. [2] Takashima Y, Era T, Nakao K, et al. Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 2007; 129:1377–88. [3] Morikawa S, Mabuchi Y, Niibe K, et al. Development of mesenchymal stem cells partially originate from the neural crest. Biochem Biophys Res Commun 2009;379:1114–9. [4] Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 1987;20:263–72. [5] Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of ﬁbroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 1970;3:393–403. [6] Phinney DG, Kopen G, Isaacson RL, et al. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem 1999;72:570–85. [7] Anjos-Afonso F, Siapati EK, Bonnet D. In vivo contribution of murine mesenchymal stem cells into multiple cell-types under minimal damage conditions. J Cell Sci 2004;117:5655–64.

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Contents lists available at ScienceDirect

Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha

3

Prospective isolation of human MSC Abhishek Harichandan, MSc, Hans-Jörg Bühring, PhD, Head of Laboratory for Stem Cell Research * University Clinic of Tübingen, Department of Internal Medicine II, Division of Haematology, Immunology, Oncology, Rheumatology, and Pulmonology, Laboratory for Stem Cell Research, Otfried-Müller-Str. 10, 72076 Tübingen, Germany

Keywords: mesenchymal/stromal stem cells MSC prospective isolation surface antigens MSC subsets

Conventionally, mesenchymal/stromal stem cells (MSC) are functionally isolated from primary tissue based on their capacity to adhere to the plastic surface. This isolation procedure is hampered by the unpredictable inﬂuence of secreted molecules or interactions with co-cultured hematopoietic and other unrelated cells as well as by the arbitrarily selected removal time of non-adherent cells prior to expansion of MSC. Early removal of non-adherent cells may result in the elimination of a late adhering MSC subsets and late removal increases the inﬂuence of undesired cells on the growth and differentiation of MSC. Finally, in conventional protocols MSC are co-expanded together with macrophages, endothelial cells and other adherent cells. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSC based on the selective expression or absence of surface markers. Here we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow-derived MSC. Ó 2011 Elsevier Ltd. All rights reserved.

Characteristics of bone marrow-derived MSC Mesenchymal stem/stromal cells (MSC) are multipotent cells, which are able to form ﬁbroblast-like colonies (CFU-F) [1,2]. After expansion in culture, bone marrow-derived MSC express the surface markers CD29, CD73, CD90, CD105, CD106, CD140b, and CD166 but lack CD31, CD45, CD34, CD133, and MHC class II expression [3–5]. They are not only able to differentiate into osteoblasts, adipocytes, and chondrocytes, but also into cells of non-mesodermal lineages including hepatocytes, neuron-like and pancreatic-like cells [6–11]. Because of their microenvironment forming ability and multi-lineage * Corresponding author. Tel.: þ49 7071 2982730; Fax: þ49 7071 292730. E-mail address: [email protected] (H.-J. Bühring). 1521-6926/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2011.01.001

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differentiation capacity, they present an attractive cell source for co-transplantation with hematopoietic stem cells as well as for replacement therapy of damaged tissues in patients with osteoarthritis, spinal cord injury, cardiovascular, neurological, and immunological diseases [12–15]. MSC from other tissues Originally, MSC were derived from cultured plastic-adherent bone marrow cells but meanwhile, a number of other tissues have been identiﬁed that contain MSC at varying frequencies and with varying differentiation capacities. Additional sources with MSC potential include placenta, adipose tissue, peripheral blood, umbilical blood, amniotic ﬂuid, fetal hepatic and pulmonary tissue, skin, and prostate [3,16–25]. Although cultured MSC from all sources appear to be negative for CD31, CD45, CD80, and uniformly express CD9, CD10, CD13, CD29, CD73, CD90, CD105, and CD106, a more tissue-speciﬁc expression of other surface antigens has been reported. For example, only adipose tissue-derived MSC express high levels of CD34, and only placenta-derived MSC but not bone marrow-derived MSC are positive for SSEA-4, and TRA-1-81 [17,26]. In contrast, bone marrow-derived MSC but not placentaderived MSC express high levels of CD271 and tissue non-speciﬁc alkaline phosphatase (TNAP) [6,27–29]. MSC from different sources do not only display differential expression patterns of surface antigens but also vary in their differentiation capacity. In a recent publication it was demonstrated that bone marrow-derived MSC display a better chondrogenic differentiation potential compared with MSC of other sources [30]. As MSC represent an attractive tool for cartilage tissue repair strategies, bone marrow is considered as the preferred MSC source for these therapeutic approaches [30]. Isolation procedures of MSC Functional isolation of MSC Conventional procedures to prepare MSC for research and clinical purposes rely on the expansion of unselected bone marrow cells based on their capacity to adhere to the plastic surface in culture dishes. These functionally isolated MSC are expanded in deﬁned media in the presence of platelet lysate or other growth factor compositions [4,31–33]. This isolation procedure is accompanied by several limitations including i) undesired interactions of MSC with hematopoietic cells and their released growth factors in the ﬁrst culture period, ii) the challenging decision to deﬁne the optimal time point of removal of nonadherent cells and replacement with fresh media, and iii) the co-expansion of other adherent cells – mainly macrophages and endothelial cells – during the expansion period. In addition, functionally isolated MSC do not provide any information about the antigenic composition of the starting population. As a consequence, most of publications describe retrospective antigen expression proﬁles of MSC progeny but not of the initiating cells. Not surprisingly, a variety of surface markers such as CD109, CD166 (however, this molecule is expressed on CD56þ MSC in primary BM), and CD318, are exclusively found on cultured MSC but not on their primary counterparts [6,34]. Vice versa, other markers like CD271 or CD56, which are known to be highly expressed on primary MSC or MSC subsets, are rapidly downregulated in culture [6]. Despite the limitations of functional isolation protocols, these procedures are still prevalent for large-scale MSC preparations in clinical settings because expensive GMP-manufactured antibodies for immunoselection are not required. Prospective isolation of MSC In contrast to functional isolation procedures, the prospective isolation of MSC allows a precise deﬁnition of the starting population. This isolation procedure also precludes the potential adverse effect of co-cultured hematopoietic cells and avoids the potential removal of important MSC subsets together with other non-adherent cells. In addition, no other adherent cells are co-cultured that may interfere with the expansion of MSC. To isolate MSC from primary bone marrow or other tissue, several markers were identiﬁed, which are suitable to enrich for these cells. These markers include antibodies against a variety of surface molecules including CD49a, CD63, CD73 (SH3/SH4), CD105 (SH2), CD106, CD140b, CD271, TNAP, Hsp90-beta, as well as orphan antigens deﬁned by antibodies STRO-1, W3D5,

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Table 1 List of antigens/antibodies used for the prospective isolation of MSC. Markers used A) Known antigens for positive selection CD9 (MRP-1; MIC3) CD10 (Neprilysin; CALLA) CD26 (DPP4) CD34 (MY10; gp105-120)) CD44 (PGP-1; ECMR-3) CD49a (Integrin a1) CD49e (Integrin a5) CD56 (NCAM) CD63 (MLA1; TSPAN30) CD73 (NT5E) CD90 (Thy-1)

CD105 (Endoglin)

CD106 (VCAM-1) CD117 (C-Kit) CD130 (gp130) CD140b (PDGFRB) CD146 (MCAM)

CD166 (ALCAM)

CD200 (MRC, OX2) CD271 (LNGFR)

CD309 (Flk-1; VEGFR-2) CD349 (Frizzled-9) ALDH GD2 (Neural Ganglioside) HSP90beta Integrin alphaV/beta5 SSEA-4 TNAP B) Unknown (antibody-deﬁned) antigens for positive selection. 3G5 D7-FIB STRO-1 W5C5a C) Known antigens for negative selection CD3 (T-cell surface glycoprotein) CD14 (LPS receptor) CD31 (PECAM-1)

Tissue

References

Synovial membrane Placenta Placenta Adipose tissue Bone marrow Bone marrow Bone marrow Bone marrow Bone marrow Bone marrow Adipose tissue Bone marrow Synovial membrane Endometrium Synovial membrane Bone marrow Cartilage Endometrium Wharton’s jelly Bone marrow Umbilical cord Amniotic ﬂuid Bone marrow Endometrium Bone marrow Adipose tissue Endometrium Synovial membrane Cartilage Bone marrow Fetal membranes Bone marrow Amnion Bone marrow Chorion Adipose tissue Bone marrow Placenta Bone marrow Bone Marrow Umbilical chord Bone marrow Bone marrow Bone marrow Bone marrow

[44] [17] [17] [22–24,45–47] [48] [49–53] [54] [6,27,28] [52] [55–57] [23,24] [58] [44] [59] [60] [48,56,57,61–63] [64] [65] [66] [67,68] [21] [36] [55] [69] [55,70] [16,71] [59,65,69] [44] [64] [52] [3] [55] [3] [6,27,28,33,37,63,72–75] [3] [45] [76] [17] [35] [77] [20] [78] [55] [79] [6,27–29]

Adipose tissue Bone marrow Bone marrow Adipose tissue Bone marrow

[16] [74,80] [29,49,52,67,68,81–87] [16] [37]

Peripheral blood Peripheral blood Bone marrow Adipose tissue

[19] [19] [76] [22–24] (continued on next page)

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Table 1 (continued ) Markers used

Tissue

References

CD34 (Hematopoietic progenitor cell antigen)

Bone marrow Peripheral blood Bone marrow Lung Adipose tissue Adipose tissue Adipose tissue Adipose tissue Bone marrow Bone marrow

[29,76,87–89] [19] [49,54,62,74,81,87,88,90] [18] [23] [23,24] [22] [23,24] [49,81,83,87,90] [33,63,90]

CD45 (Leukocyte common antigen)

CD105 (Endoglin) CD144 (Cadherin-5) CD146 (MCAM) CD235a (GlycophorinA) Lin (various antigens) a

Manuscript in preparation.

W5C5 (Table 1A and B). In some cases, MSC were not or not only selected by their immunophenotype but rather by functional features like the enzymatic activity of aldehyde dehydrogenase, which is known to be increased in stem cells of many tissues [35]. Employing Aldeﬂuor as a speciﬁc dye to monitor this enzyme activity, a 9.5 fold enrichment of bone marrow-derived MSC in the Aldeﬂuorbright population was achieved (Table 1A) [35]. In other approaches, bone marrow-derived MSC were enriched by negative selection, employing markers such as CD14, CD34, CD45, and/or CD235 (glycophorin A) and other “lineage-negative” markers (Table 1C). Distinct markers are required for the selection of MSC from other sources than bone marrow because of some unique phenotypic peculiarities. For example, placenta-derived MSC are preferentially isolated using antibodies against CD349 (frizzled-9), SSEA-4, and TRA-1-81, which is in contrast to their bone marrow-derived counterparts that are preferably isolated by CD271 or TNAP selection [17]. Other markers such as CD34 and CD117 are more suitable to select adipose- and amniotic ﬂuid-derived MSC, respectively (Table 1A) [36]. The CFU-F assay is the most frequently used test to analyze the clonogenic potential of prospectively isolated MSC [1,2]. Candidate antibodies selective for MSC can be evaluated by screening their reactivity with cell populations that express established key MSC markers such as CD271 or STRO-1. An example is shown in Fig. 1, showing bone marrow cells double stained of with antibodies against CD271 and CD140b (PDGF receptor-beta). The FACS plot demonstrates that only CD271bright but not CD271dim cells give rise to clonogenic MSC and that these populations differ considerably in their morphological appearance. Giemsa staining shows that CD271bright cells are characterized by a relatively bright nuclear staining and a high cytoplasmic content, compared to the immature lymphoblastoid appearance with darker nuclear staining of CD271dim cells. The plot additionally shows that CD140b is a more selective marker for MSC isolation than CD271, because this molecule is expressed only on CD271bright but not CD271dim cells. As expected, clonogenic cells (CFU-F) were exclusively found in the CD140bþ population. Using this screening approach, additional antibodies with speciﬁcity for MSC have been identiﬁed [6,17,27,28,37] and may be discovered in the future. Prospective isolation of MSC subsets Several groups have reported that MSC are heterogeneous with respect to their growth and differentiation potential [34,38,39]. However, little information exists about markers that discriminate between developmentally, functionally, and morphologically distinct MSC subsets. Recently, we introduced a monoclonal antibody against CD56 that recognizes a distinct MSC subset with high selectivity [6]. This antibody (termed 39D5) detects a CD56 epitope, which is not expressed on NK cells (Fig. 2A) but is highly expressed on about 0.5–15% of CD271bright cells (Fig. 2B). Giemsa staining revealed that CD56 cells contain a large bright cytoplasm with vacuoles, whereas cells of the CD56þ subset contain a smaller cytoplasm with basophilic granules (Fig. 2C). Interestingly, cells of the CD56þ population were about two times more clonogenic than cells of the CD56 subset (Fig. 2D). Further analysis has shown that this increased frequency of clonogenic cells is correlated with an increased proliferation rate [6]. Surface marker expression analysis of sorted CD56þ and CD56 cells revealed that

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Fig. 1. Morphological features and clonogenic capacity of sorted CD271brightCD140bþ and CD271dimCD140b bone marrow cells. Cells were stained with anti-CD271 and anti-CD140b, gated on the indicated populations, and sorted by ﬂow cytometry. Fourteen days after culture in serum-free, b-FGF containing medium, the resulting colonies were enumerated and CFU-F numbers normalized to 5.000 plated cells. Note that CFU-F were exclusively found in the CD140bþ subset and only cells of this subset gave rise to ﬁbroblast-like cells. The morphology of CD271dim and CD271bright cells was evaluated by staining of sorted cells with Giemsa.

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Fig. 2. CD56 deﬁnes a subset of MSC. A) CD56 epitope NCAM16.2 but not 39D5 is expressed on natural killer cells from peripheral blood. B) CD56 is expressed on cells of a CD271bright MSC subset. C) CD271brightCD56þ and CD271brightCD56 BM cells are clonogenic. CFU-F derived from 500 FACS-sorted cells were stained and scored as described. Data represent the mean CFU-F numbers of three different experiments (* ¼ p < 0.01). D) Morphology of CD271brightCD56 and CD271brightCD56þ cells. Subsets were sorted, cytocentrifuged, stained with Giemsa solution, and scored on a Zeiss Axiovert 200 microscope. Note the presence of basophilic-like granules in cells of the CD271brightCD56þ population.

only CD56þ cells but not CD56 cells coexpress CD166, and only a subset of CD56 cells but not CD56þ cells express CD349 (frizzled-9) [6]. However, when these cells were put into culture, both cell types expressed high levels of CD166 and moderate levels of CD349 (Fig. 3). The tumor antigen CDCP1 (CD318), which is not expressed on primary MSC, was upregulated after culture of cells from both MSC populations, whereas CD271 and CD56 were rapidly downregulated. When cultured cells of both populations were induced to differentiate into deﬁned cell lineages, only MSC derived from the CD56 population were able to differentiate into adipocytes [6]. This population was also the major source for

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Fig. 3. Expression of selected markers on cultured MSC derived from sorted CD271brightCD56 and CD271brightCD56þ cells. Sorted MSC were cultured on gelatine-coated ﬂasks and cultured in b-FGF containing serum free medium. After reaching conﬂuence, cells were stained with antibodies against the indicated antigens. Note the rapid downregulation of CD56 and CD271 after culture and the upregulation of CD318.

MSC with osteogenic differentiation potential. In contrast, only MSC from the CD56þ subset effectively gave rise to chondrocytes, suggesting that this subpopulation is the preferred source for therapeutic approaches in the ﬁeld of cartilage tissue repair [6]. We have previously shown that CD56þ MSC express low levels of TNAP, a molecule that is upregulated during osteogenic differentiation [6]. In a model proposed by Gronthos et al. cell surface expression of TNAP is absent on early STRO-1þ stem cells but upregulated during osteogenic differentiation [40]. This STRO-1þTNAP population may correspond to the recently described CD56þTNAP/ dim subset, which was identiﬁed by our group. In agreement with this hypothesis, cells of the CD56þ subset mature at a later time point into osteoblasts compared to CD56 cells. We therefore propose an extended model, in which STRO-1þCD56þTNAP/dim MSC represent an immature precursor with multi-lineage differentiation capacity. Cells committed to the chondrocyte lineage diverge at very early (CD56þ) stages of MSC differentiation. This chondrogenic potential, which is rapidly lost upon differentiation into TNAPþCD56 cells, is accompanied by the induction of the adipogenic differentiation potential and by the enhancement of the osteogenic differentiation capacity. Several reports underline the important role of CD56 expression on ﬁbroblasts to support the growth of hematopoietic stem cells [41–43]. The contribution of CD56 was initially described in mouse and monkey [41,42], but a more recent report showed that CD56 expressed on a mouse stromal line plays a crucial role to support human hematopoiesis in vitro and in vivo [43]. The authors showed that co-culture of CD34þCD38 cord blood cells with a CD56þ stromal cell line resulted in a signiﬁcantly greater expansion rate of CD34þ hematopoietic cells compared to stromal cells, which did not express CD56. This enhancing effect could be blocked by the addition of an inhibitory anti-CD56 antibody, suggesting that direct interactions between CD56 molecules from different cells are essential. It remains open, whether CD56 on human MSC plays a similar hematopoiesis supporting activity. As human hematopoietic stem cells do not express CD56, it is unlikely that a potential supporting effect is caused by homotypic interactions between CD56 molecules on stromal and hematopoietic cells. Rather, CD56þ stromal cells may interact with extracellular matrix components such as heparin sulfate or chondroitin sulfate proteoglycans. We have recently shown that SSEA-3, but not SSEA-4, TRA-1-60 and TRA-1-81, is a candidate marker for MSC derived from primary femur derived bone marrow [27]. To verify this assumption, bone marrow cells were stained with CD271, SSEA-3, and CD56 and gated on CD271brightSSEA3CD56, CD271brightSSEA-3þCD56, and CD271brightSSEA-3CD56þ cells (Fig. 4A). The clonogenic potential and the differentiation capacity of the sorted populations were determined by CFU-F assays and appropriate differentiation protocols. As shown in Fig. 4, clonogenic cells were about 44-fold enriched for CFU-F in the CD271brightSSEA-3þCD56 population, 83-fold in the CD271brightSSEA3CD56þ population, but only about 2-fold in the CD271brightSSEA-3CD56 fraction. Not surprisingly,

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Fig. 4. SSEA-3 deﬁnes a subset of MSC complementary to CD56. A) Display of CD56 versus SSEA-3 of triple stained bone marrow cells gated on the CD271bright cells. Note that CD56þ MSC lack coexpression of SSEA-3. B) Culture of sorted bone marrow cells gated on the indicated populations (Passage 0). Cells were sorted on a FACS Aria cell sorter (Becton Dickinson) and transferred into T25 ﬂask and cultured for 12 days. C) Osteogenic differentiation of MSC. After 10 days of culture in differentiation medium, osteoblasts were stained for alkaline phosphatase activity. D) Adipogenic differentiation of MSC. After 21 days of culture in differentiation medium, adipocytes were stained for neutral lipids with Oil Red O dye and visualized on a Zeiss Axiovert 200 microscope. Note the exclusive presence of adipocytes in the CD56- subsets. E) Clonogenic capacity of sorted BM cells. Deﬁned cell numbers of the sorted BM cells were plated on gelatine-coated ﬂasks and cultured serum free in b-FGF containing medium. 12 days after culture, the colonies were enumerated. CFU-F numbers were normalized to 2000 plated cells.

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CD271brightSSEA-3þCD56 MSC gave rise to osteoblasts and adipocytes (Fig. 4C and D), but not to chondrocytes (not shown). In contrast, CD271brightSSEA-3CD56þ cells were able to differentiate into chondrocytes (not shown) but not into adipocytes. Collectively, SSEA-3 is a suitable and selective marker for the isolation of osteoblast and adipocyte precursors, whereas CD56 is the more appropriate target for the isolation of chondrocyte precursors. Isolation of single MSC We have recently described the prospective isolation of MSC subsets from primary tissue using antibodies against molecules, which are selectively expressed on the surface of these subsets [6,28]. Although phenotypically distinct MSC subsets exhibit properties, which are unique with regard to their proliferation and differentiation capacity, there is still a broad heterogeneity at the clonal level [6]. Heterogeneity of individual MSC clones has been reported by several groups, who demonstrated that the developmental and proliferative potential was highest in cells giving rise to large colonies, whereas small-sized colonies were derived from cells with limited differentiation and proliferation capacity [6]. Sorting of single cells into culture plates does not only provide information about the growth characteristics of individual MSC clones but also about the frequency of MSC clones with deﬁned differentiation potential. Pittenger et al. described that almost 100% of colonies derived from single bone marrow cells underwent osteogenic differentiation, about 80% of the colonies revealed adipogenic differentiation potential, but only 30% of the colonies showed chondrogenic differentiation potential. Our group was successful to isolate clones with capacity for osteoblast but not adipocyte differentiation as well as for adipocyte but not osteoblast differentiation [6]. Further experiments are required to determine the frequency of MSC with multipotent differentiation capacity as well as of MSC with restricted differentiation potential. These analyses may contribute to customize complex models of MSC maturation and differentiation, similar to those proposed for cells of the hematopoietic system. Concluding remarks Conventionally, MSC are functionally isolated by their capacity to adhere to the surface of culture plates. The resulting cells are poorly deﬁned and give rise to a heterogeneous mixture of cells including MSC, reticular cells, macrophages, and endothelial cells. To gain information about the starting population, several markers have been introduced to prospectively isolate MSC and their subsets. A similar degree of hierarchy and progenitor cell heterogeneity may exist among MSC as described for the hematopoietic system. The identiﬁcation of MSC subsets and clonal analysis of individual MSC may contribute to gain more insight in the heterogeneity of MSC from different tissues.

Practice points Bone marrow-derived CD56þ MSC may be considered as a promising starting population for therapeutic approaches in the ﬁeld of cartilage tissue repair. Bone marrow-derived SSEA-3þ MSC may be considered as a promising starting population for therapeutic approaches in the ﬁeld of bone repair.

Research agenda Novel MSC-reactive antibodies combined with clonal analysis of individual MSC may contribute to establish a developmental scheme similar to the hematopoietic system.

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Conﬂict of interest None to declare. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich SFB-685 (Immunotherapy: Molecular Basis and Clinical Applications) project C10: Development of therapeutic antibodies for the elimination of tumor stem cells; by the DFG project BU 516/2-1: Identiﬁzierung und funktionelle Untersuchung von MSC-speziﬁschen Molekülen; and by the DFG project SK49/10-1: Identiﬁcation and functional analysis of human adult spermatogonial and germ line stem cells. The authors would like to thank Kavitha Sivasubramaniyan for her critical revision of the manuscript and acknowledge Drs. Uwe Ochs and Peter de Zwart for supply of bone marrow samples. References [1] Friedenstein AJ, Deriglasova UF, Kulagina NN, et al. Precursors for ﬁbroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 1974;2:83–92. [2] Friedenstein AJ, Gorskaja JF, Kulagina NN. 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Best Practice & Research Clinical Haematology 24 (2011) 37–47

Contents lists available at ScienceDirect

Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha

4

Osteoprogenitors and the hematopoietic microenvironment Paolo Bianco, Professor of Pathology a, b, *, Benedetto Sacchetti, Ph.D Postdoctoral Fellow a, b, Mara Riminucci, Associate Professor of Pathology a, b a b

Department of Molecular Medicine, La Sapienza University, 00161 Rome, Italy Biomedical Science Park San Raffaele, 00128 Rome, Italy

Keywords: Hematopoietic stem cell Hematopoietic niche Mesenchymal stem cell Bone marrow stromal cell

The identiﬁcation of skeletal progenitor cells in the human bone marrow (so-called mesenchymal stem cells) by anatomy and phenotype (CD146-expressing, adventitial reticular cells) has coincided with the recognition that the ability to transfer the hematopoietic microenvironment is an inherent property of skeletal progenitor cells. Inasmuch as these cells generate osteoblasts, associate with sinusoids (the assembly of which they dynamically direct), and coincide with, and self-renew into, stromal reticular cells, these cells are pivotal organizers of the hematopoietic microenvironment. Their nature as osteogenic cells and sinusoidal location reconcile the dual view of endosteal surfaces and sinusoidal walls as the hematopoietic stem cell “niches”, and highlight the dynamic nature of a niche/microenvironment essentially maintained by cells with properties of progenitors/stem cells for skeletal tissues. This view brings the long recognized, and somewhat mysterious, interaction between bone and bone marrow into a new perspective, where two stem cells interact with each other at the same niche. Ó 2011 Published by Elsevier Ltd.

A seminal experiment marking the birth of two key concepts A single seminal experiment [1] stands at the date and place of birth of two lines of investigation that were to become, and currently are, the focus of extensive attention. The ﬁrst line of investigation lead to the large wealth of ex vivo and in vivo studies aiming at elucidating the role of microenvironmental cues in regulating not only hematopoiesis, but also hematopoietic stem cell function – what

* Corresponding author. Department of Molecular Medicine, La Sapienza University of Rome, Division of Pathology, Viale Regina Elena 324, 00161 Rome, Italy. Tel.: þ3906 444 1049; Fax: þ3906 4940896. E-mail address: [email protected] (P. Bianco). 1521-6926/$ – see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.beha.2011.01.005

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is presently often referred to as the hematopoietic “niche”. The second line was to develop into the concept that bone marrow includes a second type of stem cell, in addition to hematopoietic stem cells (HSCs), referred to as “osteogenic” [2], “stromal” [3], or “skeletal” [4,5] stem cells, prior to their rediscovery as “mesenchymal stem cells” [6,7]. The experiment [1] consisted in transplanting bone-less fragments of mammalian bone marrow at heterotopic sites. The general biological question being pursued by Tavassoli and Crosby was to determine why hematopoiesis is restricted to bone as the dominant site in post-natal mammals. Within this general context, the speciﬁc question tackled by a speciﬁc experimental approach (heterotopic transplantation) was whether hematopoiesis can indeed be transplanted to a non-hematopoietic, nonskeletal site. The answer that nature provided was, at a glance, cryptic and obscure, seemingly irrelevant to the question being asked – can hematopoiesis be established heterotopically? That answer was – “there is an osteogenic potential in the bone marrow”. Since the time of these ﬁndings, investigators have failed to grasp the relevance of nature’s answer to the investigators’ quest. Hematopoiesis can indeed be transferred to heterotopic sites, if the osteogenic potential of bone marrow is also transferred. Many years later, we can now understand the meaning. It is cells endowed with that potential (hence, a population of skeletal progenitors), which establishes the hematopoietic microenvironment. Indeed, a most conspicuous result of the Tavassoli and Crosby experiment was that heterotopic transplantation of marrow leads to formation of bone. Actually, of an architecturally sound “ossicle,” with a true shell of cortical bone, a cavity, and hematopoietic tissue contained within it. This observation provided the foundation for subsequent work by Friedenstein and others [8–10], who assigned the osteogenic potential revealed by the Tavassoli and Crosby experiment to non-hematopoietic, stromal cells contained within the bulk of hematopoietic tissue. It was the work of Friedenstein and coworkers that established the concept that bone marrow stromal cells would include a population of non-hematopoietic, skeletogenic, multipotent progenitors able to generate, in vivo, histology-proven skeletal tissues. This concept was much later popularized, and to a large extent corrupted, into the quite different concept of “mesenchymal stem cells”. Read through the most common interpretation of the concept, “mesenchymal stem cells” would be ubiquitous and equipotent cells found throughout the body and capable of generating virtually every mesoderm derivative (beyond skeletal tissues, including also muscle, endothelial cells, cardiomyocytes, etc.) and even non-mesoderm derivatives such as liver cells (endoderm derivatives) and neurons (neuroectoderm derivatives) (reviewed in [11]). While revealing the osteogenic potential of bone-free marrow, Tavassoli and Crosby’s work established at the same time the notion that indeed, hematopoiesis can be established at heterotopic sites, but that it is dependent on the establishment of a bone structure ﬁrst. Again, this observation emphasized that close link between bone and marrow that had attracted the investigators’ attention, and before that, the attention of philosophers and poets alike (from Aristotle to Cervantes and Shakespeare), long before the simple observations of a young unsettled Italian fellow (Giulio Bizzozero) and an established German professor (Ernst Neumann) would ascribe to bone marrow the function of making blood cells [12]. This sets the stage for the concept of a hematopoietic “niche” in bone, ﬁrst proposed by Schoﬁeld in 1978 [13], and inherited by present-day experimental work, however, leaving unsolved the problem of which cell(s) would in fact provide the “niche”. The multiple meanings of a niche In spite of its universal usage, the term niche does not actually convey a univocal meaning. In Schoﬁeld’s view, it meant the components that would ensure constant regeneration of hematopoietic tissue (and hematopoietic progenitors) in bone through renewal of stem cells. Inherited by what was to become a stem cell ﬁeld encompassing a much broader array of stem cell types and dependent systems, a “niche” is the set of spatially deﬁned environmental cues dictating self-renewal (hence, asymmetric division) of stem cells, in any tissue where stem cells exist and operate [14]. In a modiﬁed view (e.g., [15]), which has gained wide acceptance in hematology, a “niche” is the site where hematopoietic stem cells are kept quiescent (rather than self-renew; that is, the site where they do not divide rather than the site where they divide asymmetrically). Finally, in most studies attempting to grasp the microanatomy of a microanatomically deﬁned entity, it is the place where one can localize bona ﬁde HSCs (whether functionally or phenotypically deﬁned, and regardless, of course, of any consideration

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of their kinetics) [16–19]. As related to the speciﬁc biology of hematopoiesis, therefore, the “niche” remains a quite effective and readily communicated word, but also a quite elusive concept of an elusive entity. In contrast, one faces much less uncertainty when trying to deﬁne the “hematopoietic microenvironment.” By this, one means the set of local cues provided by cells of non-hematopoietic origin and dictating the development of long-term hematopoiesis – either in vitro, as per the seminal experiments of Dexter and Allen [20,21], or in vivo, as per a line of investigation that goes from Tavassoli and Crosby (and their largely unknown XIX century antecedent, Goujon [22]), through the work of Friedenstein and coworkers, to our very day. Heterotopic transfer of the HME Transplantation has long been the mainstay of in vivo experimental dissection of crucial components of the hematopoietic microenvironment, through a series of experiments sharing the histology-proven establishment of hematopoietic tissue at heterotopic sites as the critical readout. Progressive reﬁnement of the same fundamental approach over time led from transplantation of entire bone marrow fragments [1] to transplantation of non-hematopoietic, adherent stromal cells, to transplantation of single clones of stromal cells [8]. These studies clariﬁed that the osteogenic potential associated with bone marrow demonstrated by Tavassoli and Crosby is in fact associated with non-hematopoietic stromal cells that can be grown in vitro, and indeed with single clonogenic stromal cells (Colony Forming Unit-Fibroblast, CFU-F) capable of giving rise to multiple non-hematopoietic cell types (the osteogenic stem cells of Friedenstein and Owen). At the same time, these experiments progressively narrowed the focus on cells that actually transfer the hematopoietic microenvironment. In all of these experiments, bone of donor origin is established along with hematopoietic tissue of host origin, giving rise to an artiﬁcial bone/bone marrow organ that exhibits reverse chimerism (“reverse” bone marrow transplantation) as compared to the chimeric bone marrow generated by transplantation of hematopoietic progenitors. Distinguishing individual stromal cells hidden amongst hematopoietic cells within the heterotopic ossicles, in order to determine their donor or host origin, remained essentially unfeasible, or perhaps unaddressed, until recently when their donor origin was unequivocally demonstrated [23]. In all of these experiments, bone is formed before a network of sinusoids is established, and hematopoiesis does not appear until such network is developed. One of the appealing aspects of heterotopic transplantation systems rests indeed with their ability to recapitulate a temporal sequence of events in which signiﬁcant hints can be encrypted. In such systems, as well as in individual bone rudiments developing orthotopically in vivo, hematopoietic colonization is the result of seeding of blood-borne hematopoietic progenitor/stem cells (Fig. 1). Hence, the need for sinusoids is not surprising, and yet, the speciﬁc anatomy and ﬂow characteristics of sinusoids, as opposed to capillaries, are often not appreciated. Slow ﬂow and marginal, laminar ﬂow of blood-borne cells are maximized in a sinusoid-type vessel, facilitating vascular egress of blood-borne cells. The apparent requirement for sinusoids in order for hematopoiesis to develop heterotopically ﬁnds here a simple explanation in simple laws of ﬂuid dynamics. What remains less readily understood is what turns newly forming blood vessel into sinusoids, rather than capillaries. In heterotopic systems, the endothelial lining is provided by neighboring host vessels, and indeed, sinusoids that are established in the developing chimeric bone marrow are made of the same kind of endothelial cell that lines host capillaries just outside the boundaries of the developing ossicle. Hence, a local cue(s) must be operating, and must be provided at least in part by the transplanted, donor cells. In spite of the temporal link between bone formation and establishment of the hematopoietic microenvironment within heterotopic ossicles, the latter is not at all an obligate consequence of the former. Of note, bone marrow stromal cells from caPPR mice (constitutively active PTH/PTHrP receptor driven by the bone-speciﬁc type I collagen promoter [24], one of the two original disclosers of the osteoblastic niche [25]) are inefﬁcient in transferring the hematopoietic microenvironment. In humans, several kinds of cell strains that can generate genuine osteoblasts and bone upon in vivo transplantation fail to establish a hematopoietic microenvironment. This is the case of cells derived in culture from bone surfaces rather than from bone marrow proper, such as trabecular bone cells, or periosteal cells [23], and ms in preparation. Lack of support of the hematopoietic microenvironment also occurs when cells are pushed to extensive in vitro proliferation through mitogens such as FGF-2

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Fig. 1. (A) Heterotopic transplant of human CD146-puriﬁed osteoprogenitors. 4 weeks post-transplant, demonstrating formation of bone and mesenchymal tissue, within which a nascent hematopoietic spot is seen, around a developing, sinusoidal type vessel (sin) coated with human CD146-expressing adventitial reticular cells. ost, osteoid tissue (unmineralized early bone). Top, hematoxylin/ eosin staining. Bottom and right, hCD146 immunostaining. (B) Heterotopic transplant at 7 weeks, showing clusters of granulopoiesis (h) and megakaryocytes (mk) around a sinusoid (sin), coated with CD146-expressing, human adventitial reticular cells (arrows). hac, hydroxyapatite/tricalcium phosphate carrier.

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[23]. Interestingly, human cell strains that form bone, but do not transfer the hematopoietic microenvironment, regularly include a low proportion of CD146-expresing cells, and yet express another marker of “MSCs”, CD63, at high levels. CD146 and adventitial reticular cells For a long time, the identity of the single cells comprised within the bone marrow stroma that are endowed with properties of multipotent skeletal progenitors, and are able at the same time to transfer the hematopoietic microenvironment, remained elusive. This was largely due to the lack of markers suited not only for isolation and enrichment of CFU-Fs (which can be accomplished using multiple markers, ﬁrst and foremost STRO-1 [26], but also many others including, for example, endoglin (CD105) or alpha1 integrin [27,28]), but rather, and more importantly, for identifying the anatomy of those cells that are explanted as CFU-Fs from intact tissues through correlative studies. Likewise, a marker suited at the same time for CFU-F isolation and histology and for following their fate over culture and after in vivo transplantation is essential. In both cases, the marker should be suited for direct histological analysis of tissues comprising hard phases such as bone, or even harder phases such as the inorganic mineralized scaffolds used to load cells to be transplanted. This, of course, applies with speciﬁc relevance to studies on human cells and tissues, which necessarily elude more stringent and sophisticated, genetic approaches of lineage tracing. A breakthrough came when CD146/MCAM (Melanoma Cell Adhesion Molecule [29]), a cell adhesion molecule of the immunoglobulin super family, turned out to represent one such marker. CD146 is expressed at high levels in all human bone marrow stromal cells that can be assayed as CFU-Fs, and therefore in all human skeletal progenitors, prior to seeding in culture and throughout culture of the progeny of seeded cells. In situ, osteoblasts and bone-lining cells do not express CD146. CD146/MCAM expression is restricted to adventitial cells in the bone marrow microvasculature, particularly in cells known from classical histology as adventitial reticular cells. First identiﬁed by SEM studies in rodents and lagomorphs [30], adventitial reticular cells were later noted for their characteristic expression of alkaline phosphatase in rodents [31,32] and humans [33,34]. Long suspected of being implicated with hematopoietic support based on morphological evidence alone, adventitial reticular cells produce large amounts of stromal cell-derived factor-1/CXCL12 (the ligand for CXCR4) both in humans [23] and in the mouse (CAR cells [35]), and on this updated basis, they have been suggested to contribute to an HSC niche. The term adventitial alludes to their position in the sinusoid wall, where they reside abluminally to endothelial cells. The term reticular, in turn, signiﬁes the branched morphology, which results from a wealth of thin processes projected from the cell body away from the sinus wall into the adjacent hematopoietic tissue (Fig. 2). Once isolated based on immuno selection for CD146, adventitial reticular cells behave as clonogenic progenitors (CFU-Fs), and generate a clonal progeny of cells, which in turn express CD146, and can be induced to osteogenic differentiation. Of note, upregulation of osteoblastic markers is coupled with down regulation of CD146 in vitro. Upon transplantation in vivo, CD146 positive cells generate bone and CD146 negative osteoblasts, guide the formation of sinusoids, and also selfrenew into CD146 positive human adventitial reticular cells that line the sinusoids, around which hematopoiesis is established. Hence, CD146-expressing adventitial reticular cells are skeletal progenitor/stem cells. Not only do they transfer, but they generate and organize the hematopoietic microenvironment (sought by blood-borne hematopoietic progenitors). They generate osteoblasts (and bone), direct the formation of sinusoids, and establish themselves along their walls. All three cell types that have been implicated with the “niche” affair, in fact, depend on one, which generates two of the three (osteoblasts and reticular cells), and organizes the third (sinusoidal endothelium). Three players for a dual niche Until very recently, the dominant view of the HSC niche entertained in the relevant literature has been polarized to a dual identity of HSC niches; i.e., an osteoblastic niche, and a sinusoidal or endothelial niche (reviewed in [36]). It was the development of two murine models in the last decade that potently revived interest in the hematopoietic “niche”, while at the same time projecting osteoblasts at center stage of its conceptual view [25,37]. In both models, a measurable excess of functional HSCs was coupled to a measurable excess in bone tissue and in osteoblast numbers. Of note, beyond this

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Fig. 2. Cartoon depicting the position of osteoprogenitors (adventitial reticular cells) at the wall of sinusoids, and their physical contact with both immigrant, and maturing, hematopoietic cells. obs, osteoblasts.

correlation, one of the two models (caPPR mice) features additional phenotypic changes. The latter point adds some complexities to the overall interpretation of how the interplay between bone and hematopoiesis is portrayed. For example, the development of marrow cavities, and therefore of hematopoietic tissue proper, is delayed in caPPR mice due to exuberant bone formation within presumptive marrow space [38]. For this reason, the biological age of the bone marrow in each bone of caPPR mice is younger than in wild-type mice. This circumstance, and the physical constraint placed by occupancy of space in the bone marrow cavity might contribute to explain an increased number of assayable HSCs in these mice, as complementary to an inhibited development of hematopoietic tissue, and to a reduced total hematopoietic cell mass. In addition, upon heterotopic transplantation, the bone marrow stroma from caPPR mice results in establishment of heterotopic bone and osteoblasts with the same efﬁciency as wild-type stroma, but quite paradoxically, it fails to establish a heterotopic microenvironment able to support hematopoiesis proper, at variance with wild-type stromal cells [38]. This is associated with a reduced content in stromal progenitors (CFU-Fs) in caPPR vs. wild-type mice, and an enhanced expansion of their progeny through culture, suggesting that the “mesenchymal” stem cell compartment, besides the HSC compartment, also undergoes major changes in size and kinetics in caPPR mice, suggesting a potential interplay of the two stem cell compartments. To account for the existence of two putative niches, one then has to envision different functional models, in which different niches are either equivalent, or functionally distinct. In the ﬁrst scenario, HSCs would reside both at endosteal surfaces, where they would rely on cues provided by osteoblasts, and at sinusoidal surfaces, where they would be exposed to endothelial cues. In this case, there would be no deﬁning cell type of the HSC niche, and it would be difﬁcult to explain why hematopoiesis occurs speciﬁcally in bone, given the ubiquitous existence of endothelial cells. In the second scenario, in contrast, one would have to invoke dynamic exchanges between the endosteal and sinusoidal niche, and migration of stem cells in both directions as events underpinning the alternative behaviors of HSCs at either microanatomical site. Finally, one could postulate that each endosteal niche is also, at the same time, a sinusoidal niche, a view that careful consideration of the true anatomy of the microcirculation in bone would thoroughly support. However, the reverse would not be true, as sinusoids may in fact exist at a signiﬁcant distance from any endosteal surface. Osteoblasts vs. osteogenic lineage cells Plausible, and even possible, as at least some of these models can be, they all rely on a rather loose deﬁnition of the term, “osteoblast”, and an even more casual use of the term. An “osteoblast” is deﬁned

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as primarily a non-mitotic cell of a characteristic morphology lying on a bone-forming surface, in the process of producing copious amount of matrix that becomes physiologically mineralized [39]. In the relevant literature, endosteal surfaces and osteoblastic surfaces are used interchangeably, as if every square micron of the endosteum was covered by osteoblasts. In fact, proportions of bone surfaces covered by osteoblasts vary remarkably at different sites, ages, and across species. Whereas w90% of the “endosteal” surface of the bone trabeculae in a mouse primary spongiosa is covered by osteoblasts, only 0.1–7.3% of the trabecular surface of the adult human cancellous bone that host’s hematopoiesis is lined by osteoblasts [40]. In addition, osteoblasts only exist, at any bone surface in adult human bone, for about 3 weeks, the time window of a single bone-remodeling event. This would imply that taking the notion of an “osteoblastic” niche literally, not the HSCs, but the niche itself, would migrate from one site to another constantly. While again conceivable, this view would remain at odds with the very fundamental idea of a “niche” as a steady state, anatomical environment. In fact, not all cells that line the endosteal surface are osteoblasts. The vast majority of them, referred to as “bone-lining cells”, are attenuated cells that belong to the same lineage as osteoblasts, share the same type of anatomical location at bone surfaces as osteoblasts, and yet are not engaged in the deﬁning function of an osteoblast – deposition and mineralization of bone matrix [39]. Bone-lining cells are established at bone surfaces at the end of a bone formation cycle, and can be seen as a further differentiation stage of cells that once was osteoblasts. In this respect, they are equivalent to cells that at the end of a bone formation cycle remain entombed within the bone matrix – osteocytes. Bone-lining cells can be distinguished from osteoblasts based on their morphology (ﬂat and elongated, rather than plump), lack of expression of alkaline phosphatase, and reduced biosynthetic activity. This can be assessed, histologically, by the low abundance of mRNAs encoding major collagenous (COL1A1, COL1A2) and noncollagenous proteins (small leucine-rich proteoglycans, osteonectin, osteopontin, bone sialoprotein, osteocalcin) in bone matrix, all expressed at high levels, albeit with local variation reﬂecting speciﬁc functional stages in mature, active osteoblasts. Nonetheless, osteoblasts remain deﬁned essentially by their morphology and position, which are indeed necessary and sufﬁcient hallmarks of osteoblasts, and provide the key criteria whereby osteoblastic activity is measured by histomorphometry as relevant to the clinical assessment of bone metabolism [40]. All osteoblasts are cells of osteogenic lineage, but the reverse does not apply. Besides osteocytes and bone-lining cells, cells of osteogenic lineage include osteoprogenitor cells, found within the bone marrow stroma. At variance with the hematopoetic lineages, the osteogenic lineage does not feature rigorously discrete differentiation stages, and arrays of markers otherwise used to denote such stages are not always precisely deﬁned. Alkaline phosphatase, for example, the best known marker of “osteoblasts”, is not unique to osteoblasts either in the organism (the very same isoenzyme is expressed in liver and kidney) or within the bone/bone marrow organ (where perivascular cells also express it, as do subsets of cells transitioning from the osteoblastic state to an osteocyte or a bonelining cell). Whereas there is usually no ambiguity in deﬁning an osteoblast in histological sections, markers are necessary to distinguish osteoblasts from other cells in the same lineage ex vivo. Deﬁnition of markers apt to distinguish, in such settings, osteoblasts from osteoprogenitors are particularly meaningful when one aims to dissect the relative merit of osteoblasts or their progenitors with respect to the hematopoietic function. Stromal cells are osteoprogenitors, and are vascular cells Of note, all the views of the dual niche, their conceptual interpretations, and even their diagrammatic representations in the literature, invariably do include non-osteoblastic, non-endothelial, stromal cells, but fail to assign them a role or even a deﬁned anatomical position. Cartoons aiming at conveying the concept of a dual niche have repeatedly depicted HSCs as localized to either endosteal surfaces covered by osteoblasts, or to sinusoidal surfaces. However, stromal cells are envisioned and commonly represented as physically residing away from any deﬁned tissue landmark such as the endosteum or sinusoids, and they are even drawn, perhaps inadvertently but probably not by chance, in the way that best conveys in a drawing our subliminal idea of undeﬁnedness – neither black nor white, nor truly colorful – just gray [36]. In fact, stromal cells are as little in focus in current views of the dual niche, as they are in the concepts that are the object of pictorial representation. Difﬁcult to

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visualize due to both their inherent, unique morphology and the limited awareness of markers suited for their visualization, stromal cells in the bone marrow have remained the most elusive anatomical component of the bone/bone marrow organ. This is certainly uncanny considering how popular their use has been in bone biology and experimental hematology for at least 3 decades. The single most important fact that has remained elusive about the identity of “stromal cells” is, speciﬁcally, their perivascular location. Indeed, it is the whole structure of the bone marrow, and its unique vascular pattern, that is commonly overlooked whenever “stroma”, “niches” and “microenvironments” are considered – anatomically, the whole bone marrow is but a hugely extended, branched perivascular space adjacent to a hugely extended, branched sinusoidal network. Hints, nonetheless, are abundant in the earlier literature. The very name “adventitial” recalls the association of bone marrow “reticular” cells with sinusoidal walls. Once the nature of adventitial reticular cells as osteoprogenitors (i.e., “mesenchymal stem cells”, or “skeletal stem cells”) is ﬁnally recognized, it then follows that the sinus wall is the site (the niche, for some) where mesenchymal/skeletal stem cells reside. Given that HSCs also reside at the sinus wall [41], it also follows that the two kinds of stem cells – hematopoietic and stromal – known to reside and function within the bone marrow, share one and the same “niche”. It also follows, that the conceptual puzzle arising from the “duality” of HSC niches – endosteal vs. sinusoidal, osteoblastic vs. endothelial – can perhaps be solved. The sinus wall does not include endothelial cells only, but also osteogenic cells. Osteogenic cells (not osteoblasts, but progenitors of osteoblasts) are not found at bone surfaces only, but also at sinus walls, next to endothelial cells. Stated in a different way, it is not an osteoblast, but osteoblast progenitor, and not endothelial, but subendothelial cells that actually establish and transfer the hematopoietic microenvironment. Of bone and fat and blood Assuming that hematopoietic tissue is the product of HSCs, bone marrow adipocytes and bone itself is the product of skeletal (mesenchymal, stromal) stem cells. The two systems coexist physically in the bone/bone marrow organ, and adapt to each other. Noted examples of this adaptation are Neumann’s law (the replacement of inactive bone marrow territories with fat as an age-dependent phenomenon), the porotic hyperostosis seen in congenital hemolytic anemias (where bone is displaced by hematopoiesis) or even osteoporosis (where bone is displaced by marrow fat) [42]. Of note, adventitial reticular cells are the direct progenitors of bone marrow adipocytes, both in rodents and lagomorphs and in humans, so that adipocytes themselves are also perivascular (perisinusoidal) cells in the bone marrow [30]. Accumulation of lipid in adventitial reticular cells follows any reduction of hematopoietic cell mass, either simply age-related, or, for example, induced by myeloablation [43]. Not only is fatty marrow hematopoietically inactive, but also adipocytes themselves may be negative regulators of hematopoietic stem cell function [44]. Conversion of adventitial reticular cells into adipocytes replaces a cell with a thin, imperceptible body with a conspicuous, space-occupying fat cell. This event collapses the sinusoid and therefore alters blood ﬂow in the bone marrow, mediating the correlation between blood ﬂow and hematopoietic activity. In this way, adventitial reticular cells (the specialized mural cells of the specialized vessels we refer to as bone marrow sinusoids) mediate a mechanism for remodeling the microvascular network that is likely unique to the bone marrow. In all other tissues, loss of pericytes leads to vessel pruning, an irreversible simpliﬁcation of the vascular network active in the maturation stage following any angiogenic event, whether developmental or post-natal. In post-natal bone marrow, adipose conversion of reticular cells (sinus pericytes) permits a reversible pruning of the sinusoidal network: sinusoids collapse due to the formation of adipocytes at their wall, but subsequent loss of lipid at the single cell level will restore the phenotype of adventitial reticular cells; the sinusoid will then become patent again, an hematopoiesis at one particular perisinusoidal location will resume. Reversibility of the adipose-reticular phenotype shift permits reversibility of sinusoid patency, blood ﬂow and dependent hematopoietic activity. The microvascular connection As hematopoiesis is ultimately directed by blood-borne cells that immigrate to the bone marrow, and culminates with blood-borne cells that egress the bone marrow, it seems even trivial to consider

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that the bone marrow microvasculature would be at center stage of hematopoiesis, and therefore of any niche or microenvironment related to it. The connection between vascularity and hematopoiesis, if one looks at development and disease, is even more complex and more clear: intravascular erythropoiesis in birds and in human idiopathic myeloﬁbrosis, Wolff’s islands and the hemogenic endothelium of the Aorta-Gonad-Meseonephron [45–47], the sinusoidal hematopoiesis in fetal liver and the colonization of liver sinusoids in leukemia and other hematologic malignancies, all reveal, indirectly, a general, multi-faceted, microvascular connection. Because of their position, adventitial reticular cells are the ﬁrst cells in the extravascular space of the bone marrow seen by any immigrant cell. They are contractile cells, ideally positioned to regulate trafﬁc across the sinusoidal wall in both directions. Their contractile nature is very well in keeping with their nature as a local adaptation of microvascular mural cells/pericytes, otherwise found in all tissues, and long surmised to represent local tissue progenitors [48–51]. Whereas the lineage, nature, and potential diversity of pericytes across tissues remains to be deﬁned (any reference to “pericytes” as a uniform category, or even a uniform cell type [52,53] is simply unwarranted), each and every feature so far indicated as characteristic of a “pericyte” is indeed found in bone marrow osteoprogenitors. Expression of a host of smooth muscle gene products, of extracellular matrix proteins characteristic of basement membrane, and of growth factor receptors characteristic of mural cells is uniquely blended, in the transcriptome of bone marrow osteoprogenitors, with the expression of markers signifying a commitment to the osteogenic lineage, but not a mature osteoblastic phenotype [23]. Factors that act as mitogens for pericytes, such as FGF-2 or sphingosine-1-phosphate signaling through EDG receptors 1–3 (reviewed in [54,55]), are potent stimulators of proliferation of bone marrow skeletal progenitors, and inhibitors of pericyte proliferation such as TGF-beta1 [56], act likewise in cultures of skeletal progenitor cells. This suggests that the role that these and other factors play in the recruitment of mural cells to the nascent microvascular wall in many settings in development and post-natal growth, may be directly similar to the role that the same factors play in the recruitment of skeletal progenitors to the sinusoidal wall in the bone marrow. Mesenchymal cells recruited to a pericyte fate, enter a state of quiescence – they stop proliferating, do not become apoptotic, and do not differentiate into mature cell types of mesoderm lineages. Establishment of adventitial reticular cells at the sinusoidal wall thus represents a special case of a general paradigm. The best-characterized function of mural cells/pericytes is the stabilization and remodeling of vascular plexuses. Recruitment of pericytes prevents pruning of primitive vessels, stabilizes endothelial cells, and stabilizes pericytes themselves [54,55,57,58]. One of the key effectors of this function is Angiopoietin-1, the ligand of the receptor tyrosine kinase, Tie-2, expressed in endothelial cells. Angiopoietin-1 is a product of mesenchymal cells en route to becoming mural cells, and its ablation in mice results in a lethal vascular phenotype noted for the loss of remodeling of primary vascular lattices [59]. Of note, Tie-2 is also expressed in HSCs, and Ang-1 has been reported to induce HSC quiescence [15]; i.e., to retain HSCs within a functional, if not anatomical, “niche”. Therefore, the observation that skeletal progenitors are the prime producers of Ang-1 in human bone marrow [23] links directly their mural cell nature and function with a role in HSC regulation. More importantly, expression of Ang-1 in skeletal progenitors is itself tightly regulated in relation to cell differentiation. Once induced to differentiate to mature osteoblasts in vitro, or to proliferate, skeletal progenitors down regulate production of Ang-1 [23]. This posits a simple, and yet overlooked, scenario. Any property that functionally deﬁnes the HSC “niche”, like for example Ang-1, is of course itself the subject of regulation. As applied to gene products that are expressed in skeletal progenitors, regulation of the HSC niche or microenvironment, and regulation of proliferation and differentiation of skeletal progenitors are linked to one another. The long-recognized interaction of bone and its strange bedfellow, the bone marrow, thus turns into an interaction of two stem cells: both residing at sinusoidal walls, both subject to regulation, and both directing the growth and differentiation and physiology of bone and hematopoietic marrow. A tale of two tissues, and a tale of two stem cells. Conﬂict of interest None to declare.

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Acknowledgments This work was supported in part by grants from Associazione Italiana Ricerca sul cancro AIRC, Fondazione Roma, European Union (GENOSTEM), and Ministry of University and Research of Italy to PB.

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Adhesion of CD34 þ marrow precursors to human stroma is related to alphaSM actin expression by human marrow myoﬁbroblasts. J Hematother Stem Cell Res 2001;10(2):291–302. [29] Shih IM. The role of CD146 (Mel-CAM) in biology and pathology. J Pathol 1999;189(1):4–11. [30] Weiss L. The hematopoietic microenvironment of the bone marrow: an ultrastructural study of the stroma in rats. Anat Rec 1976;186(2):161–84. [31] Westen H, Bainton DF. Association of alkaline-phosphatase-positive reticulum cells in bone marrow with granulocytic precursors. J Exp Med 1979;150:919–37. [32] Bianco P, Riminucci M, Bonucci E, et al. Bone sialoprotein (BSP) secretion and osteoblast differentiation: relationship to bromodeoxyuridine incorporation, alkaline phosphatase, and matrix deposition. J Histochem Cytochem 1993;41(2):183– 91. [33] Bianco P, Boyde A. Confocal images of marrow stromal (Westen-Bainton) cells. Histochemistry 1993;100(2):93–9. [34] Bianco P, Bradbeer JN, Riminucci M, et al. 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Contents lists available at ScienceDirect

Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha

5

Mesenchymal stem cells and autoimmune diseases Francesco Dazzi, MD, Professor of Stem Cell Biology, Consultant Haematologist a, *, Mauro Krampera, MD, PhD, Assistant Professor, Consultant Haematologist b a b

Stem Cell Biology, Haematology Centre, Department of Medicine, Imperial College, London, UK Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Italy

Keywords: mesenchymal stem cells autoimmune diseases inﬂammation cell therapy

Mesenchymal stem cell (MSC) immunosuppressive properties offer a potentially attractive therapeutic modality for autoimmune diseases. MSC inhibit virtually all types of immune responses in vitro and prevent the induction of disease in several experimental models of autoimmunity. However, the processes involved in the pathogenesis of human diseases are more complicated and treatment cannot be administered before disease induction. In autoimmune diseases persistent antigenic stimulation recruits endogenous MSC to the site of lesion that contribute to the ﬁbrotic evolution. Therefore, administering MSC to a chronic inﬂammatory disorder may not be desirable. In fact, MSC are not constitutively immunosuppressive but require a ‘licensing’ step provided by molecules of acute phase inﬂammation, like IFNg and TNF-a, or toll-like receptor (TLR) ligands. Conversely, different cytokines and/or the stimulation of selective TLR make MSC to become immunostimulatory. Therefore, dissecting the inﬂammatory environment in autoimmune diseases will identify the best conditions amenable to successful MSC therapy. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

Chronic inﬂammatory autoimmune diseases impart a massive burden on health services worldwide. Historically, therapeutic strategies have centred on non-selective systemic immune suppression. The clinical trials investigating the effects of depleting T cells turned out to be disappointing [1,2], whilst the depletion of CD20 expressing B cells has been more promising [3,4] with durability of clinical responses relating to the efﬁciency of the depletion phase and the timing of the re-emergence of pathogenic clonotypes. A number of targeted therapies interfering with key pathogenetic events like cytokines or homing receptors have been developed with considerable success in some diseases. * Corresponding author. Haematology Centre, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail address: [email protected] (F. Dazzi). 1521-6926/$ – see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2011.01.002

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However, these approaches are expensive and none of them consistently lead to prolonged periods of drug-free remission [5,6]. Thus, curative therapy remains a major unmet need in the management of chronic inﬂammatory disorders. Cell therapies: options for autoimmune diseases In alternative to conventional immunosuppression, cell therapy-based approaches have more recently been tested with a view of restoring the integrity of immuno-regulatory networks and, at the same time, preserving a pool of memory cells capable of responding to environmental pathogens. Haematopoietic stem cell transplantation (HSCT) is a treatment aimed at resetting the deregulated immune system of patients with severe autoimmune diseases. HSCT induces alterations of the immune system which are beyond the effects of a dose-escalating immunosuppressive approach by creating space for a new immunological repertoire, generated as a result of the reinfused and/or residual haematopoietic stem cells [7]. Although the conditions to reset patient immune repertoire may also restore immune regulation, alternative tactics have been proposed to speciﬁcally transfuse cells with immunomodulatory activity. Regulatory T (Treg) cells play a crucial role in the maintenance of peripheral tolerance and modulate susceptibility to autoimmune disease [8,9]. The infusion of Treg cells can ameliorate immune mediated diseases in pre-clinical model but the hurdles of their ex vivo expansion has prevented their application in humans. Natural killer T (NKT) cells use an invariant T-cell receptor (invariant NKT, iNKT) which interact with synthetic glycolipids such as a-galactosylceramide in the context of the monomorphic CD1d antigenpresenting molecule. iNKT cells have the unique capacity to rapidly produce large amounts of both T helper (Th) 1 and Th2 cytokines through which they regulate autoimmune, allergic, antimicrobial, and antitumour immune responses [10]. The activation of iNKT cells with a-galactosylceramide has been tested with some success in animal models. Also monocytes/macrophages can negatively regulate immune functions when exposed to particular environments [11,12]. Similarly, myeloid cells with immunosuppressive properties have been originally identiﬁed in the tumour setting – characterised by the expression of CD11b and Gr-1 – which involves arginase and nitric oxide synthase as the main effector pathways [13]. Much attention has been paid to the potential exploitation of the immunosuppressive properties of mesenchymal stromal cells (MSC). As extensively reviewed in this issue, MSC have been described to inhibit a variety of cell types mediating both adaptive and innate immunity. The effects on T lymphocytes are probably the best characterised. MSC non-selectively and non-speciﬁcally suppress CD4þ and CD8þ T lymphocytes independently of whether they are naïve or antigen-experienced [14], their functional state [15] or the type of T-cell receptor expressed [16]. Similarly, MSC appear to inhibit the differentiation and antibody production of B cells [17–19] as well as the activation and expansion of natural killer (NK) cells [20]. Although MSC have no effect on neutrophil phagocytosis and chemotaxis, they protect them from apoptosis and inhibit their production of reactive oxygen species [21]. A further inhibitory activity is directed at pro-inﬂammatory monocytes/macrophages and the differentiation of monocytes into mature dendritic cells [22]. The anti-inﬂammatory properties of MSC can be partly ascribed to a potent anti-proliferative activity that is associated with the inhibition of cyclin D2. The capacity of T cell to proliferate appears to be rescued by the addition of exogenous interleukin-2 (IL-2) [23], thus indicating that the inhibition is reversible. The evidence that T cells are only temporarily inhibited in their proliferative and functional capacity suggests that MSC do not cause cell death [24,25]. There is evidence demonstrating that MSC protect from apoptosis not only cells of the immune system but a number of cell types of different tissue origin, including malignant cells. Such an activity is well in accord with the notion that MSC exhibit a cytoprotective effect which explains their ability to promote tissue repair. MSC for the treatment of autoimmune diseases The ﬁrst evidence that MSC could exert an immunosuppressive activity in vivo was obtained in the setting of organ transplantation in non-human primates, whereby the administration of MSC at the

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time of skin allograft proved able to prolong its survival [26]. This and other similar pre-clinical experiences paved the way to their exploitation in the clinical setting in acute graft-versus-host disease (GvHD) after allogeneic HSCT. The spectacular results obtained in an otherwise untreatable and fatal disease [27] have fuelled an enormous interest to better understand the principles of MSC therapies and test their properties in other diseases. Because of the immune mediated pathogenesis and the remarkable social burden, autoimmune diseases are considered a priority target. However, the circumstances for the use of MSC in these conditions remains to be established, not only because the prognosis of these disorders is not so poor to ethically justify a potentially harmful experimental treatment, but especially because there are a number of issues, which the animal models have highlighted, that need to be clariﬁed. One of the ﬁrst issues is the source of MSC. MSC for the treatment of steroid-refractory acute GvHD has unavoidably to come from a third-party donor because the severity of the condition would not consent sufﬁcient time to produce MSC from the original donor unless this is done before the transplant. Furthermore, the profound immunocompromised status of these patients makes allogeneic MSC unlikely to be rejected [28]. In contrast, the choice of treating a chronic condition is not generally under any similar clinical pressure and the ability of autoimmune disease patients to respond at least to alloantigens is certainly fully preserved. Therefore, the choice of using the patient both as a donor and recipient of MSC is fully justiﬁed. However, there is not sufﬁcient information to deﬁnitely choose the autologous strategy. First, there is no proof that MSC need to engraft or at least stay in the host for long time to produce the therapeutic efﬁcacy. After injection, MSC home to the site of inﬂammation where they act via paracrine mechanisms and cannot be detected any longer only a day or two after their infusion [29]. In accord, a few pre-clinical models have employed MSC originating from different species of the recipient and still observed a remarkable therapeutic efﬁcacy [30]. In the perspective of using autologous MSC, an important question that must be addressed is whether MSC from patients with autoimmune diseases maintain their ability to exert the beneﬁcial activity. The behaviour of MSC obtained from patients with a range of autoimmune diseases of rheumatological origin does not seem to differ from those of normal healthy controls [31]. Further studies conducted on MSC from patients with Crohn’s disease [32] and multiple sclerosis [33] conﬁrm this notion. In contrast, MSC from patients with aplastic anaemia have been reported to lack in immunosuppressive abilities [34]. Unfortunately, the assays employed to assess MSC immunosuppression have not been proven to essentially reﬂect their in vivo therapeutic activity thus making it difﬁcult to draw any conclusion. In pre-clinical models, MSC have been observed to delay the onset of diabetes in non-obese diabetic mice (NOD) if MSC are obtained from healthy but not NOD mice [35]. Despite the several aspects that still require attention, the experience of using MSC in pre-clinical models of autoimmune diseases has been encouraging since the very ﬁrst demonstration that MSC can prevent the generation of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis [36]. In this model, MSC determine a substantial decrease in the number of inﬂammatory inﬁltrates and reduce demyelination and axonal loss. In the ﬁrst published study the therapeutic efﬁcacy was maximal when MSC were administered at disease onset but no effect was documented after disease stabilisation. However, adipose tissue-derived MSC appear to be able to prevent the onset of the disease as well as to decrease the severity of the neurological defect after disease stabilisation [37]. This effect may relate to the peculiar capability of adipose tissue-derived MSC of migrating to the inﬂammatory sites of the central nervous system, thus controlling local inﬂammatory and sustaining glial cell regeneration. The results in the clinical setting, according to a very recent publication, are very promising. Autologous bone marrow-derived MSC, given intrathecally in 10 patients with advanced multiple sclerosis, produced clinical improvement in nearly 50% of the patients although no beneﬁt could be documented at radiologic investigations [38]. However, the experience of a previous pilot study was less enthusiastic [39]. Chemically induced (trinitrobenzene sulfonic acid) autoimmune colitis may be signiﬁcantly improved by the systemic infusion of adipose tissue-derived MSC, as a consequence of the inhibition of T helper 1-type immune response and the expansion of T regulatory cells [40]. In a model of multiorgan autoimmunity caused by a deﬁciency in regulatory T cells, MSC produced a remarkable improvement in the histopathology of the distal ileum [41]. Also in this case, the animal studies have been followed by

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encouraging clinical results in which third-party adipose-derived MSC were administered locally for the treatment of complex perianal ﬁstula in patients with Crohn’s disease [42]. More recently, autologous bone marrow-derived MSC were tested in a phase I study for the treatment refractory luminal Crohn’s disease [43]. It is too early to draw any conclusions but inﬂammatory bowel diseases appear to be a responsive target and consequently a number of groups worldwide are concentrating their efforts in this direction. The initial clinical experience in systemic lupus erythematosus (SLE) might support the exploitation of MSC in autoimmune diseases but also highlight the issue of the necessity of choosing the appropriate type of MSC. An initial study investigated the effect of autologous MSC in two patients with SLE. Despite an increased in regulatory T cells no beneﬁcial effect on disease activity was observed [44]. A few month later two studies were published by another group utilising allogeneic bone marrow MSC [45] or MSC obtained from umbilical cord [46]. In both cases MSC produced an improvement in clinical and laboratory parameters, thus suggesting that, at least in some forms of SLE, MSC function could be impaired by the underlying disease. Interesting information has been learnt from the studies in collagen induced arthritis (CIA). The results of the initial studies were in complete disagreement, whereby whilst in one case MSC effectively protected from the disease [47], in the other study the administration of MSC was in fact worsening the clinical parameters [48]. The differences could be partly reconciled with the fact that MSC were administered at different times after disease induction when changes in the inﬂammatory environment may inﬂuence MSC activities. The same concept could also explain why, in the majority of cases, MSC can prevent but not necessarily affect an established disease. The studies on MSC in GvHD have produced important results to clarify the modalities of MSC therapeutics and interpret some of the discrepancies. One of the ﬁrst study observed that the infusion of MSC is not sufﬁcient to prevent the development of GvHD [49]. Another study, using MSC of human umbilical cord blood origin, observed that the GvHD produced by human T cells in NOD/SCID mice could not be prevented when MSC were given right at the beginning of the lymphocyte infusion. In contrast, GvHD pathology and symptoms were fully prevented if MSC were given in multiple doses at weekly intervals although no effect was detectable if MSC were administered too late in the course of the disease [50]. These apparently conﬂicting data were resolved when MSC were tested for their ability to prevent GvHD when given at different times in one single dose. It was observed that only when MSC were infused at day þ2 or þ20, they signiﬁcantly increased the survival of recipient mice [51]. At these times, the levels of IFNg were particularly high in this model and therefore, as we will extensively discuss in the next section, likely to promote MSC immunosuppressive activity. The clinical studies are fully in accord with these data. Whilst the administration of MSC at the time of HSC transplant did not change the frequency of acute or chronic GvHD [52], their administration at the time of full-blown disease resulted in the control of the disease in a large proportion of cases [27]. Inﬂammatory environment and plasticity of MSC immunomodulatory activity At this stage, to better understand the results described in the previous section, it becomes fundamental to discuss more extensively whether and how the surrounding environment affects the MSC immunosuppressive activity. The immunosuppressive activity of MSC was initially observed when MSC were added to cultures of activated immune cells. However, MSC are not constitutively inhibitory, but they acquire their immunosuppressive functions after being exposed to the inﬂammatory environment. This important principle stemmed from the seminal observation that anti–IFN-g receptor antibodies can revert the suppressive effect of MSC. The various techniques to activate an immune response in vitro invariably elicit both T cells and monocytes thus generating the release of high concentrations of IFNg [18] which in turns activate the immunosuppressive activity of MSC (Fig. 1). This ‘licensing’ step is a fundamental requirement to induce MSC mediated immunosuppression [53]. However, the role of IFNg is more complex than just being an activating agent tout-court. Its level and the contemporary presence of other inﬂammatory cytokines can change the functional proﬁle of MSC. In fact, IFNg can enable MSC to act as antigen-presenting cells [54,55]. Mice immunised with ovalbumin-pulsed IFNg-treated MSC developed antigen-speciﬁc cytotoxic CD8þ T cells. Similarly, IFNg-treated human MSC induce signiﬁcant IL-2 production when co-cultured with HLA-restricted

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Fig. 1. MSC immunosuppressive activity is modulated by cytokines and Toll-like receptor (TLR) ligands. Upper panel: The inhibitory activity of MSC on T cell proliferation requires a ‘licensing’ step that can be provided by IFNg. In fact, the presence of anti-IFNreceptor antibodies in culture impairs the immunosuppressive activity (from Ref. [18]). Lower panel: MSC activity on T cell proliferation can be polarised by stimulating different TLR. In condition of T cell activation (A) MSC acquire the ability to suppress. Similarly, the stimulation of TLR3 stimulates such activity (C), whilst the use of TLR4 agonists prevents the acquisition of MSC mediated inhibitory properties (from Ref. [66]).

inﬂuenza-speciﬁc T-cell hybridomas. More interestingly, other data indicate that the antigen-presenting property of MSC develop only within a narrow window, at low levels of interferon-gamma. As IFNg levels increase, decrease in MHC class II molecule expression on MSC decreases with the loss of alloreactive inducing activity [54]. More recent studies demonstrated that MSC, like dendritic cells, can even cross-present exogenous antigen and induce an effective CD8þ T-cell immune response [56]. However, the physiological relevance of MSC as antigen-presenting-cells has to be elucidated and whether this is a property that could be exploited for cellular vaccination remains equally unclear. There are other inﬂammatory cytokines, such as TNF-a or IL-1b, that can license MSC immunosuppression [57] and determine substantial changes in their immunophenotypic proﬁle. IFNg, TNF-a or IL-1b induce the upregulation of HLA-class I, ICAM-1 and VCAM-1 and the de novo expression of both HLA-class II and the inhibitor ligand B7-H1. Although these changes may promote APC-like function, they are also critical for MSC immunosuppressive properties [57]. Some of these discrepancies can be partly explain by the observations that different cytokine combinations can produce different effects. Whilst IFNg alone is sufﬁcient to induce IDO and B7-H1 upregulation, when in combination with TNFa it induces HGF production and the two cytokines act synergistically in the induction of COX2 [58]. In addition, MSC can secrete SOD3, an anti-inﬂammatory enzyme involved in the catabolism of superoxide anion, which is regulated following exposure to IFNg and TNF-a [59]. Although in a typical inﬂammatory environment MSC acquire an immunosuppressive function, not all inﬂammatory milieux produce the same outcome. One of the very ﬁrst observations in MSC immunobiology was that MSC can indeed acquire immunosuppressive or immunostimulating properties [60]. More recent studies have indicated an important role for toll-like receptors (TLR). TLR are membrane-spanning, non-catalytic receptors that recognise structurally conserved molecules derived from microbes and mediate the activation of immune responses of both innate and adaptive type. MSC express a large number of TLR the stimulation of which has been shown to profoundly affect MSC immunomodulatory properties as well as their migratory phenotype. This aspect is of particular relevance in the therapeutic setting because TLR activation has been implicated in the pathogenesis of

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a number of inﬂammatory diseases whereby they can initiate or sustain the chronic inﬂammatory process. Therefore, depending on the pattern of TLR stimulation, MSC can acquire different functional properties (reviewed in Ref. [61]). It is well established that MSC express a number of TLR both at RNA and protein level. High mRNA expression of TLR 1, 2, 3, 4, 5, and 6 has been consistently detected, whilst TLR2, TLR3, TLR4, TLR7, and TLR9 has been reported by ﬂow cytometry. The expression of some TLR is affected also by the oxygen level. The expression of TLR on MSC is certainly functional. It has been shown that TLR3 and TLR4 binding antagonises MSC immunosuppressive activity by interfering with the Jagged1-Notch1 loop [62]. However, if TLR 3 and TLR4 activation is effected on isolated MSC before they are added to the T-cell response cultures, an opposite effect has been observed whereby TLR stimulation boosts MSC immunosuppressive activity [63]. In addition, exposure of MSC to poly(I:C) – a TL3 ligand – or LPS – a TLR4 ligand – induces the production of pro-inﬂammatory mediators, such as IL-1, IL-6, IL-8, and CCL5 together with the expression of inducible NO synthase (iNOS), and TNF-related apoptosis-inducing ligand (TRAIL). TLR activation of MSC might generate an inﬂammatory site to attract innate immunity cells, such as neutrophils, thus promoting defences against pathogens [64]. More recently, it has been proposed that the complexity of TLR-mediated stimulation of MSC could be revisited by an analogy with the functional status of monocytes/macrophages. Although in a simpliﬁed approach, it has been established that stimulation of monocytes with speciﬁc cytokines or TLR agonists polarises them into a classical M1 pro-inﬂammatory phenotype, while others promote their alternate M2 phenotype associated with anti-inﬂammatory and tissue repair activity [65]. Utilising low-level, short-term TLR-priming protocol, Waterman and colleagues noted nearly opposite effects on human MSC following stimulation of TLR3 or TLR4. TLR4-primed MSC exhibit a mostly proinﬂammatory proﬁle with increased levels of molecules like IL-6, IL-8, or TGFb (MSC1), whilst TLR3primed MSC develop the characteristics of immunosuppressive cells producing IL-10, IDO and PGE2 (MSC2) [66]. As a consequence of MSC polarisation, T cell inhibition may be achieved only following MSC2 co-culture, while T lymphocyte activation may occur only in co-culture with MSC1. It should be noted however that longer exposure to TLR ligands and/or the concomitant presence of other cytokine is likely to add layers of complexity to this simpliﬁed classiﬁcation (Fig. 1). The network of stimuli affecting MSC activity is largely unknown but the notion that MSC are functionally plastic has fundamental implications to understand their physiology, its role in pathological processes and the factors that inﬂuence their therapeutic exploitation. In fact, some of the molecules that modulate MSC immunological properties also affect the ability to differentiate along the mesenchymal lineage, thus regulating stem cell renewal and tissue repair. Should MSC be used for autoimmune diseases? The rationale for using MSC in autoimmune disease would be to deliver a potent but local immunosuppressive and anti-inﬂammatory activity. The lesson we have learnt from the concept of MSC polarisation towards an immunosuppressive or immunostimulating phenotype has profound implication on the strategies we should adopt to identify the patients who are more likely to respond to MSC therapies. The inﬂammatory environment to which MSC are exposed is in fact capable of shaping their properties in completely opposite directions. It is important to remember that the same molecules (cytokines and TLR ligands) that modulate MSC activity are prominently involved in the pathogenesis of autoimmune diseases. We believe that a careful characterisation of the patients’ microenvironment is fundamental to identify the ‘signature’ of those more likely to beneﬁt of this cell therapy. Another factor to be considered is that in chronic inﬂammatory autoimmunity, the disease is often associated with ﬁbrotic changes that result from the recruitment of mesenchymal stromal cells to the site of the lesion. Interestingly, these cells often share the same properties of bone marrow MSC because they exhibit multipotential differentiation ability and once expanded they can also immunosuppress [67]. However, the fact that despite their accumulation the disease persists, suggests that the microenvironment impairs their functions. Studies in mice have shown that mesenchymal cell targeting by TNF is a key pathogenic mechanism in chronic inﬂammatory joint and intestinal diseases. Therefore, the adoptive transfer of MSC to patients with disorders characterised by ‘mesenchymal cell pathologies’ could eventually add fuel to the pathological process and exacerbate the disease.

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Fig. 2. The inﬂammatory microenvironment in autoimmunity conditions MSC plasticity. The inﬂammatory environment associated with autoimmune diseases is supposed to modify the way by which MSC intervenes on the inﬂammatory process. If MSC are infused during acute inﬂammation, the microenvironment (M1-type monocytes) actively ‘licenses’ MSC to inhibit the effector T cells responsible for tissue damage. In case of antigen persistence the players involved in inﬂammation change and produce ﬁbrosis. We propose that, if MSC are infused to patients during this phase the microenvironment does not provide the ‘licensing’ and/or favours the recruitment of MSC to the ﬁbrotic process.

Fortunately, the limited clinical experience reported so far seem to exclude overt aggravation of the underlying condition. Several investigations are currently in progress to identify molecules with the ability to revert the process of ﬁbrosis which could eventually restore the original tissue with or without the intervention of exogenous MSC. In conclusion, as summarised in Fig. 2, the inﬂammatory environment to which MSC are exposed is a fundamental factor inﬂuencing MSC functions. However, it should also be noted that under some circumstances the lack of therapeutic effects could be ascribed to a weak activity of MSC on the underlying disease and/or to the fact that the disease, when established, is completely insensitive to the immunosuppressive activity. Conﬂict of interest statement None to declare.

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Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha

6

Neuroprotective features of mesenchymal stem cells Antonio Uccelli, MD a, b, c, *, Federica Benvenuto, PhD a, b, Alice Laroni, MD a, Debora Giunti, PhD a a

Department of Neurosciences, Ophthalmology and Genetics, University of Genoa, Via De Toni 5, 16132 Genoa, Italy Center of Excellence for Biomedical Research, University of Genoa, Italy c Advanced Biotechnology Center (ABC), Genoa, Italy b

Keywords: mesenchymal stem cells (MSC) experimental autoimmune encephalomyelitis (EAE) central nervous system (CNS): neurons microglia oligodendrocytes neuroprotection

Bone marrow (BM) derived mesenchymal stem cells (MSC) differentiate into cells of the mesodermal lineage but also, under certain experimental circumstances, into cells of the neuronal and glial lineage. Their therapeutic translation has been signiﬁcantly boosted by the demonstration that MSC display signiﬁcant also anti-proliferative, anti-inﬂammatory and anti-apoptotic features. These properties have been exploited in the effective treatment of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis where the inhibition of the autoimmune response resulted in a signiﬁcant neuroprotection. A significant rescue of neural cells has been achieved also when MSC were administered in experimental brain ischemia and in animals undergoing brain or spinal cord injury. In these experimental conditions BM-MSC therapeutic effects are likely to depend on paracrine mechanisms mediated by the release of growth factors, anti-apoptotic molecules and anti-inﬂammatory cytokines creating a favorable environment for the regeneration of neurons, remyelination and improvement of cerebral ﬂow. For potential clinical application BM-MSC offer signiﬁcant practical advantages over other types of stem cells since they can be obtained from the adult BM and can be easily cultured and expanded in vitro under GMP conditions displaying a very low risk of malignant transformation. This review discusses the targets and mechanisms of BM-MSC mediated neuroprotection. Ó 2011 Elsevier Ltd. All rights reserved.

* Corresponding author. Department of Neurosciences Ophthalmology and Genetics, University of Genoa, Via De Toni 5, 16132 Genoa, Italy. Tel.: þ39 0 103537028; Fax: þ39 0 103538639. E-mail addresses: [email protected], [email protected] (A. Uccelli). 1521-6926/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2011.01.004

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Introduction Mesenchymal stem cells (MSC) were ﬁrstly identiﬁed by Friedenstein as stromal cells from the bone marrow (BM) and described as spindle-shaped cells in culture [1]. In this organ MSC represent a very rare population, less than 0,1% of nucleated cells representing progenitor cells of the stromal lineage at different stages of differentiation. They take part to the bone marrow microenviroment within the haematopoietic niche, supporting the mainteinance of hematopoietic stem cell (HSC) pool and the differentiation programs of blood born cells [2,3]. Beside their trophic role in the haematopoeitic niche, MSC are likely to play a similar role in other tissues where they display also an anti-proliferative activity resulting in tissue homeostasis [4]. In addition to adult bone marrow, MSC have been identiﬁed in several tissues and organs of either fetal or adult origin [5–7]. BM-derived MSC differentiate into fat, bone, and cartilage but can also transdifferentiate into embriological unrelated tissues [8]. They are easily cultured in vitro under appropriate conditions, grow as adherent cells until conﬂuence resulting in yields sufﬁcient for clinical exploitation for cell therapy or gene therapy strategies [9]. MSC for the tratment of experimental autoimmunity of the central nervous sytem Several studies showed that MSC possess immunomodulating properties exerted in vitro on cells populations of both adpative and innate immunity [10]. These features were together with the reported ability of MSC to transdifferentiate into neural cells [11] and migrate, although to a limited extent, to the central nervous sytem (CNS) [12] induced researchers to exploit them for the treatment of experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis (MS). Intravenous (i.v.) infusion of MSC improved the clinical course of EAE [13,14]. In this model injected MSC on one side induced peripheral T cell tolerance to myelin proteins thus reducing migration of pathogenic T cells to the CNS and, on the other side, homed themselves to the CNS where they preserved axons and reduced demyelination. In other studies it was observed that following administration in mice with EAE, MSC exert an effect on oligodendrocytes enhancing remyelination possibly through the release of neurtrophins such as Brain Derived Neurotrophic Factor [15–17]. Immunomodulating properties of MSC are not the only mechanisms that could explain their therapeutic plasticity. The heterogenity of MSC, expressing a large number of regulatory proteins, may explain their wide therapeutic features and their capacity to respond differently to injuries depending on the microenvironment and despite their low engraftment in vivo [18]. MSC produce cytokines and a variety of soluble factors regulating several biological activities as demonstrated by their transcriptome analysis [19], which suggests their ability to support survival of non-proliferating hematopoietic stem cells (HSC) within the niche, thus playing a major role in the maintenance of local homeostasis [3]. Neuroprotective properties of MSC MSC initially attracted interest for their presumed ability to home into injured tissues and differentiate into multiple cellular phenotypes in vivo. This prediction was challenged by recent observations indicating that only small numbers of the infused cells engraft into tissues, even if damaged and they quickly disappear quickly [20] and that just MSC supernatant sufﬁces to block fulminant hepatic failure [21]. These observations together with in vitro studies showing that signiﬁcant biological effects on target cells could be achieved in several experimental conditions without the need of cell contact have led to the hypothesis that MSCs therapeutic plasticity relies greatly on the paracrine release of molecules. Several groups have demonstrated that MSC can rescue neurons from apoptosis in vitro [22– 24], a mechanism that together with their ability to inhibit immune-mediated damage, can explain the reduced axonal loss observed in mice with EAE treated with MSC [14,25,26]. This anti-apoptotic effect, together with the reported capacity of releasing neurotrophic molecules may well explain the remarkable effect obtained with the administration of MSC, either i.v. or locally, in experimental models of stroke and spinal cord injury. For example, direct transplantation of bone

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marrow pluripotent cells has been reported to promote remyelination[27]. In another study the injection of MSC into the lumbar spine of rats led to the inﬁltration of the spinal cord parenchyma following complete transection and motor improvement [28]. An improvement of motorneurons survival was observed following transplantation into the lumbar spinal cord in transgenic SOD1 (G93A) mice, a model for human amyotrophic lateral sclerosis [29]. In this study, authors observed a delay of death and improved motor performances upon MSC transplantation. In a model of stroke the injection of MSC signiﬁcantly improved functional outcome in rats reducing apoptosis and promoting the induction of local neurogenesis [30]. MSC secrete a number of growth factors and cytokines, which normally support hematopoietic progenitors to proliferate and differentiate [31]. Besides, MSC may be directly involved in promoting plasticity of the ischemic damaged neurons or in stimulating glial cells to secrete neurotrophins such as BDNF and NGF, in the reduction of apoptosis in the penumbral zone of the lesion and the proliferation of the endogenous cells in the subventricular zone [32]. An indirect effect involving the recruitment of local progenitors was suggested also by the implantation of human MSC in the mouse hippocampus, which stimulated proliferation, migration and differentiation of the endogenous neural stem cells that survived as more differentiated neural cells [33]. The effects produced by the MSC are presumably explained by their secretion of different trophic factors including NGF, VEGF, CNTF and FGF-2 and BMI-1 that probably account for the increase in neurogenesis acting directly on local precursors but also activating nearby astocytes, which independently may increase neurogenesis [33].

Fig. 1. The main neuroprotective effects are depicted. Dashed lines indicate the current lack of strong evidence for that phenomenon.

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In a mouse model of global ischemia the administration of human MSC resulted in the improvement of neurological function and prevented neuronal cell death in the hippocampus. Authors claimed an indirect effect mediated by local microglia, which, following MSC administration, displayed an enhanced expression of the neuroprotective factor Ym1 and other trophic molecules including IGF and Gal-3 [34]. The observation that MSC neuroprotective effect may occur through the interaction with local neural cells is consistent with experiments addressing the interaction between MSC and microglia. The latter, upon activation, produces a variety of inﬂammatory molecules, including nitric oxide (NO), tumor necrosis factor (TNF), interleukin 1-b (IL1- b), and reactive oxygen species (ROS) that have been closely associated with the pathogenesis of neural damage resulting from ischemia, inﬂammation and primary neurodegeneration. Zhou et al demonstrated that human MSC are able to inhibit LPS-stimulated microglia activation and the production of inﬂammatory factors through diffusible molecules and that human MSC sense inﬂammatory molecules, released by the activated microglia, thus increasing signiﬁcantly the production of neurotrophic factors, which are likely to be involved in neuroprotection [35]. Consistent with these results, Kim and colleagues demonstrated that cell death of dopaminergic neurons induced by activated microglia is inhibited by the addition of human MSC [36,37]. Regardless of the role of inhibition of microglia activation in the neuroprotective effects observed following MSC transplantation in several experimental models, it has been clearly shown that i.v. administration of MSC sufﬁces to signiﬁcantly revert the upregulation of molecules involved in oxyradical detoxiﬁcation occurring during the acute phase of EAE [38]. Interestingly, a similar turnaround of the increase of oxidative stress associated proteins was observed following coculture of neurons exposed to H2O2 in the presence of MSC [38] suggesting that proinﬂammatory and oxidative stress molecules released by macrophages and microglia and known to damage neurons during neuroinﬂammation can be inhibited by MSC. Microglia is not the only neural cell type targeted by MSC. In fact it has been shown in vitro that MSC can instruct neural precursor cells (NPC) to enter an oligodendrogenic program while inhibiting their ability to differentiate into astrocytes [36,39]. These results support the in vivo observation that in EAE MSC administration leads to some degree of remyelination [15,16,39]. Conclusions In conclusion, experimental evidence in preclinical model of neurological diseases suggests that MSC are a promising approach to achieve neural repair and protection. However, current data do not support the possibility that most of the reported effects occur through cell replacement. Many other paracrine mechanisms, including a potent anti-inﬂammatory capacity, the direct release of antiapoptotic and neurotrophic factors, the ability to induce other cells, such as microglia, to acquire a protective phenotype and to induce proliferation of local neural progenitor cells possibly leading to remyelination, are likely to sustain the protective effects observed in preclinical models (Fig. 1). Because the clinical translation into humans has been based so far mainly on their given immunomodulatory functions [40] or on their ability to support hematopoiesis [41], properly designed and carefully controlled clinical studies will have to conﬁrm the therapeutic relevance of these neuroprotective features. Conﬂict of interest statement Authors do not have any conﬂict of interest concerning the topic of this review. Some of the results discussed here were obtained from research supported by grants from the Fondazione Italiana Sclerosi Multipla (FISM) (AU), the Italian Ministry of Health (Ricerca Finalizzata) (AU), the Italian Ministry of the University and Scientiﬁc Research (MIUR) (AU), the ‘Progetto LIMONTE’ (AU) and the Fondazione CARIGE (AU). Funding The funding sources were not involved in any decision about how to perform research described in the article or in the writing of the article.

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[22] Scuteri A, Cassetti A, Tredici G. Adult mesenchymal stem cells rescue dorsal root ganglia neurons from dying. Brain Res 2006;1116(1):75–81. [23] Crigler L, Robey RC, Asawachaicharn A, et al. Human mesenchymal stem cell subpopulations express a variety of neuroregulatory molecules and promote neuronal cell survival and neuritogenesis. Exp Neurol 2006;198(1):54–64. [24] Wilkins A, Kemp K, Ginty M, et al. Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro. Stem cell res 2009;3(1):63–70. [25] Zhang J, Li Y, Lu M, et al. Bone marrow stromal cells reduce axonal loss in experimental autoimmune encephalomyelitis mice. J neurosci res 2006;84(3):587–95. [26] Kassis I, Grigoriadis N, Gowda-Kurkalli B, et al. Neuroprotection and immunomodulation with mesenchymal stem cells in chronic experimental autoimmune encephalomyelitis. Arch Neurol 2008;65(6):753–61. [27] Akiyama Y, Radtke C, Honmou O, Kocsis JD. Remyelination of the spinal cord following intravenous delivery of bone marrow cells. Glia 2002;39(3):229–36. [28] Satake K, Lou J, Lenke LG. Migration of mesenchymal stem cells through cerebrospinal ﬂuid into injured spinal cord tissue. Spine 2004;29(18):1971–9. [29] Vercelli A, Mereuta OM, Garbossa D, et al. Human mesenchymal stem cell transplantation extends survival, improves motor performance and decreases neuroinﬂammation in mouse model of amyotrophic lateral sclerosis. Neurobiol disease 2008;31(3):395–405. [30] Chen J, Li Y, Katakowski M, et al. Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat. J neurosci res 2003;73(6):778–86. [31] Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J cellular biochem 2006;98(5):1076–84. [32] Li Y, Chen J, Chen XG, et al. Human marrow stromal cell therapy for stroke in rat: Neurotrophins and functional recovery. Neurology 2002;59:514–23. [33] Munoz JR, Stoutenger BR, Robinson AP, et al. Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. Proc Natl Acad Sci USA 2005;102(50):18171–6. *[34] Ohtaki H, Ylostalo JH, Foraker JE, et al. Stem/progenitor cells from bone marrow decrease neuronal death in global ischemia by modulation of inﬂammatory/immune responses. Proc Natl Acad Sci USA 2008;105(38):14638–43. [35] Zhou C, Zhang C, Chi S, et al. Effects of human marrow stromal cells on activation of microglial cells and production of inﬂammatory factors induced by lipopolysaccharide. Brain res 2009;1269:23–30.

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[36] Rivera FJ, Couillard-Despres S, Pedre X, et al. Mesenchymal stem cells instruct oligodendrogenic fate decision on adult neural stem cells. Stem Cells 2006;24(10):2209–19. [37] Kim YJ, Park HJ, Lee G, et al. Neuroprotective effects of human mesenchymal stem cells on dopaminergic neurons through anti-inﬂammatory action. Glia 2009;57(1):13–23. [38] Lanza C, Morando S, Voci A, et al. Neuroprotective mesenchymal stem cells are endowed with a potent antioxidant effect in vivo. J neurochem 2009;110(5):1674–84. [39] Bai L, Caplan A, Lennon D, Miller RH. Human mesenchymal stem cells signals regulate neural stem cell fate. Neurochem res 2007;32(2):353–62. *[40] Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versushost disease: a phase II study. The Lancet 2008;371(9624):1579–86. [41] Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000;18(2):307–16.

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Mesenchymal stem cells for treatment of acute and chronic graft-versus-host disease, tissue toxicity and hemorrhages O. Ringden, MD, PhD, Professor in Transplantation Immunology *, K. Le Blanc, MD, PhD, Professor in Clinical Stem Cell Research Center for Allogeneic Stem Cell Transplantation, Division of Clinical Immunology, and Hematology Center, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, F79, SE-141 86 Stockholm, Sweden

Keywords: Mesenchymal stem cells Hematopoietic stem cell transplantation Graft-versus-host disease Hemorrhagic cystitis Tissue toxicity

Mesenchymal stem cells (MSCs) have immunomodulatory effects and low immunogenicity. MSCs inhibit T-cell alloreactivity in vitro. Immune inhibition is caused by soluble factors. MSCs affect almost all cells of the immune system. They are safe to infuse in humans with no acute toxicity and no ectopic tissue formation. We treated patients with life-threatening acute graft-versus-host disease (GVHD) not responding to conventional immunosuppressive therapy with MSCs. Approximately half of the patients responded. HLA-identical or third party MSCs were equally effective. Children tended to have a better response compared to adults. MSCs have also been used for chronic GVHD with positive effects. MSCs also reversed tissue toxicity such as hemorrhagic cystitis, pneumomediastinum and colon perforation with peritonitis. A patient with extensive hemorrhages was successfully treated with repeated doses of MSCs pooled from two donors. This may indicate that MSCs apart from wound healing may stimulate clotting and vasoconstriction. To conclude, MSCs is a novel treatment that may be used for GVHD, tissue toxicity and hemorrhages because of its immune inhibitory and anti-inﬂammatory effects. Ó 2011 Published by Elsevier Ltd.

Graft-versus-host disease and toxicity in hematopoietic stem cell transplantation Allogeneic hematopoietic stem cell transplantation (HSCT) is mainly used to treat hematopoietic malignancies such as leukemia, but can also be used for severe aplastic anemia and some rare inborn

* Corresponding author. Tel.: þ46 8 585 826 72; Fax: þ46 8 746 6699. E-mail address: [email protected] (O. Ringden). 1521-6926/$ – see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.beha.2011.01.003

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errors of metabolism [1–3]. Graft-versus-host disease (GVHD) in its acute or chronic form causes considerable morbidity and mortality after HSCT [4,5]. T-cells from the graft are responsible for triggering GVHD and proliferate after activation by recipient major histocompatibility class I or class II antigens, and minor antigenic peptides [6]. Antigen-presenting cells, including dendritic cells of host origin, present the antigens to CD4þ T-cells in association with human leukocyte antigen system (HLA) class II molecules. Cytokines including IL-1 stimulate CD4þ T-cells, which release IL-2. IL-2 stimulates CD8þ T-cells, which react with HLA class I positive targets. Natural killer (NK-cells) and macrophages also participate in the development of acute GVHD. Activated CD4þ cells produce interferon-g which enhances HLA class II expression on epithelial cells and macrophages, which further stimulates T- and NK-cell activation. The main target organs for acute GVHD are skin, gastrointestinal tract, liver and the lymphohematopoietic system. GVHD may be abolished if the graft is T-cell depleted [7,8]. Using non-manipulated grafts, GVHD needs to be prevented by immunosuppressive drugs. The most commonly used drugs are cyclosporine in combination with a short arm of methotrexate [9,10]. Mild or moderate acute GVHD is easily reversed using high doses of steroids. However, more severe acute GVHD grade III is more difﬁcult to overcome and grade IV is life-threatening. If high-dose steroids fails, second line therapy including anti-IL-2 receptor antibodies, monoclonal anti-CD3 antibodies, antibodies against tumor necrosis factor-a, recombinant human IL-1 receptor antibodies, methotrexate, rapamycin and a large variety of treatments have been tried [11–15]. Responses to these therapies are generally poor with a high mortality of hemorrhages, multiorgan failure, and infectious complications [16]. Chronic GVHD may appear as a continuation of the acute form, but may also be quiescent [4,17]. If often appears later in the course, three months or more, after HSCT. Chronic GVHD resembles autoimmune disorders and skin disease, keratoconjunctivits sicca, oral mucositis, strictures, malabsorption, liver disease, obstructive bronchiolitis, may appear. Chronic GVHD is also associated with immunodeﬁciency with frequent bacterial and viral infections. Chronic GVHD may be treated with steroids, cyclosporine, tacrolimus, extracorporeal photopheresis, azathioprine, 1 Gy of total body irradiation, thalidomide, mycophenolate mofetil, sirolimus, and anti-B-cell antibodies [4,18–22]. Acute and especially chronic GVHD have a pronounced antileukemic and antitumor effect [23–25]. The allogeneic graft-versus-tumor effect is one of the reasons to perform HSCT in patients with malignancies. The other reason is that high doses of chemotherapy and irradiation can be given disregarding hematopoietic bone marrow aplasia, which is rescued by HSCT from a healthy donor. Conditioning by chemo radiotherapy prior to HSCT may also induce toxicity to other tissues than bone marrow, such as mucositis, gastroenteritis, liver toxicity, pneumonitis and hemorrhagic cystitis, among other complications. Rationale to use mesenchymal stem cells for graft-versus-host disease and tissue toxicity after hematopoietic stem cell transplantation Mesenchymal stem cells (MSCs) have raised interest in regenerative medicine because they can differentiate to several mesenchymal tissues and participate in wound healing, support hematopoiesis and participate in the marrow microenvironment [26]. MSCs have immunomodulatory effects and also low immunogenicity, which may make them useful for transplantation. MSCs inhibit T-cell alloreactivity in mixed lymphocyte cultures (MLCs) or by mitogens [27–29]. MSCs inhibited allogeneic speciﬁc cytotoxic T-cell lysis if they were added early in the MLC, but not when they were added in the cytotoxic phase [30]. Inhibition was caused by soluble factors including hepatocyte growth factor and transforming growth factor-a, prostaglandin E2, IL-10 and indoleamine2,3-dioxygenase [31–34]. MSCs affect almost all cells of the immune system, T-cells, B-cells, dendritic cells and NK-cells [26]. Mesenchymal stem cells for treatment of severe acute graft-versus-host disease It was demonstrated that MSCs were safe to infuse in humans with no acute toxicity or no ectopic tissue formation [35–37]. Furthermore, MSCs prolonged skin allograft survival in baboons [27]. Because of these ﬁndings and the suppression of alloantigens in vitro, we decided to treat patients with lifethreatening acute GVHD not responding to conventional immunosuppressive therapy with MSCs

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[38,39]. In the ﬁrst patient with grade IV acute GVHD, we observed a dramatic response in the gastroenteral tract and liver. However, when cyclosporine was discontinued, acute GVHD reappeared. Reinstitution of cyclosporine and a second infusion of MSCs again reversed acute GVHD [40]. Based on the in vitro ﬁndings that MSCs were equally suppressive in MLC whether they were HLA-identical, haploidentical or completely HLA mismatched, we used third party MSCs for practical reasons. Third party MSCs can be stored frozen and be ready available for patients who develop steroid-resistant acute GVHD. After this initially successful case, we found that MSCs improve acute GVHD in some, although not all patients. In our ﬁrst series of eight patients with grades III-IV steroid-refractory acute GVHD, responses were seen in six of the patients [38,39]. Two patients died soon after MSC treatment with no obvious response. In one of them, MSC donor DNA was detected in colon and a mesenteric lymph node. Survival in patients treated with MSCs was signiﬁcantly better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSCs during the same time period (p ¼ 0.03). The results from this pilot study inspired a European phase II study in which 55 patients with therapy-resistant acute GVHD were treated with MSCs [41]. The patients received a median dose of bone marrow-derived MSCs of 1.4 106 (0.4–9 106) cells/kg body weight. Twentyseven patients received one dose, 22 received two doses, and six patients 3–5 doses. MSCs were obtained from HLA-identical sibling donors (n ¼ 5), haploidentical donors (n ¼ 18), and third party HLA-mismatched donors (n ¼ 69). Complete response was obtained in 30 patients and nine had a partial response. Response rate was not related to MSC donor and recipient HLA match. Complete responders had an overall survival two years after MSC infusion of 52%, as opposed to 16% for partial and non-responders (p ¼ 0.018). Children tended to have a better response, 21/25 (68%), compared to 18/30 (43%, p ¼ 0.07) in adults. Fang and co-workers used human adipose tissue-derived MSCs for steroid refractory acute GVHD [42]. They treated six patients with a dose of 1 106/kg of adipose-derived MSCs. No side-effects were noted. Two patients received HLA-haploidentical MSCs, and four patients received third party MSCs. Acute GVHD disappeared completely in 5/6 of the patients. One of the responders died from leukemic relapse. The patient who did not respond to MSC therapy died from multiorgan failure. Ho and co-workers reported three patients treated with MSC using a dose ranging from 0.92 to 1.34 106/kg. Two patients showed a response, but one patient died twelve days after MSC infusion [43]. Kebriaei and co-workers did a randomized study where grades II-IV acute GVHD patients were randomized to receive two treatments of MSCs (ProchymalÒ) in a dose of either 2 or 8 106 MSCs/kg [44]. Median age was 52 years in the 32 adult patients enrolled in the study. Twenty-one patients had a grade II, eight had a grade III, and three had grade IV acute GVHD. An initial response to MSC therapy was seen in 94% with a complete response in 77%. There were no differences in safety or efﬁcacy between the low and the high MSC dose groups. As an alternative to MSCs expanded in fetal calf serum, platelet lysate medium has also been used. von Bonin and co-workers reported experience of 13 adult patients treated with MSCs expanded in platelet lysate medium for steroid-refractory acute GVHD. Complete response was initially seen in two patients (15%). The remaining 11 patients received additional immunosuppressive therapy and further MSC infusions, and partial response was seen in 5/11 of those additional patients [45]. In another study on platelet-lysate generated MSC, eleven children were treated for severe resistant acute or chronic GVHD [46]. Median dose was 1.2 106 MSCs/kg. Overall response was obtained in 71% of the children with a complete response in 24%. Four patients had GVHD recurrence between two and ﬁve months after MSC infusion. A recent study conﬁrmed encouraging results using MSCs in pediatric patients [47]. They used Prochymal in twelve children 0.4 to 15 years of age, treated for therapy-resistant grade III and IV acute GVHD on compassionate use basis. The MSC dose was 8 106 cells/kg in two patients and 2 106 cells/kg in the remaining patients. The cells were given twice a week for four weeks. Overall, 7 (58%) patients had complete response, 2 (17%) partial response and 3 (25%) mixed response. The cumulative incidence of survival at 100 days from the initiation of Prochymal therapy was 58%. Complete resolution of gastrointestinal symptoms occurred in 9 (75%) of the children. All these studies with varying numbers of patients treated for acute GVHD suggest that complete and partial responses can be obtained in a majority of patients. HLA-compatibility between MSC donor and recipient does not seem to be of major importance. A cell dose between 1 and 2 106 cells/kg

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appears as effective as higher doses. Whether a particular source of stem cells is superior, bone marrow versus adipose-derived MSCs or MSCs cultured in medium containing fetal calf serum or platelet lysate medium, cannot be concluded from the available data. Furthermore, if the number of passages of MSCs is of importance for response or not remains to be answered. A higher response rate in children than adults may be explained by a better healing capacity and that children can tolerate severe acute GVHD better, compared to adults. From the data of Kebriaei and co-workers [44], it seems that there is a better response if patients are treated for milder acute GVHD. Otherwise, we do not know why some patients respond and others do not. To answer this question, a better understanding how MSC repair damaged tissue is needed. For instance, is the effect direct or indirect? Are accessory cells needed for this process? MSC donor DNA has been detected in target tissues of patients with GVHD at the site of injury [39]. This may suggest that MSCs exert their immunomodulatory and healing effect at the site of injury. However, in some patients MSC donor DNA cannot be detected after infusion. More data from MSC responders and non-responders is required as well as an understanding of how MSC are distributed after intravenous infusion, Furthermore, studies regarding optimal dosing, timing and how to match different donors with the different kinds of patients are required. Future research and the ongoing randomized trials will hopefully shed light on these issues. Mesenchymal stem cells for treatment of chronic graft-versus-host disease As for acute GVHD, there is a variety of treatments used for chronic GVHD because of the poor response in refractory disease. Due to the difﬁculty to evaluate clinical responses, there is a scarcity of prospective randomized studies of the various treatments [19,48]. Because of the difﬁculties in the evaluation of response and outcome regarding chronic GVHD, the National Institutes of Health (NIH) formed a consensus on how to measure response in different organs with chronic GVHD involvement [49]. Chronic GVHD resembles autoimmune disorders. Furthermore, MSCs showed positive effects in several experimental autoimmune models [50–53]. There is also an association between acute and chronic GVHD [54–56]. Some risk-factors, target organs and pathophysiology is similar and MSCs also showed effects in treatment of acute GVHD [38,39,41,42,44,46]. Therefore, it seems logical to try MSCs for chronic GVHD [57]. We ﬁrst used MSCs for treatment in extensive chronic GVHD in a patient with lichenoid changes all over the skin and slight liver disease [39]. He was given 1 106 MSC/kg on day 153 after HSCT. The lichenoid skin changes did not improve, but the liver enzymes declined. He later died of Epstein-Barr virus post transplant lymphoproliferative disorder (PTLD). Subsequently, Zhou and co-workers reported on four patients with sclerodermatous chronic GVHD, who gradually improved after treatment with MSCs [58]. More recently, Weng et al presented their experiments using MSCs in the treatment of 19 patients with refractory chronic GVHD [59]. The median dose of MSCs was 0.6 106 cells/kg and 14/19 showed partial or complete responses (34%). Immunosuppression given for chronic GVHD could be discontinued in ﬁve patients within a median of 324 days after MSC infusion. The response rates were graded according to the NIH scoring system [49]. The cumulative response rate for skin was 78%. Among three patients with scleroderma, one had a partial response. Cumulative responses in oral mucosa, liver and gastrointestinal tract were between 90 and 100%. Two-year survival rate was 78%. B-cells are involved in chronic GVHD [60]. The effects of MSCs on B-cells may therefore be of importance. It was reported that MSCs inhibited B-cell proliferation in vitro in an MSC/lymphocyte ratio of 1:1 [61]. However, it may be questioned if this concentration of MSCs compared to lymphocytes is relevant for the in vivo situation. Anyhow, another study found that MSCs arrested B-cell proliferation in the G0/G1 phase of the cell cycle [62]. We studied an ultimate B-cell function, IgG secretion, and found that MSCs stimulated blood and splenic B-cell IgG production [63]. Thus, the value of in vitro data for prediction of in vivo responses in chronic GVHD is likely to be limited, since T-cells are inhibited and B-cells may be activated. In vivo studies in experimental animals and in patients treated with MSCs should address this point. Mesenchymal stem cells for treatment of tissue toxicity and hemorrhages Because MSCs localize to target organs and heal acute GVHD, we also treated patients with severe tissue toxicity with infusions of MSC [64]. Life-threatening grade 5 hemorrhagic cystitis was the

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indication for treatment with MSCs in the ﬁrst two patients [65]. The two patients needed massive transfusions of erythrocytes, platelets and fresh frozen plasma. After infusion of MSCs, both patients had dramatically reduced transfusion requirements [64]. In spite of this, both patients died of multiorgan failure. One of them received MSCs consecutively from two donors. At autopsy, donor DNA from the two donors could be demonstrated in the bladder tissue. This patient had a bladder perforation, which seemed healed at autopsy. In total, we have now treated 12 patients for severe hemorrhagic cystitis after HSCT. Seven of these patients have been reported previously [64]. As mentioned above, two patients died of multiorgan failure. Two patients did not respond to MSC infusions. However, in the remaining eight patients, gross hematuria disappeared after median 3 (1-14) days. The two most recent responders received MSCs pooled from two donors. The reason to pool MSCs from several donors was that when we pooled MSCs from several donors in vitro, we could improve the response rate in mixed lymphocyte cultures [66]. Therefore, this may suggest that pooling of MSCs from several donors might improve the response rate. In two HSCT patients, pneumomediastinum resolved after treatment with MSCs [64]. Colon perforation and peritonitis was reversed in an elderly female with severe gastrointestinal GVHD. Peritonitis and abdominal muscle defense developed on day 70 after HSCT. X-ray showed intra-abdominal air. The patient refused surgery. She was treated with antibiotics and 1 106 MSCs/kg from a third party donor. Six days after MSC infusion peritonitis and abdominal muscle defence disappearted. X-ray showed that abdominal gas had markedly decreased and had completely disappeared 11 days after MSC infusion. As second colon perforation was also reversed by MSCs. Unfortunately, the patient died of invasive fungal infection. A 61-year old male with myeloﬁbrosis developed extensive hemorrhages from the proximal jejunum during the neutropenic phase after HSCT using an unrelated donor [67]. Due to multi-speciﬁc anti-HLA antibodies, he was refractory to platelet transfusions. Surgery was impossible for technical reasons and platelet refractoriness. He was given 2 106 MSCs/kg pooled from two donors. Prior to MSC infusion, he was daily given several erythrocyte units, platelets from HLA-identical donors and several units of fresh frozen plasma. After MSC infusions, hemorrhages stopped and he only required occasional erythrocyte and platelet transfusions. He experienced two additional hemorrhages, which were stopped by MSC infusions. Due to hematopoietic graft failure, he was regrafted with a new donor. Subsequent to this, he again had hemorrhage during the pancytopenia that was treated by MSCs. Due to poor graft function, despite complete donor chimerism, he was given a T-cell depleted bone marrow boost from the same donor. He is now six months after retransplantation, alive and well with stable hematopoietic engraftment. From this patient and the experience using MSCs for the treatment of hemorrhagic cystitis, it is obvious that MSCs can heal damaged tissue and stop hemorrhages. One may speculate if MSCs, apart from wound healing, stimulate clotting and also induce vasoconstriction. Adverse effects All studies have uniformly shown that it is safe to infuse MSCs i.v. and no side-effects have been observed. It has been debated whether MSCs with their immunomodulatory effects increase the risk of leukemic relapse, following HSCT. One study indicated that patients co-infused with MSCs at the time of HSCT had an increased risk of leukemic relapse [68]. However, it is expected that any treatment that decreases acute and especially chronic GVHD also will decrease the graft-versus-leukemia effect [23–25]. Patients with acute and chronic GVHD are susceptible to opportunistic infections. MSCs also decrease the immune function of all cells of the immune system [26]. It has therefore been discussed if MSCs may increase the risk of infection in these already severely immunocompromised HSCT patients. However, MSCs did not decrease the effects of virus-speciﬁc T-cell responses to cytomegalovirus and Epstein-Barr virus, in contrast to the effects on alloantigen cytotoxic T-cells [69]. Conclusion The dramatic effects seen after i.v. infusion of MSCs in certain patients have opened up the ﬁeld to treat patients with acute and chronic GVHD, tissue toxicity and hemorrhages after HSCT. Based on these ﬁndings, MSCs may also be used for autoimmune disorders such as inﬂammatory bowel disease

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and other indications. MSCs may also be tried in patients with gastrointestinal perforations and peritonitis, where surgery is not possible for various reasons. Pilot studies have shown that MSCs may be an effective tool. Now it is time for randomized studies to prove its efﬁcacy. Randomized studies have been performed for the treatment of severe acute GVHD and others are ongoing. Randomized studies are also needed to prove its efﬁcacy in patients with chronic GVHD and tissue toxicity. Studies are also needed how to optimize the use of MSC. Conﬂict of interest statement None to declare. Acknowledgments We thank Inger Hammarberg for excellent typing of this manuscript. The study was supported by grants from the Swedish Cancer Society, the Children’s Cancer Foundation, the Swedish Research Council, the Tobias Foundation, the Cancer Society in Stockholm, the Swedish Society of Medicine, the Stockholm County Council, the Sven and Ebba-Christina Hagbergs Foundation, and Karolinska Institutet. References [1] Ringden O, Remberger M, Svahn BM, et al. Allogeneic hematopoietic stem cell transplantation for inherited disorders: experience in a single center. Transplantation 2006;81:718–25. [2] Storb R, Thomas ED, Weiden PL, et al. Aplastic anemia treated by allogeneic bone marrow transplantation: a report on 49 new cases from Seattle. Blood 1976;48:817–41. [3] Thomas ED, Buckner CD, Banaji M, et al. One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 1977;49:511–33. [4] Ringden O, Deeg HJ. Clinical spectrum of graft-versus-host disease. 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Contents lists available at ScienceDirect

Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha

8

Ex vivo expansion of mesenchymal stromal cells Maria Ester Bernardo, MD, PhD a, Angela Maria Cometa, PhD a, Daria Pagliara, MD a, Luciana Vinti, MD a, Francesca Rossi, MD a, b, Rosaria Cristantielli, PhD a, Giuseppe Palumbo, MD, PhD c, Franco Locatelli, MD, PhD a, d, * a Dipartimento di Ematologia ed Oncologia Pediatrica, IRCCS Ospedale Pediatrico Bambino Gesù, P.le Sant’ Onofrio, 4, 00165 Roma, Italy b Seconda Università di Napoli, Italy c Clinica Pediatrica, Università degli Studi di Roma "Tor Vergata", Italy d Università degli Studi di Pavia, Italy

Keywords: mesenchymal stromal cells ex vivo expansion fetal calf serum platelet lysate malignant transformation cellular therapy

Mesenchymal stromal cells (MSCs) are adult multipotent cells that can be isolated from several human tissues. MSCs represent a novel and attractive tool in strategies of cellular therapy. For in vivo use, MSCs have to be ex vivo expanded in order to reach the numbers suitable for their clinical application. Despite being efﬁcacious, the use of fetal calf serum for MSC ex vivo expansion for clinical purposes raises concerns related to immunization and transmission of zoonoses; the standardization of expansion methods, possibly devoid of animal components, such as those based on platelet lysate, are discussed in this paper. Moreover, this review focuses on the search of novel markers for the prospective identiﬁcation/isolation of MSCs and on the potential risks connected with ex vivo expansion of MSCs, in particular that of their malignant transformation. Available tests to study the genetic stability of ex vivo expanded MSCs are also analyzed. Ó 2010 Elsevier Ltd. All rights reserved.

Introduction In addition to hematopoietic stem cells (HSCs), the bone marrow (BM) also contains mesenchymal stromal cells (MSCs). These cells were ﬁrst recognized more than 40 years ago by Friedenstein et al. who described a population of adherent cells from the BM which were non-phagocytic, exhibited

* Corresponding author. Dipartimento di Ematologia ed Oncologia Pediatrica, IRCCS Ospedale Pediatrico Bambino Gesù, P.le Sant’ Onofrio, 4, 00165 Roma, Italy. Tel.: þ39 06 68592129; Fax: þ39 06 68592292. E-mail address: [email protected] (F. Locatelli). 1521-6926/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2010.11.002

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a ﬁbroblast-like appearance and could differentiate in vitro into bone, cartilage, adipose tissue, tendon and muscle [1]. Moreover, after transplantation under the kidney capsule, these cells gave rise to the different connective tissue lineages [2]. In general, MSCs represent a minor fraction in BM and their exact frequency is difﬁcult to calculate because of the different methods of harvest and separation. However, the frequency in human BM has been estimated to be in the order of 0.001–0.01% of the total nucleated cells; moreover, this frequency declines with age, from 1/104 nucleated marrow cells in a newborn to about 1/2 106 nucleated marrow cells in a 80-year old person [3]. After their ﬁrst identiﬁcation in the BM [1], human MSCs (hMSCs) were isolated from a variety of other human tissues, including periosteum, muscle connective tissue, perichondrium, dental pulp, adipose tissue and fetal tissues, such as lung, BM, liver and spleen [4–7]. Amniotic ﬂuid and placenta have been found to be rich sources of MSCs [8,9]; both fetal and maternal MSCs can be isolated from human placenta [8]. Although present at low frequency, MSCs have been also identiﬁed in umblical cord blood (UCB); in this respect, quality criteria for the selection of UCB units are considered critical for the successful isolation of MSCs from this source [10,11]. One of the hallmark of MSCs is their multipotency, deﬁned as the ability to differentiate into several mesenchymal lineages, including bone, cartilage, tendon, muscle, marrow stroma and adipose tissue (AT) [7,12]. Usually, trilineage differentiation into bone, adipose tissue and cartilage is taken as a criterium for multipotentiality [12,13]. MSCs display unique immunological properties that modulate the responses, in vitro and in vivo, of all cells involved in the immune response, including T and B lymphocytes, dendrtic cells and natural killer (NK) cells [14–16]. However, the exact mechanisms by which MSCs exert their functions are still poorly understood. Whether MSCs mainly mediate their effect through soluble factors or cell-to-cell contact is still a matter of active investigation [14,17,18]. Moreover, the mechanisms by which MSCs display their immunosuppressive effect are largely restricted to in vitro studies; the in vivo biological relevance of the in vitro observations needs to be elucidated [14–16]. Thanks to their ability to home to inﬂamed sites and repair-injured tissues, and to their immunomodulatory properties, MSCs are today considered a promising tool in approaches of immunoregulatory and regenerative cell therapy [19]. Thus far, MSCs have been employed in phase I/II clinical trials [20–23], mainly addressing the issues of feasibility and safety of infusion; and to date no adverse effects have been registered after MSC administration. In particular, MSCs, usually harvested at passage 2 to 3 of in vitro culture, have been successfully employed in the clinic to enhance hematopoietic stem cell engraftment in HLA-haploidentical, T cell-depleted allografts [20] and UCB transplantation [21], as well as to treat the most severe and refractory forms of acute graft-versus-host disease [22]. Moreover, local injections of adipose tissue (AT)-derived MSCs have already been successfully employed in the clinical setting to treat complex perianal ﬁstulas resistant to conventional treatments in Crohn’s Disease (CD) patients [23]. Ex vivo isolation and characterization of MSCs Most of the information available on MSC phenotypic and functional properties are derived from studies performed on cells cultured in vitro [13–15]. To date, MSC isolation/identiﬁcation has mainly relied on their morphology and adherence to plastic, resulting in a heterogeneous population of cells, referred to as MSCs [13]. Immunephenotyping by ﬂow cytometry is also applied to characterize ex vivo expanded MSCs and to deﬁne their purity. However, at present, no speciﬁc marker has been shown to speciﬁcally identify true MSCs and ex vivo expanded cells are currently stained with a combination of positive (CD105, CD73, CD90, CD166, CD44 CD29) and negative (CD14, CD31, CD34 and CD45) markers, at least in case of BM-derived cells (indeed, a proportion of AT-derived MSCs express CD34) [5,12,13]. Therefore, little is known about the characteristics of the primary precursor cells in vivo, since it has not yet been possible to isolate the most primitive mesenchymal cell from bulk cultures. One of the hurdles has been the inability to prospectively isolate MSCs because of their low frequency and the lack of speciﬁc markers. Recently, some groups have reported the identiﬁcation and prospective isolation of the most primitive mesenchymal progenitors, both in murine and human adult BM, based on the expression of speciﬁc markers [24–34].

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Anjos-Afonso et al. have reported the identiﬁcation, isolation and characterization of a novel multipotent cell population in murine BM, based on the expression of the stage-speciﬁc embryonic antigen-1 (SSEA-1). This primitive subset can be found both directly in the BM and in mesenchymal cell cultures and can give rise to SSEA-1þ MSCs [24]. In human cells, with the aim to prospectively isolate MSCs, surface markers such as SSEA-4, STRO-1 and the low afﬁnity nerve growth factor receptor (CD271) [25–28], which enrich for MSCs, have been employed. Moreover, Battula et al. have recently isolated by ﬂow cytometry MSCs from human BM, using antibodies directed against the surface antigens CD271, mesenchymal stem cell antigen-1 (MSCA-1), CD56 and SSEA-3, and identiﬁed novel MSC subsets with distinct phenotypic and functional properties [29,31]. In particular, CD271 has been reported to deﬁne a subset of MSCs with immunosuppressive and lymphohematopoietic engraftment-promoting properties in vivo [33]. Plateletderived growth factor receptor-beta (PDGF-RB; CD140b) has been also proposed as a marker for the isolation of clonogenic MSCs [29] and other reports have demonstrated a 9.5-fold enrichment of MSCs in human BM cells with prominent aldehyde dehydrogenase activity [30]. A STRO-4 monoclonal antibody has been also demonstrated to be speciﬁc for mesenchymal precursors cells from human and ovine tissues, being capable to enrich colony-forming ﬁbroblasts when employed for MSC isolation from BM [34]. Table 1 details the published markers and/or combination of antigens identifying mesenchymal precursors. Despite the identiﬁcation of these new MSC markers [24–34], none of the available has demonstrated to be singularly capable to identify the true mesenchymal progenitors. Indeed, MSCs may be composed by different cell subsets which might be responsible for speciﬁc functions and characterized by different cell surface markers. Therefore, further research in this ﬁeld is warranted in order to identify an MSC-speciﬁc marker; this will hopefully allow to dissect the developmental hierarchy of MSCs and will facilitate the generation of homogenous cellular products. Novel techniques, such as proteomic approaches and microarray analysis, have also been proposed and might be useful for performing comparisons of expression proﬁles between different MSC populations (i.e. MSCs from different tissue sources, cultured in different conditions, at different in vitro passages) and, therefore, to deﬁne new MSC surface antigens that can be used to identify cell subsets [35–37]. For example, NOTCH3, JAG2 and ITGA11 transcripts have been recently demonstrated to be expressed on ex vivo expanded BM-MSCs through genome-wide, gene expression analysis [38]. Table 1 Antigens expressed on primary and culture-expanded MSCs. Antigen

Expanded/primary MSCs

Human/murine MSCs

CD105 (endoglin, SH2) [12,13] CD73 (ecto-5’ nucleotidase, SH3, SH4) [12,13] CD166 (ALCAM) [12,13] CD29 (b1-integrin) [12,13] CD44 (H-CAM) [12,13] CD90 (Thy-1) [12,13] TRA-1-81 Sca-1 STRO-1 [27] CD349 (frizzled-9) [27] SSEA-3; SSEA-4 [25,31] Oct-4

Expanded Expanded Expanded Expanded Expanded Expanded Expanded Expanded Primary Primary Primary Primary

Nanog-3 SSEA-1 [24] CD271 (low afﬁnity nerve growth receptor) [30,33] MSCA-1 [30] CD140b (PDGF-RB) [29] STRO-4 [34] NOTCH3, JAG1, ITGA11 [38]

Primary Primary Primary

Human, murine Human Human Human, murine Human, murine Human Human (placenta) Murine Human (BM) Human (BM, placenta) Human þ/- (BM, placenta) Human þ/- (BM, placenta, fetal tissues) Human þ/- (BM, placenta) Murine (BM) Human

Primary Primary Primary Expanded

Human (BM) Human Human, ovine (BM) Human (BM)

Modiﬁed from Bernardo et al. Ann NY Acad Sci. 2009; 1176:101.

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Ex vivo expansion of MSCs Due to the low frequency of mesenchymal progenitors in human tissues, in vivo use of MSCs requires that the cells be extensively ex vivo manipulated to achieve the numbers that are necessary for their clinical application [20–22]. Indeed, MSCs can be expanded in vitro to hundreds of millions of cells from a 10 to 20 ml BM aspirate [12,13]. In this regard, the cells are mainly cultured, either under experimental or clinical-grade conditions, in the presence of 10% fetal calf serum (FCS) [12–14,19–22] and serum batches are routinely pre-screened to guarantee both the optimal growth of MSCs and the bio-safety of the cellular product. Most of the clinical experience that has been gained on MSCbased cellular therapies both in the context of hematopoietic stem cell transplantation (HSCT) with the aim to promote engraftment [20,21] and to treat acute GvHD [22], and in strategies of Regenerative Medicine [23], has been obtained with FCS-expanded cells. Differences in isolation methods, culture conditions, media additives greatly affect cell yield and possibly also the phenotype of the expanded cell product [39–45]. For these reasons, efforts have been made within the MSC Developmental Committee of the European Group for Blood and Marrow Transplantation (EBMT) for the standardization of MSC isolation and expansion procedures [20–22,46]. This organization, including European centers interested in the biology and clinical application of MSCs, has deﬁned a common MSC expansion protocol based on the use of 10% pre-screened FCS, in order to facilitate comparisons between cell products generated at different sites and to run large-scale clinical studies. Nonetheless, the use of FCS raises concerns when utilized in clinical-grade preparations, because it might theoretically be responsible for the transmission of prions and still unidentiﬁed zoonoses or cause immune reactions in the host, especially if repeated infusions are needed, with consequent rejection of the transplanted cells [39–42]. In this regard, Horwitz et al. reported sensitization in a child with Osteogenesis Imperfecta treated with repeated infusions of MSCs [47]. This sensitization was characterized by the formation of alloantibodies directed against MSCs and was responsible for their rejection, already after the second infusion of ex vivo expanded allogeneic MSCs [47]. In view of these considerations, serum-free media, appropriate for extensive expansion and devoid of the risks connected with the use of animal products, are being investigated. Both autologous and allogeneic human serum have been tested for the in vitro expansion of MSCs and one group showed that autologous serum was superior to both FCS and allogeneic serum in terms of proliferative capacity of the expanded MSCs [39]. The reduction of bovine antigens by a ﬁnal 48-hour incubation with medium supplemented with 20% human serum, to prepare hypoimmunogenic MSCs, has also been proposed [40]. Several serum-free media, based on the use of cytokines and growth/ attachment factors, such as basic ﬁbroblast growth factor (b-FGF) and trasforming growth factor beta (TGF-b), have been also tested in experimental conditions [48,49]. Recently, platelet lysate (PL) has been demonstrated to be a powerful substitute for FCS in MSC expansion, offering a signiﬁcative advantage in terms of proliferative capacity of MSCs, thanks to its high concentration of natural growth factors (GFs) [41–45]. Indeed, human PL contains all GFs that are secreted by platelets to initiate wound healing, including PDGFs, b-FGF, vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and TGF-b [41,42]; moreover, it can be easily obtained from apheresis products of healthy volunteers [42], as well as from buffy coats [45]. Immediately after apheresis, PLT products are frozen at 80 C, subsequently thawed to obtain the release of PLT-derived GFs and centrifuged to eliminate platelet bodies; PL preparations from several healthy donors are pooled to be used as a culture supplement for the generation of MSCs [42]. Doucet et al. ﬁrst demonstrated that growth factors contained in PL are able to promote MSC expansion in a dosedependent manner [41]. This was further substantiated by the data published by our group and showing that a culture medium additioned with 5% PL is superior to 10% FCS in terms of clonogenic efﬁciency and proliferative capacity of MSCs, therefore providing more efﬁcient expansion, together with a signiﬁcant time saving [42]. Moreover, the in vitro immune regulatory properties of PLexpanded MSCs resulted to be comparable to those of MSCs cultured in the presence of FCS in terms of capacity to decrease alloantigen-induced cytotoxic activity, to promote differentiation of CD4þ T cell subsets expressing a Treg phenotype and to induce augmentation of IL-6 production in culture supernatant [42]. On the contrary, the suppressive effect in vitro on alloantigen-induced lymphocyte

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subset proliferation was greater in the presence of FCS-expanded MSCs, as compared to MSCs cultured in PL [42]. Moreover, gene expression changes in long term cultured PL-MSCs have been studied by microarray analysis and resulted to be similar to those of MSCs expanded in the presence of FCS, suggesting that replicative senescence modiﬁcations develop in both cases (in the absence of malignant transformation) and are independent of the culture conditions [50]. Altogether, these data suggest that PL could be a suitable substitute for FCS in the generation and expansion of MSCs; however, further studies are needed to better understand the characteristics and functional properties, in vitro and in vivo, of PL-expanded MSCs, as compared to those cultured in FCS-based medium. This information needs to be acquired before introducing this culture supplement in the routine preparation of cellular products to be employed for clinical application. Moreover, clinical data on the safety and efﬁcacy of MSCs have been obtained, so far, mainly with cells expanded in the presence of FCS, whereas relatively little in vivo experience is available with MSCs cultured in PL. Other safety issues related to ex vivo expanded MSCs The utilization of ex vivo expanded MSCs for clinical application is associated not only with the potential risk of the immunogenicity of the cells or with the bio-ssafety of medium components, but also with the potential in vitro transformation of the cells during expansion. It has been shown by some groups that the ex vivo manipulation of both human and murine MSCs may alter the functional and biological properties of the cells, leading to the accumulation of genetic alterations [51–55]. In particular, the groups of Rubio and Røsland published that human AT- and BM-derived MSCs are prone to undergo malignant trasformation after long term ex vivo expansion; transformed cells showed an increased proliferation rate, displaied an altered morphology and immunephenotype and carried cytogenetic abnormalities. Moreover, these cells induced tumor formation when injected into immunideﬁcient mice [51,54]. A high susceptibility to malignant transformation was also described in murine BM-derived MSCs by different groups [53,55]. These latter cells, probably due to their animal origin, display a high degree of chromosome instability, characterized by the development of both structural and numerical aberrations even at early culture passages [53], and induce tumor formation when infused into irradiated allogeneic recipients [55]. On the contrary, our own group and other laboratories did not conﬁrm a propensity of human MSCs to develop morphological and genetic changes [40,42,56,57]. In particular, our group has investigated the potential susceptibility of human MSCs, derived from both BM and UCB and expanded in the presence of FCS or PL, to undergo transformation after in vitro culture prolonged to either passage 25 or cell senescence [40,42,56]. We found that both BM- and UCB-MSCs can be cultured for long term without loosing their usual phenotypical and functional characteristics. Using genetic studies, performed through both conventional and molecular (array-Comparative Genomic Hybridization, array-CGH) karyotyping, the absence of chromosomal abnormalities was documented [40,42,56]. Telomere length decreased along the different culture passages and telomerase activity was not found to be present. Moreover, in case of UCB-MSCs, p16ink4a , a protein regulating cell cycle and involved in the process of senescence, was found to be normally expressed on expanded cells and anchorage growth independence in soft agar was never observed [44]. Similar ﬁndings were obtained in BM-MSCs cultured until passage 3 following the FCS-based expansion protocol developed within the EBMT Developmental Committee in the perspective of being employed for clinical application [20–22]. Recently, French researchers have reported the presence of aneuploidy in a number of MSC preparations for clinical application, after cultivation both in the presence of FCS þ Fibroblast Growth factor2 (FGF-2) and PL [57]. To further characterize the genetic abnormalities, quantitative analysis of genes related to transformation and senescence was performed. Normal and stable expression of c-myc, p53 and p21 was demonstrated, whereas human telomerase reverse transcriptase (hTERT) was never expressed. Moreover, MSCs normally expressed p16ink4a and anchorage growth independence in soft agar was never obtained [57]. These data suggest that, although aneuploidy can occur during MSC

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expansion, it does not reﬂect cell transformation, but rather senescence of the cells. Based on these results, the French MSC clinical trials which were temporary interrupted due to the potential risks associated with the infusion of transformed cells, have been re-opened. Very recently three research groups, who previously reported the propensity of human MSCs to undergo malignant transformation after long term in vitro culture, have now discovered that the cancer-like cells they described were unrelated to the original MSCs and belonged to tumor cell lines that they were using for other projects [58,59]. The risk of cross-contamination and its consequences should always be considered when culturing concomitantly primary cells for long term and immortalized cells lines and it is highly recommended that cell line veriﬁcation with DNA ﬁngerprinting be performed to conﬁrm experimental results before publication. In light of these discrepant observations, phenotypic, functional and genetic assays, although known to have limited sensitivity, should be routinely performed on MSCs before in vivo use to demonstrate whether their biological properties, after ex vivo expansion, remain suitable for clinical application. In particular, the absence of transformation potential in cultured cells has to be documented before infusion into patients, particularly into immune-compromised subjects where impairment of immune surveillance mechanisms might further favor the development of tumors in vivo. In particular, from a practical point of view, karyotyping should be performed on expanded cells and be included in the quality controls and release criteria for MSC administration into patients. Indeed, a precise characterization of the genetic proﬁle of MSCs could allow to identify phenomena of senescence, developing in cells at the end of their life-span, versus transformation of cells, due to the occurrence of genetic alterations. Conclusions At present, MSCs are extensively expanded ex vivo before being used in the clinical setting [13,20–22]. The adoption of different isolation methods and culture conditions may lead to multiple MSC populations with slightly different biological and functional characteristics. For instance, differences in culture medium or supplements (FCS, human serum, PL, addition of GFs), plating density, level of conﬂuency at cell detachment may inﬂuence their proliferative capacity, expression of surface markers or differentiation capacity, leading to the commitment towards a speciﬁc phenotype or tissue lineage [13,19,41,45]. This supports the need for the deﬁnition and validation of common isolation and expansion protocols for the preparation of MSCs both for experimental and clinical purposes. The use of a uniform expansion method, possibly devoid of animal components, facilitates the comparisons between cell products generated at different sites and allows to perform large multicenter collaborative studies. Platelet lysate has been recently proposed as alternative culture supplement for MSC ex vivo manipulation [41–45]; despite the fact that expansion procedures using PL have been implemented in different laboratories, deﬁnitive standards to produce clinical-grade PL-MSCs are lacking and need, therefore, to be deﬁned. Moreover, it remains to be studied whether the clinical safety and efﬁcacy proﬁle of PL-expanded MSCs is similar to FCS-expanded MSCs. Given the reports of potential transformation of adult human MSCs after ex vivo culture [51– 55], phenotypic, functional and genetic assays should be routinely performed on MSCs before in vivo use to demonstrate whether, after ex vivo expansion, they remain suitable for clinical application. Moreover, surface markers and functional assays to speciﬁcally identify MSCs should be further investigated; these tools could allow for the prospective isolation of MSCs from human tissues, as well as to facilitate the generation of homogenous cell products in different laboratories. In particular, novel techniques, such as proteomic approaches and microarray analysis, might be of help for improving the knowledge on MSC expression proﬁle and functional properties and be employed with the aim to identify the true and most primitive mesenchymal progenitors. Once more largely deﬁned in their biological properties and culture-expanded state, MSCs could be properly employed as a therapeutic strategy in various areas of clinical application including modulation of alloimmune responses in the setting of allogeneic HSCT and organ transplantation and promotion of tissue repair in degenerative and inﬂammatory diseases.

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Practice points Ex vivo expanded MSCs are currently employed to promote hematopoietic engraftment and to treat steroid-refractory acute Graft-versus-Host Disease in patients given an allograft of hematopoietic progenitors, as well as to repair ﬁstulas in patients with Crohn’s Disease. Usually MSCs to be employed in the clinic are cultured in the presence of 10% FCS and harvested at passage 2 to 3 of culture. PL is a suitable alternative culture supplement for MSC ex vivo expansion, able to abrogate the risk of sensitization to heterologous proteins and of transmission of known and unknown zoonoses. The proliferative capacity of MSCs expanded in the presence of PL is superior to that of FCScultured MSC. Characterization of genomic stability is a mandatory requirement for releasing MSC for clinical use.

Research agenda Biological and functional studies aimed at characterizing PL-expanded MSCs, in comparison to FCS-cultured MSCs, are warranted. Clinical trials comparing the safety and efﬁcacy of MSCs obtained in the presence of either FCS or PL must be performed in order to better deﬁne the optimal culture medium supplement. Future studies should invest on the characterization of novel markers for the prospective identiﬁcation/isolation of mesenchymal progenitors in the different human tissues. Extensive investigations on gene expression proﬁle by microarray analyses and proteomic approaches could unravel peculiarities of MSC populations obtained with different culture methods.

Funding This work has been partly supported by grants from Istituto Superiore di Sanità (National Program on Stem Cells), MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca, Progetti di Rilevante Interesse Nazionale, PRIN), from Associazione Italiana per la Ricerca sul Cancro (AIRC) IG6071 and from the special project “5per mille” from AIRC to F.L.; and from AIRC IG9062 to M.E.B. Conﬂict of interest The authors have no conﬂict of interest to disclose.

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Contents lists available at ScienceDirect

Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha

9

Mesenchymal stem cells in ex vivo cord blood expansion Simon N. Robinson, Ph.D., Senior Research Scientist, Department of Stem Cell Transplantation and Cellular Therapy a,1, Paul J. Simmons, Ph.D., Professor and Director, C. Harold and Lorine G. Wallace Distinguished Chair, Professor and Director, Center for Stem Cell Research, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston b, 2, Hong Yang, Ph.D., MD, Senior Research Scientist, Department of Stem Cell Transplantation and Cellular Therapy a, 3, Amin M. Alousi, MD, Assistant Professor, Department of Stem Cell Transplantation and Cellular Therapy a, 4, J. Marcos de Lima, MD, Associate Professor, Department of Stem Cell Transplantation and Cellular Therapy a, 5, Elizabeth J. Shpall, MD, Professor, Stem Cell Transplantation and Cellular Therapy; Medical Director, Cell Therapy Laboratory; Director, Cord Blood Bank a, * a

Department of Stem Cell Transplantation and Cellular Therapy, University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA The Centre for Stem Cell Research, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 1825 Pressler Street, Houston, Texas 77030. USA b

Keywords: cord blood (CB) transplantation ex vivo expansion mesenchymal stem cells (MSC)

Umbilical cord blood (CB) is becoming an important source of haematopoietic support for transplant patients lacking human leukocyte antigen matched donors. The ethnic diversity, relative ease of collection, ready availability as cryopreserved units from CB banks, reduced incidence and severity of graft versus host disease and tolerance of higher degrees of HLA disparity between donor and recipient, are positive attributes when compared to bone

* Corresponding author. Tel.: þ1 713 745 2161; Fax: þ1 713 794 4902. E-mail addresses: [email protected] (S.N. Robinson), [email protected] (P.J. Simmons), [email protected] (H. Yang), [email protected] (A.M. Alousi), [email protected] (J. Marcos de Lima), [email protected] (E.J. Shpall). 1 Tel.: þ1 713 563 1897; Fax: þ1 713 563 1898. 2 Tel.: þ1 713 500 3427; Fax: þ1 713 500 2424. 3 Tel.: þ1 713 563 1885; Fax: þ1 713 563 1898. 4 Tel.: þ1 713 745 8613; Fax: þ1 713 794 4902. 5 Tel.: þ1 713 745 3219; Fax: þ1 713 794 4902. 1521-6926/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2010.11.001

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marrow or cytokine-mobilized peripheral blood. However, CB transplantation is associated with signiﬁcantly delayed neutrophil and platelet engraftment and an elevated risk of graft failure. These hurdles are thought to be due, at least in part, to low total nucleated cell and CD34þ cell doses transplanted. Here, current strategies directed at improving TNC and CD34þ cell doses at transplant are discussed, with particular attention paid to the use of a mesenchymal stem cell (MSC)/CB mononuclear cell ex vivo coculture expansion system. Ó 2010 Elsevier Ltd. All rights reserved.

Background Since the ﬁrst CB transplant (CBT) was performed by Gluckman et al.[1] in 1988, >20,000 patients have received this procedure to support treatment for a variety of malignant and non-malignant diseases.[2–16] The reported event-free survival rates for such patients are comparable with those achieved following the transplantation of unrelated allogeneic bone marrow (BM), or mobilized peripheral blood progenitor cells (PBPCs).[15] In addition, there are many reports of lower rates of graft versus host disease (GvHD) than are commonly observed with BM and PBPC transplantation, particularly in pediatric patients. This reduced incidence of GvHD is observed despite the use of CB grafts with greater donor-recipient human leukocyte antigen (HLA) mismatching than would be tolerated by recipients of BM,[4,5,9,12] or PBPC allografts.[17–19]

Challenges One major challenge associated with the use of CB for transplantation is the relatively low cell dose available. This is thought to contribute, at least in part, to the slower engraftment and an elevated risk of engraftment failure that is associated with CBT.[20–24] For example, the time required for a patient receiving CBT to achieve an absolute neutrophil count (ANC) of 0.5x109/L can range from 23 to 41 days. Similarly, the median time for a CBT patient to achieve a transfusion-independent platelet count of 20x109/L can range from 56 to >100 days. Further, while engraftment failure rates for CBT recipients as a whole (pediatric and adult CB recipients) can range from 12–20%,[5,9,13,14] those data for adult patients (>18 years old and/or >45 kg) are particularly poor with engraftment failure rates reaching of 20% or higher having been reported.

Evidence of threshold doses for effective transplantation Data from studies performed by Gluckman et al.[9] demonstrated that engraftment and survival were superior in CBT patients who received a transplant dose of 3.7x107 TNC/kg. These data suggest that there is a threshold CB total nucleated cell (TNC) dose above which time to engraftment is improved and graft failure rate reduced and below which time to engraftment is prolonged and graft failure rate increased. However, it is rare that a CBT cell dose of 3.7x107 TNC/kg is achieved. This is particularly true for CBT patients of >45 kg. Additional analyses of these data revealed that a lower, more readily realized target CBT dose ‘threshold’ of 1.0x107 TNC/kg was still associated with favorable engraftment rates and could be applied for this patient population.[2,9] However, analysis of data from patients who received myeloablative therapy and a single CB unit in North America or Europe through 2005 at the world’s three largest CBT registries: Center for International Blood and Marrow Transplant Research (CIBMTR), National Cord Blood Program (NCBP) and Eurocord,[25] revealed that a 100-day treatment-related mortality of approximately 44% was closely correlated with a CBT dose of <2.5x107 TNC/kg (P<0.0001) despite state-of-the-art care practices. These data underscore the urgent need to improve CBT strategies with the goal of improving neutrophil and platelet engraftment and reducing the risk of engraftment failure, especially for patients >45 kg.

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Strategies to enhance CBT outcome At the University of Texas M. D. Anderson Cancer Center Transplant Program, PBPC is the most commonly used source of unrelated haematopoietic support for cancer patients. PBPC remain the ‘gold standard’ against which the efﬁcacy of CBT is compared. For comparison, patients who receive unrelated PBPC transplants achieve an ANC of 0.5x109/L at a median of 11 days post-transplant as compared to 23–41 days for CBT recipients. Similarly, while PBPC recipients achieve a transfusionindependent platelet count of 20x109/L at a median of 13 days post-transplant, patients receiving CBT might not achieve platelet engraftment until >100 days. Further, while PBPC recipients have an engraftment failure rate of <1%, rates of 12-20% are common for CBT. Given that these differences are likely a consequence of the limited cell dose associated with CBT, two major therapeutic strategies are being explored by different clinical centers to increase the cell dose at transplant and thereby improve time to neutrophil and platelet engraftment and reduce engraftment failure are (i) double CBT[26-30] and (ii) ex vivo expansion.[26,27,31,32] Double cord blood transplantation While double CBT does provide signiﬁcantly more rapid neutrophil engraftment (23 days; range 1541 days),[28–30,33] when compared to single CBT, it continues to be associated with signiﬁcantly delayed engraftment and elevated engraftment failure when compared to BM or PBPC transplantation.[27] The exact mechanisms by which double CBT improves the rate of engraftment over single CBT remain to be determined, however, it is worthy of note that while the progeny of both CB units are detectable for a short period after transplant, only one CB unit will ultimately predominate.[33] Measures that are predictive of which CB unit will ultimately predominate remain to be determined. Rationale for ex vivo expansion The observation that there is a threshold dose below which CBT recipients have markedly delayed engraftment and elevated risk of graft failure suggests that suboptimal numbers of the cells responsible for rapid engraftment are being transplanted.[34] If this is the case, increasing the dose of those CB cell subpopulations responsible for rapid engraftment, should improve the time to neutrophil and platelet engraftment and reduce the risk of graft failure. This has been the rationale behind the development of ex vivo expansion strategies. However, it is important to emphasize that double CBT and ex vivo expansion are not mutually exclusive, with the hope that a combination of these strategies might provide the greatest beneﬁt to CBT recipients. Concerns associated with ex vivo expansion The goal of increasing the numbers of haematopoietic progenitors available for transplant using ex vivo expansion has been explored in a PBPC setting.[35–44] These PBPC studies set the stage for the use of this approach with CB. While there is evidence of functional and phenotypic heterogeneity within the primitive haematopoietic progenitor compartment,[45–48] one concern associated with ex vivo expansion is that short-term reconstituting, lower ‘quality’ haematopoietic progenitors will be expanded at the expense of longer-term reconstituting, higher ‘quality’ haematopoietic progenitors, thereby signiﬁcantly impacting the haematopoietic reserve of the graft.[49] Evidence primarily in animal models, suggest that this may occur under certain conditions.[50–57] Clinically, while the absence of durable engraftment from ex vivo expanded CD34þ cells has been reported in some cases,[58] durable engraftment has been reported in other patients who received expanded autologous products as the sole source of haematopoietic support following high dose therapy.[51] This suggests that the primitive haematopoietic progenitor cell compartment in general,[59–64] properties of homing[65] and the basic biologic and genetic characteristics of the primitive haematopoietic progenitor cells[66] are preserved following ex vivo expansion.

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Methodologies Static liquid cultures Static liquid culture was shown to ﬁrst require the puriﬁcation of CD34þ (or CD133þ) cells from fresh or frozen tissue.[44,67,68] Although clinical grade immunomagnetic devices can be used for the isolation procedure,[68] the puriﬁcation process is limited by low recovery of the target cells (median CD34þ cell recovery of 35%, range, 4–70%). Despite this loss, isolated CB CD34þ (or CD133þ) cells have been expanded ex vivo and used in clinical trials at the M. D. Anderson Cancer Center.[27,69] Initially, positively selected CD133þ cells were cultured in medium supplemented with 100 ng/ml each of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF) and thrombopoietin (TPO),[70] for 10 days which resulted in a 56-fold expansion of total nucleated cells (TNCs) and a 4-fold expansion of CD34þ cells. A two-step, 14 day ex vivo CB expansion protocol was subsequently developed[71] where the CD133þ cells were cultured for one week with the same SCF-G-CSF-TPO regimen in a small volume of 50 ml and then transferred to a larger 800 ml volume on day 7 with fresh media/growth factors and continued culture for the second week. This procedure yielded a >400-fold increased TNCs and >20-fold increased CD34þ cells. It should be noted that in some cases, due to the upfront cell losses incurred by the immunomagnetic selection procedure, even marked ex vivo expansion only gives rise to a cell product whose numbers are not markedly different from that of the original CB unit. However, the argument can be made that exposure to the ex vivo expansion cytokine milieu generated a cell product that is different in ‘quality’ to the original CB unit with cells possibly ‘primed’ by the ex vivo exposure to growth factors and subsequently better able to home, engraft and proliferate when transplanted. Mesenchymal stem cell based cultures Ex vivo liquid culture removes the primitive haematopoietic cells from molecular cues provided by the haematopoietic microenvironment. As a consequence the addition of exogenous cytokines is required to prevent apoptosis and stimulate proliferation. An alternative approach is the ex vivo coculture of haematopoietic cells with components of their haematopoietic microenvironment. The haematopoietic microenvironment contains the putative stem cell ‘niche’ and is composed of haematopoietic and non-haematopoietic, cellular and extracellular components thought to provide the complex molecular cues that direct primitive haematopoietic progenitor self-renewal, proliferation and differentiation.[72–80] This would be consistent with the observation that ex vivo contact between primitive haematopoietic progenitors and stromal components of the haematopoietic microenvironment preserve stem cell activity.[81–87] Mesenchymal stem cells (MSC) are one component of the haematopoietic microenvironment and can be isolated from a variety of fetal and adult tissues.[89–92] Phenotypically MSC express CD73, CD90, CD105, CD16, and HLA-ABC(I) and do not express CD31, CD34, CD45, CD80 and HLA-DR(II). MSC can be grown and expanded as adherent, contact-inhibited monolayers in tissue culture ﬂasks, although the primary nature of the cell-type limits the degree of expansion that can be achieved before senescence occurs. Ex vivo CB MNC/MSC co-culture The CB MNC/MSC ex vivo co-culture technique does not require the isolation of CD34þ (or CD133þ) cells from the CB, thereby minimizing the losses associated with this procedure. Using a co-culture strategy with bone marrow-derived MSCs and a supportive growth factor regimen which included Flt3-Ligand (FLT3-L), SCF, GCSF, and TPO, CB MNCs were cultured in 50 ml medium on stroma for 7 days at which time the non-adherent cells were removed in cultured in a larger volume (800 ml) for an additional 7 days. The ﬂasks containing the adherent cells were also re-fed with 50 ml of media and the FLT3L-SCF-GCSF-TPO regimen and cultured for the subsequent 7 days. On day 14 all of the cells from the adherent and non-adherent cultures were pooled for evaluation. Using this strategy a 10–20 fold increase in TNCs, a 7–18-fold increase in committed progenitor cells (colony-forming units, CFU), a 2–5-fold increase in primitive haematopoietic progenitors (high proliferative potential

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colony-forming cells) and a 16–37-fold increase in CD34þ cells was achieved.[88] Building on this experience, a CB MNC/MSC co-culture strategy was developed to maximize the available expanded cells dose for transplant.[89] In the research laboratory, ex vivo expansion culture was characterized by a 6-fold increase in TNC, 30-fold increase in CD133þ cells, 8-fold increase in CD34þ cells, >200-fold increase in CFU, 50-fold increase in cobblestone area-forming cells persisting in culture for 2 weeks (CAFCwk2) and thought to be representative of more mature haematopoietic progenitors, and a reduction in (0.05-fold) cobblestone area-forming cells persisting in culture for 6 weeks (CAFCwk6) and thought to be representative of more primitive haematopoietic progenitors. These CAFCwk6 data provide evidence of the expansion of the more mature haematopoietic progenitors at the expense of the more primitive haematopoietic progenitor cell population. The fold increases cited are over those numbers originally present in the CB unit prior to ex vivo expansion and therefore represent true expansion. Ex vivo CB MNC/MSC co-culture expansion trial at the M. D. Anderson Cancer Center Clinical-scale CB MNC-MSC ex vivo expansion procedures were developed[89] and validated.[90] A trial was subsequently designed to test the clinical feasibility of transplanting the expansion product from CB MNC/MSC co-cultures into patients with haematologic malignancies. The trial, approved by the MD Anderson Institutional Review Board (IRB Protocol 05-0781) and the U.S. Food and Drug Administration (FDA) (IND 13,034), details that patients will receive two CB units matched in at least 4/ 6 HLA antigens, with a minimum dose of 1x107 TNC/kg from each unit. For the initial cohort of 12 patients, a family member (matched at 2/6 antigens) serves as the third party haploidentical MSC donor.[90] Approximately 100 ml of marrow is aspirated from the donor and MSC isolated by plastic adherence. In preparation for co-culture with CB MNC, the MSC are grown to >70% conﬂuence in 10 x T175 culture ﬂasks. The CB unit with the lowest TNC dose is then thawed and divided equally between each of the 10 MSC layers, each in 50 ml of ex vivo expansion media containing 100 ng/ml each of SCF, Flt-3-ligand (Flt-3L), G-CSF and TPO. After 7 days of co-culture at 37 C in a 5% CO2-in-air fully humidiﬁed atmosphere, non-adherent cells are removed from each ﬂask and each transferred into individual 10 x 1-liter Teﬂon-coated culture bags (American Fluoroseal) with fresh ex vivo expansion medium added to generate 800 ml. This liquid culture step (in the absence of MSC) is performed for an additional 7 days (14 days total). The original co-culture ﬂasks also receive 50 mls of fresh medium. At the end of the ex vivo expansion procedure, all non-adherent cells from the culture bags and culture ﬂasks are pooled, washed and prepared for transplantation. Samples are removed for prospective ﬂow cytometric analysis and quality assurance testing. Preliminary clinical results with family member-derived MSCs Patients were admitted on day -9 for hydration and received the designated preparative regimen on days -8 through -2. On day 0, the unmanipulated CB unit was thawed and infused, followed by infusion of the expanded CB cells. Median TNC and CD34þ cell expansions were 12-fold (range, 1–13) and 12fold (range, 1-27), respectively. The mean expanded doses of 5.7x107 TNC/kg and 3.8x105 CD34þ cells/ kg representing important increases compared to those achieved in our previous expansion studies. Furthermore, when the second unmanipulated CB unit is considered, patients on this trial received a total of 9.5x107 TNC/kg and 8.2x105 CD34þ cells/kg. Recipients of myeloablative therapy, engrafted neutrophils in a median of 14.5 days (range, 12–23) and platelets in 30 days (range, 25–51). Rationale for the use of “Off-The-Shelf” MSC for the clinical protocol The complex logistics of generating MSC from a patient’s family member have limited accrual to the clinical trial. Such limitations include: (a) an appropriate family member was not always be available to donate marrow

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(b) disease progression is rapid for selected patients with for example, acute leukemia who were relapsing during the 3 weeks it took to generate sufﬁcient MSCs and 2 weeks to perform the CB MNC/MSC co-culture expansion (delaying transplant for 5 weeks total after patient enrolled on the trial). We postulated that the availability of an “off-the-shelf” source of Good Manufacturing Practice (GMP)-compliant, allogeneic MSC would alleviate this logistical problem with MSC essentially available for immediate use. The development of master cell banks from young, healthy volunteers provides an optimal source of MSC and standardization of selection and isolation procedures ensures a reproducible, readily available MSC product. The Stro-1 antibody, developed by Dr. Paul Simmons,[91] allows prospective isolation of human bone marrow MSC without plastic adherence. Angioblast Systems, Inc., acquired the technology and developed the allogeneic MSC product RevascorÔ for clinical use primarily in the treatment of ischaemic cardiovascular disease.[92–95] To date, more than 20 congestive heart failure patients have received injections of RevascorÔ with no adverse events related to the cells (Dr. Silviu Itescu, Angioblast Systems, Inc., personal communication). Angioblast has agreed to supply RevascorÔ as an ‘off-the-shelf’ product for the M. D. Anderson CB MNC/MSC co-culture expansion trial. Pre-clinical studies of Angioblast-MSC-CB expansion Pre-clinical studies have conﬁrmed that the 10 ﬂasks of MSC required for the ex vivo CB expansion protocol can be routinely generated in 4 days from a single vial (107 cells) of the Angioblast MSC product. Multiple experiments performed to compare the performance of the Angioblast MSC product with that of normal donor-derived MSC have revealed no difference in the expanded CB product generated.[96] Preliminary clinical results with Angioblast-derived MSCs With the pre-clinical Angioblast data described above, the M.D. Anderson CB MNC/MSC ex vivo expansion Protocol 05-0781 and FDA IND 13,034 were amended to include a separate cohort of patients who would be treated identically to the ﬁrst cohort, but who would receive CB cells expanded on the Angioblast product. Accrual to that cohort has been initiated. During CB MNC-MSC ex vivo expansion, a median expansion of 14-fold (range 1–30) for TNC and 40-fold (range 4-140) for the CD34þ cells was achieved. At transplant, the contribution of the unmanipulated CB included 2.35x107 (range 0.2–8.2) TNC/kg and 0.95 X 105 (range 0–4) CD34þ cells/kg, while the contribution of the ex vivo expanded CB unit was 5.8x107 (range, 0.3–14.4) TNC/kg and 8.7x105 (range, 0-93.4) CD34þ cells/kg. These were higher doses than we have ever infused into any of our recipients of unmanipulated double CBT, or CB expanded with our liquid culture system. As with the family member-derived MSCs, median time to neutrophil engraftment (500/ml) was 15 days (range 9-42) and platelet engraftment (>20,000/ ml) was 38 days (range 13–62) with 26 patients (81%) of patients becoming platelet transfusion independant. On transplant dayþ21, the chimerism assays revealed that the MSC-expanded unit contributed to engraftment with a mean of 19% of the mononuclear cell, 16% of the T cell, and 14% of the myeloid fractions due to the expanded unit. Subsequently, haematopoiesis was increasingly derived from the unexpanded unit with long-term engraftment provided exclusively by the unexpanded unit by six months post-transplant in the vast majority of patients. Summary Our initial CB expansion protocol (#02-407, IND#7166) involved culture of CD133 þ CB cells in teﬂon bags for 14 days with media containing 100 ng/ml SCF, G-CSF and TPO. In this system the CD133 þ CB cells are initially cultured in bags with 50 ml of media/growth factors for 7 days and then transferred to a bag with 800 ml of fresh media and growth factors for another 7 days at which time they are washed and infused. With this strategy our patients experienced a modest improvement in engraftment of 20 days for neutrophils and 65 days for platelets compared to recipients of double

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unmanipulated CB units who engrafted neutrophils in 22 days and platelets in 100 days. However, we experienced a loss of >70% of the CB CD34þ cells following the CD133-selection procedure, which stimulated us to investigate the MSC-based co-culture system, where we could culture the entire CB unit without need for positive selection. The use of the MSC-based CB expansion protocol (#05-0781) used a similar 14-day strategy where for the ﬁrst 7 days the cells are cultured in 50 ml, but this time in 10 ﬂasks containing 10% of the CB unit plus MSCs that are 70% or more conﬂuent (rather than the entire CD133þ fraction in one bag with the liquid culture system). The growth factor regimen for this trial included the addition of Flt3-ligand to the SCF-GCSF-TPO regimen. On day 7, the non-adherent cells in each of the 10 ﬂasks are transferred to 10 teﬂon-coated bags with 800 ml of media and the four growth factors (rather than transfer to a single bag in the liquid culture system with three growth factors). Fifty ml of media/growth factors are added to the 10 ﬂasks containing the adherent layers of MSC-CB, and both those ﬂasks and the 10 bags are cultured for another 7 days. On day 14, all of the cells in the bags and ﬂasks are pooled, washed and infused. This strategy has shown more promising results providing markedly higher TNC and CD34þ cell doses than have ever before been achieved. The improvments are likely due to the use of MSCs to recapitulate the haematopoietic milieu, the ability to culture the whole CB unit rather than the CD133þ fraction, minimizing the large upfront CD34þ cell losses due to the positive selection procedure, and possibly the addition of FLT3-ligand to the cultures. The improvement in median times to engraftment of neutrophils (14 days) and platelets (35 days) are encouraging and accrual to the trial continues. Conﬂict of interest statement None. Acknowledgements This work was supported in part by NIH Grant #RO1-CA061508-15 and Cancer Prevention & Research Institute of Texas (CPRIT) Grant # RP100469. The authors would like to acknowledge the dedicated researchers, clinicians and nurses of the M. D. Anderson Cancer Center. References *[1] Gluckman E, Devergie A, Bourdeau-Esperou H, et al. Transplantation of umbilical cord blood in Fanconi’s anemia. Nouv Rev Fr Hematol 1990;32:423–5. [2] Wagner JE. Umbilical cord blood stem cell transplantation. Am J Pediatr Hematol Oncol 1993;15:169–74. [3] Wagner JE, Kernan NA, Steinbuch M, et al. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 1995;346:214–9. [4] Wagner JE, Rosenthal J, Sweetman R, et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood 1996;88:795–802. *[5] Kurtzberg J, Laughlin M, Graham ML, et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996;335:157–66. [6] Laporte JP, Gorin NC, Rubinstein P, et al. Cord-blood transplantation from an unrelated donor in an adult with chronic myelogenous leukemia. N Engl J Med 1996;335:167–70. [7] Laporte JP, Lesage S, Portnoi MF, et al. Unrelated mismatched cord blood transplantation in patients with hematological malignancies: a single institution experience. Bone Marrow Transplant 1998;(Suppl 1):S76–7. [8] Cairo MS, Wagner JE. Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation. Blood 1997;90:4665–78. *[9] Gluckman E, Rocha V, Boyer-Chammard A, et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 1997;337: 373–81. [10] Locatelli F, Rocha V, Chastang C, et al. Cord blood transplantation for children with acute leukemia. Eurocord Transplant Group. Bone Marrow Transplant 1998;21(Suppl. 3):S63–5. [11] Locatelli F, Rocha V, Chastang C, et al. Factors associated with outcome after cord blood transplantation in children with acute leukemia. Eurocord-Cord Blood Transplant Group. Blood 1999;93:3662–71. [12] Rocha V, Wagner Jr JE, Sobocinski KA, et al. Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. N Engl J Med 2000;342:1846–54. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. [13] Long GD, Laughlin M, Madan B, et al. Unrelated umbilical cord blood transplantation in adult patients. Biol Blood Marrow Transplant 2003;9:772–80.

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Best Practice & Research Clinical Haematology Vol. 24, No. 1, pp. i1, 2011

Keyword Index autoimmune diseases, 49 Bone marrow stromal cell, 37 cell therapy, 49 cellular therapy, 73 central nervous system (CNS): neurons, 59 cord blood (CB) transplantation, 83 experimental autoimmune encephalomyelitis (EAE), 59 ex vivo expansion, 73, 83

Mesenchymal stem cell, 37 mesenchymal stem cells (MSC), 59, 83 Mesenchymal stem cells, 49, 65 mesenchymal stroma cell (MSC), 13 mesenchymal stromal cells, 73 mesenchymal/stromal stem cells, 25 microglia, 59 MSC subsets, 25 MSC, 25 multipotent differentiation, 13 neuroprotection, 59

fetal calf serum, 73

oligodendrocytes, 59

Graft-versus-host disease, 65

pericyte adventitial reticular cell, 13 platelet lysate, 73 pluripotency, 3 prospective isolation, 25

Hematopoietic niche, 37 Hematopoietic stem cell transplantation, 65 Hematopoietic stem cell, 37 Hemorrhagic cystitis, 65 inflammation, 49 malignant transformation, 73 MAPC, 3 mesenchymal progenitor cell (MPC), 13

reprogramming, 3 sca-1, 13 surface antigens, 25 Tissue toxicity, 65 vascular smooth muscle cell (VSMC), 13