Prospective identification and isolation of murine bone marrow derived multipotent mesenchymal progenitor cells

Best Practice & Research Clinical Haematology 24 (2011) 13–24

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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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