mesenchymal stem cells in vivo

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Review

Human bone marrow mesenchymal stem cells in vivo

E. Jones and D. McGonagle

Great confusion still exists amongst cell biologists, musculoskeletal and other specialists interested in regenerative medicine regarding

the in vivo identity of human bone marrow (BM) mesenchymal stem cells (MSCs). Contrary to views held in some quarters, methods for

the robust identification and purification of BM MSCs are now well established. Human BM MSCs represent a phenotypically homogeneous

cell population that share an identical phenotype with marrow adventitial reticular cells (ARCs), which are stromal cells similar in nature

to pericytes. When an extensive panel of markers is used to characterize BM MSCs, it appears that the diverse MSC markers described

in different laboratories are expressed on the same cell population. Rare cell phenotypical analysis and in vitro colony forming unit-fibroblast

(CFU-F) assays produce no compelling evidence that BM MSCs circulate in healthy man. Furthermore, although investigators speak of

a number of specific MSC markers, a true marker of MSC ‘stemness’ and multipotentiality has not yet been defined since culture-expanded

MSCs may lose some of these markers, but remain multipotential. This knowledge provides a platform for understanding MSCs in vivo

leading to novel approaches for therapy development, including in situ tissue engineering.

K EY WORDS: Mesenchymal stem cells, Surface markers, In vivo topography, MSCs in circulation.

Background

The concept of a mesenchymal stem cell (MSC) arose from thework of Friedenstein and colleagues four decades ago [1]. Theynoted that upon plastic adherence of bone marrow (BM) cells,a rare cell population developed into colony forming units thatwere fibroblastic (CFU-F) [2]. Following in vitro culture expan-sion, clonal cultures derived from individual CFU-Fs could beintroduced into diffusion chambers in experimental models wherethe formation of bone, cartilage and stromal elements wasobserved. The frequency of these marrow CFU-Fs was extremelylow, ranging from 1/10 000 to 1/100 000 BM mononuclear cells(MNCs) [3]. This is considerably lower than the frequency of

CD34

þ

haematopoietic progenitor/stem cells (HSCs) that com-prise about 1% of the MNC fraction [4]. Therefore, it was longrecognized that the study of BM MSCs in vivo would representa much tougher challenge than the study of HSCs.

The interest in MSCs increased greatly almost a decadeago with the reporting of novel markers for culture-expandedMSCs including CD73 and CD105 and the development of robustin vitro assays of MSC tripotentiality [5]. Some investigatorssuggested that these findings were erroneously celebrated by thescientific community and media as the happy outcome of anextraordinary hunt for MSCs [6]. Indeed, the studies in questiondescribed the same culture-expanded CFU-F population thatwent back as far as Friedenstein’s work, and the identity of theunknown ancestral cell remained enigmatic. The only firm clue tothe in vivo identity of BM MSCs came from the work of Simmon’s

group [7] who showed that an antibody Stro-1 could be usedto enrich CFU-Fs approximately 100-fold; however, theirpurification was still not feasible.

The ongoing confusion in the MSC field has been contrib-uted to by the assumption that any marker expressed onculture-expanded MSCs was also likely to be present in vivo.Consequently, independent laboratories have begun to usedifferent markers of expanded MSCs to search for MSCs in vivo

[8, 9]. This has resulted in the perception that these in vivo

MSCs were a heterogeneous cell population, and could be distinct

from Stro-1þ stromal cells and progenitors. Indeed, the confusionto the in vivo identity of the BM MSC has lead to difficulty withterminology whereby the MSC acronym continues to signify bothMSCs and marrow stromal stem cells [5, 6, 10]. Based on ourstudies of in vivo MSCs and related literature, the purpose of thisarticle is to reconcile these apparent contradictions and to discusstheir implications for regenerative medicine development.

Markers for the in vivo identification of MSCs

There is still a widely held perception that BM MSCs representa phenotypically heterogeneous population of cells. There area number of reasons for this. First, as far back as the pioneering

work of Friedenstein et al . [11, 12], it has been recognized thatnot all CFU-Fs were highly proliferative and multipotential.Second, many different groups have used a limited number of diverse phenotypic markers to identify in vivo MSCs/CFU-Fsusing magnetic enrichment or flow cytometry [8, 9, 13–17]. Takinga synthesis of these functional and phenotypic data to a logical(but not necessarily correct conclusion) has led to the impressionthat MSCs were both functionally and phenotypicallyheterogeneous.

To clarify this, we used multiparameter flow cytometry andcross-tested different MSC markers and purification methods,including plastic adherence for their selectivity and specificity forin vivo BM MSCs [14, 18]. We found that all these methodsidentified a phenotypically identical rare cell population that wasdistinct from BM haematopoietic cells by their very low CD45

expression and a larger cell size. The Stro-1 marker was uniformlyexpressed on these cells [14], but its cross-reaction with otherBM populations somewhat limited its utility for BM MSCidentification. Another disadvantage of the Stro-1 antibody relatedto the fact that its target antigen has not thus far been defined,despite the fact it has been over 15 yrs since its original discovery.

With this in mind, we next aimed to find the best positivemarker for BM MSCs based on the criterion of the highestexpression on MSCs and the lowest expression on all othermarrow cell populations. As a result, it was demonstrated thatthe low-affinity nerve growth factor receptor (LNGFR), nowclustered as CD271, was the most differentially expressed marker[14, 19]. Importantly, high-level expression of CD271 on BMMSCs was reported in at least four other independent studies[13, 20–22]. These observations have lead to the recent commer-cialization of CD271 as a preferred marker for the purification of

The Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK.

Submitted 18 May 2007; revised version accepted 27 June 2007.

Correspondence to: D. McGonagle, The University of Leeds, Chapel Allerton

Hospital, Leeds LS7 4SA, UK. E-mail: [email protected]

Rheumatology 2008;47;126–131 doi:10.1093/rheumatology/kem206

Advance Access publication 6 November 2007

126ß The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected]



a homogeneous population of cells that contains all the BM MSCactivity [23]. Notably, another neural molecule (gangliosidemolecule GD2) has been recently shown to be expressed on BMMSCs, both in vivo and following expansion in culture [24].

In the past, we were also able to show that both CD73 andCD105 were uniformly present on BM CD45lowCD271þ cells [18],as were many other putative MSC markers such as CD90 [14].With the exception of CD146/Muc18, which showed a wide

spectrum of positivity from low to brightly positive cells, everyother marker tested in our studies has demonstrated no apparentheterogeneity and was either uniformly positive or uniformlynegative on MSCs. Of course, with the passage of time andincreased availability of good-quality commercial antibodies,further phenotypic heterogeneity within BM CD45lowCD271þ

MSCs may be found, parallel to past discoveries in the HSC field,when CD34þ population was later subdivided into CD34þCD38À

and CD34þCD38þ fractions [25]. Nevertheless contrary to somecurrently propagated views [26, 27], a phenotypically distinct,in vivo BM MSC population has now been identified. Importantly,a striking consensus regarding the morphology of fresh MSCsis emerging, regardless of the method of isolation used. Theyappear as large cells that have prominent nucleoli and bleb-likeprojections, which extend further as MSCs adhere—this is

different from spindle-shaped morphology of typical culturedMSCs [14, 15, 21]

Based on functional assays, the presence of MSCs in extraskeletal locations including synovium, fat and even placentaltissue and umbilical cord has been firmly established [28–31].Identifying the MSC population from the much larger stromalfraction will be a more formidable challenge compared with MSCidentification in the marrow. There has been a common opinionthat CD73, CD105, CD90 and CD44 are highly specific forMSCs, and hence can discriminate multipotential cells from themore mundane tissue resident fibroblasts. More recently, how-ever, several studies showed that these markers were ubiquitouslyexpressed on stromal cells from many locations as well as on skinfibroblasts [32–34], and at best they only inform an investigatorthat the phenotyped cells are non-haematopoietic and stromal in

origin. Of note, we originally utilized a fibroblast marker termedD7-FIB to purify MSCs from the BM and showed that thesecells expressed other fibroblastic markers such as CD10 and CD13[14]. In the BM, where the overwhelming majority of cells arehaematopoietic, these markers may indeed be useful, but inconnective tissues, where most of the cells are fibroblastic, theirutility for the isolation of resident MSCs will be limited and asearch for new, more specific markers, if they indeed exist, isneeded. For the isolation of MSCs from post-partum tissues, suchas placenta, an embryonic stem cell marker SSEA-4 was found tobe useful [30] and, more recently, it was successfully appliedfor the isolation of MSCs from adult BM [35]. It remainsto be investigated whether SSEA-4 identifies all, or only asubpopulation, of CD45lowCD271þ cells.

Another important issue to bear in mind is the stability of putative MSC markers in culture. Despite the loss of certainmarkers following passaging [7, 14] and the gain of others [18],MSC cultures remain multipotential, indicating that these markersare unlikely to be reflective of the MSC’s true ‘stem cell’ natureor its multipotentiality. More likely, many markers present onMSCs in vivo may be induced by the BM microenvironment or bereflective of some other MSC function in vivo that is lost uponplastic adherence and exposure to culture media. At this stage,it would appear that the heterogeneity in the MSC proliferativeand differentiation capacities, first noted by Friedenstein et al . [1]cannot be explained on the basis of known surface markers alone.

Marrow topography of MSCs

Having defined major surface markers specific for MSCsin vivo, many investigators used immunofluorescence or

immunohistochemical techniques to identify their possiblein vivo niches [18, 36, 37]. It is well established that HSCsoccupy a specific niche in the BM close to trabeculae [38] and thatHSCs are regulated by several local cues including oxygen andcalcium gradients and other molecules. Our phenotypical andmorphological data together with data from other laboratories[6, 37] indicated that BM MSCs could be virtually identical to BMstromal supportive cells termed adventitial reticular cells (ARCs).

In this respect, it is not surprising that many investigators stillrefer to MSCs as multipotent mesenchymal stromal cells [39]or marrow stromal stem cells [6, 15]. The morphological andphenotypical similarities between MSCs and ARCs are listedin the Table 1.

Other investigators have suggested that the topography of MSCs in the BM is that of a vascular pericyte, the cell lining of theouter surfaces of blood vessels [36]. In one view, ARCs themselves‘can be seen as bona fide specialized pericytes of venous sinusoidsin the marrow’ [37] linking both cells together. Indeed, MSCssorted based on Stro-1þCD106þ or Stro-1þCD146þphenotype,expressed smooth muscle actin or were positive for 3G5 antigen,which is said to be specific for pericytes [15, 16]. These findingshave led to a broader proposition that MSCs are generallydistributed throughout the post-natal organism as vascular

pericytes [47]. At the same time, cells that meet the criteria forMSCs, have been isolated from articular cartilage [48], which isavascular, arguing that MSCs do not have to be exclusivelylocated in a perivascular niche. Indeed, bone lining cells (BLCs)as potential reservoirs of MSC activity in the BM cannot beoverlooked. The presence of MSC activity in BLCs would explainwhy bone explants enzymatically pre-treated to remove all softmarrow tissues could generate fully competent MSC cultures inmany published studies [49, 50]. Common to both ARCs andpericytes, BLCs have been shown to be negative for CD45 andpositive for smooth muscle actin [51].

In fact, all three non-haematopoietic BM cell types can possesssome degree of the MSC activity. As shown on Fig. 1, all of themare positive for CD10, a marker uniformly expressed on BMCD45lowCD271þ MSCs. Other studies showed similar distribu-

tion of BM smooth muscle actin-positive cells [52]. In general,a close lineage commonality between ARCs and pericytes [37, 40]or BLCs and ARCs [53] has been previously reported. Altogether,these findings suggest that compared with HSCs, BM MSCs mayhave a less-defined niche. In fact, the non-haemotopoietic, non-endothelial marrow compartment might form a close networkof phenotypically linked but topographically diverse cells, whichcould all have some degree of the MSC activity.

The concept of MSC as a circulating cell

It is firmly established that HSCs can be mobilized from theirmarrow niches and circulate following growth factor and/orchemotherapy administration. Furthermore, HSCs can homeback to these marrow niches following systemic reinfusion andthis forms the basis for peripheral blood autologous stem cell

TABLE 1. Shared features of mesenchymal stem cells (MSCs) and bone marrowadventitial reticular cells (ARCs)

ARCs MSCs

Morphology Reticular with longprocesses

Reticular with longprocesses [14]

LNGFR/CD271 Positive [40, 41] Positive [1 3, 1 4, 20, 21]Alkaline phosphatase Positive [37, 40] Positive [18]VCAM-1/CD106 Positive [42, 43] Positive [1 5, 1 8]

SDF-1/CXCL12 Expressing [44] Expressing [45]Stromal/fibroblast

markersSeveral including

D7-FIB, CD10 andCD13 [40]

Several including D7-FIB,CD10 and CD13 [14, 46]

Human bone marrow MSCs in vivo 127



transplantation. Indeed the concept that MSCs may also circulateis not a new one, and as noted by Prockop, was suggested exactly140 years ago—well before the advent of the molecular or even

cellular biology eras [10]. Consequently, based on this and manysubsequent studies, there is a widespread perception that MSCscirculate. But what is the recent evidence for MSCs circulating andhoming to sites of injury in man? Actually, most of the evidencecomes from experiments with rodent models where MSCs weresystemically infused and appeared to be present in greaternumbers at sites of injury [54, 55]. In our view, this scenariomight simply represent the natural and anticipated consequence of a biophysical process whereby cells artificially introduced to thecirculation get trapped at sites of injury. The criteria used to definecirculating MSCs have not been sufficiently stringent. In manysituations, single marker-based methods were used [56, 57], whichmay not be necessarily sufficient for rare cell detection. On theother hand, CFU-F assays could fail to detect MSCs consideringthe fact that MSCs could have lost their adherence propertiesfollowing entry into circulation as suggested by Khosla and

Eghbali-Fatourechi [57]. Using the latter assays, however, thepresence of rare circulating MSC in many animal species and,occasionally, in man has been demonstrated [58], but much

controversy still exists in this field.Armed with the knowledge of the in vivo phenotype of BM

MSCs, we have investigated the presence of MSCs in normalperipheral blood. We used four different pre-enrichment methodsfollowed by multiparameter flow cytometry, which in the BMhave demonstrated an excellent sensitivity for the MSC detection(1 MSC for $100000 haemopoietic cells). With a startingpopulation of between 300 and 500 million peripheral bloodMNCs (equivalent to $300–500 ml of blood), a distinctMSC population could not be detected, regardless of a pre-enrichment method used (Fig. 2A). We did detect adherentspindle-shaped cells reminiscent of fibroblasts but these did notproliferate. These cells were actively phagocytic indicating theirmonocyte/macrophage lineage nature (Fig. 2B). Importantly, nofibroblastic colonies were grown from peripheral blood of all16 donors studied. These data confirmed previous studies showing

A

B C

P

BLC

FIG. 1. MSCs may not have a specific niche in the BM. (A) 6-colour flow cytometry dot plots of gated CD45lowCD13þ MSCs from BM aspirates showing positivity for CD271,CD73 and CD10 expression. (B) Characteristic morphology of sorted BM MSCs with long projections, which is reminiscent of BM adventitial reticular cells (ARCs).(C) CD10 immunostaining of BM trephine biopsy showing the widespread distribution of CD10þ cells in vivo (original magnification 100Â). CD10þ ARC is indicated byarrow. It remains possible that ARCs, bone lining cells (BLCs) and pericytes (P), all form part of an extended stromal marrow network that has the inherent capacity to bemultipotential.

128 E. Jones and D. McGonagle



that circulating MSCs were exceptionally rare in man [58].It remains a possibility, however, that MSCs may be mobilizedfrom the marrow by growth factors [59] or following acuteinjury [60].

Although the issue of MSC circulation remains controversial,it is possible to suggest some explanations to a few unresolvedissues. First, MSC have been unequivocally shown to circulateduring the first trimester of fetal development but scarcelythereafter [61]. This could be related to the completion of theprocess of vasculogenesis during early development, consistentwith the idea of most MSCs taking a role of pericytes stabilizingblood vessels. Although MSCs are present in the cord blood [62],

higher numbers of these cells were recently found in soft tissueswithin the cord itself [63, 31], consistent with this idea. Second,if MSCs circulate in adults but are virtually undetectable, thentheir physiological role is questionable. Third, we and others havepreviously shown a very close spatial association between MSCsand fat cells in the marrow [18, 64]. It is well known that fatglobules can be released into blood following major orthopaedictrauma (termed fat embolization) [65]. Far from being a specificfeature of MSCs to egress from the BM in response to injury,this latter example illustrates that MSC circulation couldmerely represent a transient phenomenon and the unavoidableconsequence of skeletal trauma. In our view, the data showingMSC circulation in man, outside the setting of skeletal trauma,remains controversial; however, the potential circulation of BM MSCs following fracture may have a simple biophysicalexplanation.

The concept of MSC homing

A logical extension of the belief that MSCs circulate must be that,like HSCs, they possess the ability to egress from the marrow andhome to other sites. There would be a strong conceptual argumentfor MSCs trafficking from the marrow to sites of injury, if likeHSCs, the marrow was their predominant topographic location.Given the recent demonstration that MSCs are fairly ubiquitously

distributed throughout the skeleton [47], the question remains— why do they need to circulate and migrate for long distances atall? Nevertheless, if this question is to be pursued further, attemptsto fully characterize tissue-specific MSCs and then track theirmovements following an infusion would represent a first step.

There is plenty of reported evidence that MSCs show somehoming potential, which is influenced by chemokines and othermolecules [66, 67]. This does not imply an ability to circulatesystemically—in fact, it is equally plausible that MSC homing canoccur over short distances, within tissues following some localinjury. Phrased in a different way, the wide distribution of MSCsmeans that if homing occurs it may be over short distances andnot be dependent on the systemic circulation. The proposedlocation of solid tissue MSCs in a perivascular niche [47] couldgreatly facilitate such local homing without the prior need to enter

the systemic circulation.Finally, a common sense approach to the therapy of cartilage

or bone defects involves the direct injection or placement of cellsinto the defect of interest [68, 69]. At the practical level, thoseworking in the front line of clinical cellular therapy developmentfor musculoskeletal diseases are not pursuing ‘systemic infusionstrategies’, which appear to be more academically driven andderived from the HSC stem cell paradigm. It must be acknowl-edged that interest in MSC homing for the cellular therapy of congenital myopathies has been considered and there is evidencethat MSCs participate in muscle regeneration [70]. But again,this is counterbalanced by the fact that the vast majorityof infused cells seem to end up in the reticuloendothelial system[71]. The accumulation of MSCs in the muscle may be secondaryto the associated vascular damage that accompanies any muscle

injury.Regardless of whether steady-state MSC circulation represents

a natural physiological process or not, such cells may still have animportant therapeutic value when infused systemically afterlimited culture-expansion. Nowhere is this clearer than in respectto the use of MSCs in graft-vs-host disease [72], as immunomod-ulatory cells [73] or in breast cancer trials [74], where MSCs wereshown to augment haematopoietic cell recovery. These scenariosillustrate that MSCs could be therapeutically used in settingsoutside tissue regeneration and in these settings their modeof action could be different (via autocrine, paracrine and evenendocrine actions) [75].

Implications

The proposed aim of regenerative medicine strategies basedon MSCs is tissue reconstruction using, singly or in combination,MSCs, growth factors and scaffolds [76, 77]. The potential forMSC-based gene therapy has been added to this melange [78, 79].However, the concept that MSCs are functioning stromal cellsmakes it likely that following prolonged expansion in culture thatthey will exhibit cell senesence, loss of potency, genetic instabilityand even transformation. Indeed all of these concerns have beenborne out by experimental work [80–83]. Given the knowndrawbacks of gene therapy in the malignancy field [84], it wouldnot seem prudent to pursue such a strategy in cells that mayalready be rendered genetically unstable by prolonged in vitro

culture.There has been many proof-of-principle studies showing that

culture-expanded MSCs can repair bone in the experimentalsetting [77, 85], but this has not lead, in our view, to many

FIG. 2. Failure to detect MSCs in the ci rc ulati on. (A) Absence ofCD45lowD7-FIBþCD271þ MSCs in peripheral blood following selection with fourdifferent pre-enrichment methods. (B) Adherent ‘fibroblastoid cells’ from peripheralblood are 100% phagocytic as demonstrated by ferromagnetic bead ingestion. Thisindicates their monocyte/macrophage (rather than MSC) lineage nature. (C)Positive control BM MSC detection following D7-FIB-based pre-enrichment.CD45lowD7-FIBþCD271þ population of BM MSCs is encircled. (D) Adherent cellsfrom the BM contain both non-phagocytic MSCs (indicated by arrows) andphagocytic monocytic lineage cells (original magnification 400Â).




ground-breaking translational developments thus far. Indeed,orthopaedic surgeons have not favoured such a cumbersomeprocess as BM MSC expansion and re-administration, at least inthe areas of orthopaedic trauma or fracture repair, but have useda more pragmatic approach utilizing whole BM injections intofracture sites, where excellent outcomes have been reported [69,86, 87]. As few as 50 000 uncultured MSCs were found to provideeffective bone repair in one such study [88]. This may be because

BM MSCs in vivo may have much higher osteogenic differentia-tion potency compared with expanded MSCs as documented inone report [89].

In the Hernigou et al . study, CFU-F numbers in the implantedmaterial were retrospectively established based on standard14-day CFU-F assays [88]. However, the realization thatuncultured MSCs can now be enumerated by flow cytometrycould lead to the development of new strategies for fracture repair(and possibly, for other bone repair applications) whereby thenumber of fresh MSCs in the implanted cellular fractions couldbe accurately determined. Additionally, new technologies forrepairing bone based on semi-purified, uncultured BM MSCs arenow being actively developed [90]. However, in certain situations,mode of action may be more important than phenotypicalcharacterization of the cellular product. For instance, in bonerepair indications, the number of injected MSCs may be sufficient,but in applications based on immunomodulatory properties of MSCs, respective quality control procedures are likely to be morecomplicated.

Finally, a better appreciation of the in vivo biology of MSCscould lead to the emergence of in situ tissue engineering. Thisrefers to the scenario whereby the manipulation of MSCs in vivo

could be used for tissue repair without necessarily adding furtherstem cells to the joint. Indeed, ligamentization of artificialscaffolds placed in the correct orientation represented the firstexample of in situ tissue engineering in man [91]. From which jointstructures MSCs actually traffic to such scaffolds and howthey differentiate into ligaments has yet to be defined. Anotherexamples of in situ tissue engineering are the administration

of recombinant human bone morphogenetic proteins 2 or 7 forfracture repair or recombinant parathyroid hormone (PTH) forosteoporosis therapy (reviewed in [92]). All of these agents eitherlocally or systemically increase bone mass and are likely to acton resident MSCs without the need for exogenous stem cells.Understanding the cellular basis for these processes has importantimplications for musculoskeletal stem cell therapy development.

In vivo identification of MSCs in the BM opens-up new avenuesof research for understanding the nature and identity of MSCs atother sites including the synovium, periosteum [28, 93] and fat[29]. The formal identification of BM MSCs in these tissues wouldherald an exciting new era of research, not geared to expandingthese cells indefinitely in culture, but rather understanding theirin vivo biology and then devising therapeutic strategies to augmenttheir function where defective and suppress it where exaggerated.

Acknowledgements

The authors would like to thank Anne English, Karen Henshaw,Salina Jain, Andy Rawstron and David Blyth for technicalassistance, Sally Kinsey for provision of BM aspirates and PaulEmery for continuing support.

Disclosure statement: The authors have declared no conflicts of interest.

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