Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells—Potential and pitfalls

Advanced Drug Delivery Reviews 63 (2011) 342–351

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Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells—Potential and pitfalls☆ Wilhelm K. Aicher a,⁎, Hans-Jörg Bühring b, Melanie Hart a, Bernd Rolauffs a,c, Andreas Badke c, Gerd Klein d a

Center for Regenerative Medicine (ZRM), University of Tübingen Hospital (UKT), Eberhard-Karls-University, Tübingen, Germany Department of Internal Medicine II, University of Tübingen Hospital (UKT), Eberhard-Karls-University, Tübingen, Germany BG Trauma Center, Tübingen, Germany d Center for Medical Research, Section for Transplantation Immunology and Immunohematology, University of Tübingen Hospital (UKT), Eberhard-Karls-University, Tübingen, Germany b c

a r t i c l e

i n f o

Article history: Received 25 October 2010 Accepted 10 December 2010 Available online 22 December 2010 Keywords: Mesenchymal stem cell (MSC) Differentiation of MSC Functional subsets of MSC MSC niche Sources for MSC Osteogenesis Chondrogenesis

a b s t r a c t Mesenchymal stromal cells, also referred to as mesenchymal stem cells, can be obtained from various tissues. Today the main source for isolation of mesenchymal stromal cells in mammals is the bone marrow. Mesenchymal stromal cells play an important role in tissue formation and organogenesis during embryonic development. Moreover, they provide the cellular and humoral basis for many processes of tissue regeneration and wound healing in infancy, adolescence and adulthood as well. There is increasing evidence that mesenchymal stromal cells from bone marrow and other sources including term placenta or adipose tissue are not a homogenous cell population. Only a restricted number of appropriate stem cells markers have been explored so far. But routine preparations of mesenchymal stromal cells contain phenotypically and functionally distinct subsets of stromal cells. Knowledge on the phenotypical characteristics and the functional consequences of such subsets will not only extend our understanding of stem cell biology, but might allow to develop improved regimen for regenerative medicine and wound healing and novel protocols for tissue engineering as well. In this review we will discuss novel strategies for regenerative medicine by specific selection or separation of subsets of mesenchymal stromal cells in the context of osteogenesis and bone regeneration. Mesenchymal stromal cells, which express the specific cell adhesion molecule CD146, also known as MCAM or MUC18, are prone for bone repair. Other cell surface proteins may allow the selection of chondrogenic, myogenic, adipogenic or other pre-determined subsets of mesenchymal stromal cells for improved regenerative applications as well. © 2010 Elsevier B.V. All rights reserved.

Contents 1. The mesenchymal stromal cell: an introduction . . . . . . . . . . . . . . . . . . . . . 2. Characterization of human mesenchymal stromal cells . . . . . . . . . . . . . . . . . . 3. Isolation and expansion of mesenchymal stromal cells from bone marrow and solid tissues . 4. Differentiation of mesenchymal stromal cells in vitro . . . . . . . . . . . . . . . . . . . 5. Functional subsets of mesenchymal stromal cells in regenerative medicine . . . . . . . . . 6. The choice for cell-based regimen: selected or bulk MSC? . . . . . . . . . . . . . . . . . 7. Cell–matrix interactions modulate the differentiation of mesenchymal stromal cells . . . . 8. Clinical application of mesenchymal stromal cells for regeneration of bone and cartilage . . 9. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: CD, cluster of differentiation; EPC, endothelial precursor cell; FCS, fetal calf serum; GMP, good medical practice (or procedure); HSC, hematopoietic stem cell; HSPC, hematopoietic stem and progenitor cell; MSC, mesenchymal stem cell. ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “From Tissue Engineering to Regenerative Medicine—The Potential and The Pitfalls”. ⁎ Corresponding author. ZMF Research Building, University of Tübingen Medical Center, Waldhörnlestrasse 22, 72072 Tübingen, Germany. E-mail address: [email protected] (W.K. Aicher). 0169-409X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2010.12.004

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1. The mesenchymal stromal cell: an introduction More than 140 years ago the pathologist Julius F. Cohnheim presented his studies on inflammation and wound healing. He injected anilin into veins of wounded animals and observed that anilin-labelled immune cells as well as anilin-labelled fibroblast-like cells migrated to sites of inflammation and wound healing. He concluded that at least some, if not all, cells involved in wound repair might be derived from blood or bone marrow [1]. About 110 years later Friedenstein and colleagues noted that murine bone marrow contained fibroblast-like colony-forming cells, which differed from the hematopoietic stem cells and generated osteocytes in vitro [2,3]. Others extended these early studies and developed the concept of the mesenchymal stem cell (MSC) [4,5]. Arnold Caplan was among the first to propose the MSC as a therapeutic concept [6]. A more detailed investigation of MSC raised concerns regarding the term “stem cell”, as MSC did not match the criteria defined for stemness without restriction [7]. Therefore nowadays the term mesenchymal stromal cells (MSC) is preferred [8]. 2. Characterization of human mesenchymal stromal cells In 1999 the first systematic investigation of human bone marrowderived MSC confirmed the multipotent differentiation capacity of such cells and raised hopes of many patients for better or improved regimen to treat a variety of defects of locomotion tissues [9]. To be able to discriminate the bone marrow-derived MSC (bmMSC) from other bone marrow-derived stem cells including hematopoietic stem cells (HSC) or endothelial progenitor cells more precisely, a consensus suggested to define MSC according to their growth characteristics and expression of cell surface markers (Table 1, [10]). Apart from these markers (CD73, CD90, CD105), MSC express additional cell surface markers including tissue non-specific alkaline phosphatase (TNAP) or MSCA-1, [11], CD63 [12], CD146 [13–15], CD164 [16], CD271 and others [17], which may allow better definitions of MSC in the future (Table 2). But cell surface antigens selectively expressed on MSC are poorly described. Therefore it is important to further explore the expression patterns of surface antigens on MSC ex vivo and under defined in vitro conditions in more detail. However, other important characteristics of MSC, including their capacity to regenerate tissues in vivo [18], self-renewal, or generation of colonies or clusters [19] have not been included in the above-mentioned criteria. For that reason, additional tools to discriminate MSC from other cells may evolve eventually. Closely related to the bmMSC are mesenchymal stromal cells residing in different tissues [14,20–22], including placenta [15,23–26], cord blood and umbilical cord [27,28], synovial membrane [29,30], vasculature [14,31], adipose tissue [32–34], trabecular bone [35], or dental pulp [36–38]. Prima vista they seem to be almost identical to the bmMSC, although some characteristics are quite distinct [17,39] (Table 1). For instance, most bmMSC express CD146 at high intensity [22]. In contrast, many placenta-derived MSC (pMSC) express considerably less CD146 and some pMSC lack CD146 completely [15]. Osteogenic stem cells derived from dental pulp in turn express CD146 [40]. Furthermore, human pMSC express the stage-specific embryonic antigen-4 (SSEA-4), which is not found on human bmMSC [41] but on bmMSC from mice [42]. In contrast to bmMSC, adipose tissue-MSC (atMSC) express CD34 [33,43]. According to the above-mentioned consensus conference, expression of CD34 was an exclusion criteria for bmMSC (i.e., CD73+, CD90+, CD105+, but CD34-) and defined among the bone marrowderived cells the HSC, hematopoietic stem and precursor cells (HSPC), or endothelial precursor cells (EPC). Disparity in expression of cell surface antigens was also noted on MSC from different tissues for the adhesion molecules CD49d (integrin α4), CD54 (ICAM-1) and CD106 (VCAM-1) [44]. Moreover, the CD14 molecule is part of the receptor

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Table 1 Cell surface antigens on mesenchymal stromal cells isolated from different tissues. Source/tissue

CD antigens

References

Bone marrow

TNAP+, D7-fib+, STRO-1+, CD9+, CD10+, CD13+, CD15+, CD29+, CD44+, CD49+, CD56+, CD63+, CD73+, CD90+, CD105+, CD106+, CD130+, CD140b+, CD144+, CD146+, CD164+, CD200+, CD271+, CD309+, CD349+ HLA class I+, HLA class IICD11b-, CD14-, CD19-, CD20-, CD31-, CD34-, CD45-, CD50-, CD80CD13+, CD73+, CD105+, CD106+, CD146+, CD166+, CD271+ CD45low STRO-1, CD13+, CD29+, CD44+, CD73+, CD105+ CD34-, CD43-, CD45STRO-1+, CD29+, CD44+, CD73+, CD90+, CD105+ CD34-, CD45-, CD117CD44+, CD49+, CD271+ CD3-, CD14-, CD20-, CD45ALP+, STRO-1+,CD29+, CD34+, CD44+, CD54+, CD73+, CD86low, CD90+, CD105+, CD146 low, CD166+ HLA class I+, HLA class II3G5-, CD11a-, CD14-, CD31-, CD40-, CD45-, CD80-, CD106-, CD144NG2+, CD13+, CD44+, CD73+, CD90+, CD105+, CD140b+, CD146+, ALP+ ALP-, CD34-, CD45-, CD56-, CD133-, CD144SSEA-4+, TRA-1-81+, CD9+, CD10+, CD13+, CD26, CD29+, CD44+, CD49+, CD63+. CD73+, CD90+, CD105+, CD146+/-, CD164+, CD271 low, CD318+ HLA class I+, HLA class IICD11b-, CD14-, CD19-, CD34-, CD45-, CD50-, CD271-, CD349-, TNAP low CD10+, CD13+, CD29+, CD44+, CD54+, CD73+, CD90+, CD105+, CD140b+, CD166+, CD271 low, CD349+ HLA class I+, HLA class IICD3-, CD14-, CD34-, CD31-, CD45-, CD133CD29+, CD44+, CD73+, CD90+, CD105+, CD105+, CD106+ HLA class I+, HLA class IICD14-, CD34-, CD45-, CD133CD13+, CD29+, CD44+, CD73+, CD90+, CD105+ HLA class I-, HLA class IICD14-, CD34-, CD45-

[17] [10] [58] [42] [11] [63] [65] [16]

Trabecular bone

Dental pulp

Articular cartilage

Synovial membrane Adipose tissue

Perivascular sites

Term placenta

Amniotic fluid

Umbilical cord

Pancreas

[59]

[37] [38] [102]

[30] [103] [32] [33] [43] [34] [104] [14] [15]

[105] [41] [25] [65] [15]

[105] [25]

[31]

[106]

A selection of typical cell surface antigens expressed on human MSC from different sources is listed (bold, blue). Antigens possibly discriminating MSC isolated from a specific source are highlighted (yellow), human leukocyte antigens (HLA) are presented in italics and antigens not expressed by human MSC are in red letters. Depending on the methods of isolation of MSC, time of investigation (ex vivo versus after in vitro culture) or cell culture conditions, the expression of some antigens reported my differ among the studies reviewed here or elsewhere. ALP = alkaline phosphatase, NG-2 = 300 kD proteoglycan on nerve cells, SSEA-4 = stage-specific embryonic antigen 4, STRO-1 = unspecified cell surface antigen on stromal cells, TNAP = tissue non-specific alkaline phosphase, TRA-1 = teratoma-reactive antigen-1.

for lipopolysaccharides and an antigen found typically on monocytes, which is not expressed on bmMSC or pMSC, but found on human and equine atMSC [43,45]. A number of cell surface proteins are expressed on primary MSC but are lost during in vitro culture. The nerve growth factor receptor CD271 for instance is expressed at high levels on primary bmMSC but is rapidly downregulated after in vitro culture of MSC [11]. On pMSC

Table 2 Criteria defined in 2006 by a consensus conference of the International Society for Cellular Therapy to define human MSC [10]. MSC grow as adherent fibroblast-like cell in vitro. MSC express CD73, CD90, CD105, but lack antigens typical for monocytes (CD11b, CD14), B-lymphocytes (CD19, CD79), HSC or endothelial cells (CD34), leukocytes (CD45) and major histocompatibility class II antigens on their surface. (III) MSC can be differentiated at least in three lineages to generate osteocytes, chondrocytes and adipocytes. (I) (II)

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this antigen is expressed ex vivo with lower signal intensity, corroborating that MSC isolated from distinct tissues may differ considerably in their phenotype and possibly in their capacity for colony formation, proliferation and differentiation potential as well [15,16]. Comparably, the adhesion molecule CD56 (NCAM) is detected on MSC ex vivo but found hardly on any MSC in culture [16]. Other proteins are not detected on MSC ex vivo, but seem to be induced under in vitro growth conditions. In particular markers of cellular activation such as CD109, a GPI-anchored protein found on activated T-lymphocytes, platelets and endothelial cells, or CD318, a glycoprotein described originally on cancer cells and more recently detected on stem cells, are found on cultured MSC [15]. 3. Isolation and expansion of mesenchymal stromal cells from bone marrow and solid tissues From bone marrow aspirates, cord blood or alike MSC are isolated by diluting the aspirate with buffered saline, followed by density gradient centrifugation. The mononuclear cell fraction, containing among others the MSC, is enriched in the interface [46]. The cells in the interface are collected, washed and inoculated in suitable expansion media. In vitro MSC appear and behave like fibroblasts. They grow attached to tissue culture vessels in little clusters. The initial step for selection of MSC is just their growth on plastic or protein-coated surfaces. As HSC and more differentiated hematopoietic progenitors grow in suspension, the selection of adherent cells allows to simply removing unwanted HSC, HSPC or leukocytes from the preparations by aspiration of the cell culture media. But other cell types, including endothelial cells, smooth muscle cells, osteoblasts, fibroblasts, and adherent monocytic cells may contaminate the preparations depending on the source where the MSC have been harvested from. Therefore the MSC must be enriched by optimized cell culture protocols, which may include inoculation of the cells at low density (less than 10 cells.cm− 2) to generate almost clones of MSC [19]. But defined populations or sets of MSC cannot be generated by such random procedures. To obtain MSC from other sources, the cells are liberated from the tissue by mincing it by scalpel and scissors, followed by mild proteolytic degradation. Debris is removed and the mononuclear cells including MSC are enriched by density gradient centrifugation and further processes as described above. For some purposes it was shown to be beneficial to expand MSC in vessels coated with proteins, including for instance gelatin [41], different collagens [47,48], laminins [49,50], or mixes thereof (e.g., matrigel [51]). This however may influence the growth and differentiation characteristics of the evolving populations suggesting that the MSC come with a degree of plasticity influenced by the habitat in which they reside [52]. But protein coating seems not to trigger lineages determination as reported for growth factors or cytokines as in some cases coating with laminin-322 provided a chondrogenic stimulus and osteoblasts were not observed [49], whereas others reported success of osteogenic differentiation [50]. Cell culture conditions, duration of in vitro culture and other variables may explain these conflicting reports. For research purposes, MSC can be expanded in vitro in standard media enriched by serum such as fetal calf serum (FCS) [9]. Addition of FCS to cell culture media is approved by the US authorities for clinical purposes [53], but FCS cannot be utilized for culture of any cell type destined for therapy in the EU. Therefore specific protocols were developed omitting serum completely or replacing FCS by human sera, sometimes enriched by platelet lysates [54]. Moreover, for clinical applications all media, factors, stimuli, and scaffolds involved must meet the regulations set by good medical practice (GMP) [55]. 4. Differentiation of mesenchymal stromal cells in vitro When expanded in vitro, MSC can be activated to undergo differentiation to generate osteocytes, chondrocytes and adipocytes

[9]. The differentiation is initiated by addition of growth factors and low molecular weight components. For osteogenic differentiation of MSC in vitro, the presence of ascorbic acid, which is important for the expression of collagen, β-glycerophosphate as a source for phosphate during mineralization, and dexamethasone to halt cellular proliferation, are essential stimuli [56]. Others utilized parathyroid hormone, 1,25-dihydroxyvitamin D3, and bone morphogenic protein-4 (BMP-4) or BMP-6 for inducing osteogenic differentiation of MSC [57]. But it has been noted by many investigators that not all cells undergo differentiation in vitro as desired. When osteogenic or adipogenic differentiation is induced in MSC, some areas of cells in the dishes display intense staining with the von Kossa reagents marking osteocytes, or staining with the Oil Red O reagent indicating adipocytes. Other areas fail to take these dyes and the cells look rather like fibroblast (Fig. 1). This could be explained by several hypotheses. One among them claims that bulk MSC contain subsets of cells, which are predisposed to undergoing preferably a distinct differentiation pathway. It has been noted that MSC from bone marrow undergo osteogenic differentiation more efficiently when compared to pMSC [15]. Accordingly, bone marrow may contain

Fig. 1. Adipogenic and osteogenic differentiation of MSC in vitro.Bulk preparations of pMSC were expanded in vitro and adipogenic (top) or osteogenic (bottom) differentiation was induced in pMSC. After four weeks of differentiation, staining of the resulting cells with Oil Red O indicated adipogenesis (arrows), and von Kossa staining indicated mineral deposition by osteoblasts (arrows). But not all cells contain lipid vesicles after adipogenesis, and not all cells produce a mineralized extracellular matrix after osteogenesis (squares). This indicates that not all pMSC differentiated efficiently, or that the differentiation is incomplete.

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osteogenically primed subsets of MSC to a larger extent compared to other tissues. Another explanation could be the concern that MSC from different sources (e.g., adipose tissue versus bone marrow) are contaminated to a variable degree with fibroblasts, smooth muscle cells, osteoblasts, or other differentiated mesenchymal cells. Owing to the rather crude preparation techniques for isolating MSC – cell extraction, centrifugation, and adherent growth – and the lack of specific monoclonal antibodies for simple and efficient isolation or enrichment of purified MSC, contaminations with mature fibroblasts or other cells cannot be excluded. If this holds true, the better osteogenic differentiation reported for bmMSC could be explained by mature osteoblasts in the MSC preparation in the first place. Osteoblasts or fibroblasts fail to express CD146, but bmMSC do [22,34], even if to a variable degree [15,58]. Furthermore, multipotent stromal cells were also isolated from trabecular bone [35]. These cells were experimentally indistinguishable from bmMSC and they expressed CD146 and CD271 [59]. At any rate, the analysis of expression of CD146 may be one of the tools to discriminate bone marrow- and trabecular bone-derived MSC from differentiated or more mature mesenchymal cells, including osteoblasts and fibroblasts. Note that expression of CD146 is found on bmMSC ex vivo and after in vitro expansion. In contrast, CD271 is detected on bmMSC only ex vivo and lost during primary culture. Therefore the latter antigen is not suitable for discrimination of bmMSC from osteoblasts or fibroblasts under most circumstances in laboratory routine and in research.

5. Functional subsets of mesenchymal stromal cells in regenerative medicine In 1975, techniques to generate target-specific antibodies and specifically monoclonal antibodies [60] revolutionized research in biology and medicine. A plethora of monoclonal antibodies allowed not only to label cells but soon it became clear that different antibodies detected the same or closely related structures on cells whereas others reacted with distinct antigens on the same cell or with completely different types of cells. To discriminate antibodies reacting with the same molecule from antibodies reacting with different molecules (but labeling possibly the same cell), in 1981 a novel nomenclature for the human leukocyte differentiation antigens was introduced. Antibodies binding to the same molecule or even the same epitope on a given molecule were assigned to a cluster of differentiation (CD) [61]. This CD system was then adapted and utilized generally on all cell types, including the corresponding cells of other species. Human bone marrow-derived hematopoietic stem cells (HSC) for instance express an antigen numbered CD34 [62]. All types of bloodborn cells can be discriminated by distinct monoclonal antibodies. All leukocytes express a common antigen, a phosphatase called CD45. T-lymphocytes express CD3, B-lymphocytes express CD19, monocytes CD14, granulocytes CD15 and so on. Monoclonal antibodies (mAb) discriminate not only different cell types, but also individual stages of differentiation of a cell lineage. This allows the selection and enrichment of distinct subsets of cells for diagnostic or therapeutic applications. B-lymphocytes for instance can be selected by reacting the cells with an antibody to CD20, T-lymphocytes by anti-CD3, T-helper cells by anti-CD4, cytotoxic T-cells by anti-CD8, activated T-cells in addition by anti-CD25. The HSC, sometimes more precisely termed hematopoietic stem and precursor cells (HSPC), can be specifically enriched from crude bone marrow aspirations by simply reacting the cells with an antibody to CD34 [62]. The mAb discriminate even (splice-)variants or isoforms of cell surface antigens such as CD45R0, an isoform of CD45 lacking exons A, B, and C, from CD45RA, CD45RB, and CD45RC, sharing the exons A, B, or C, respectively. The expression of such isoforms of antigens on T-lymphocytes is associated with different functions or stages of activation of the respective cells.

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For MSC and their progeny such a system of cell surface antigens reactive with a distinct set of mAb to link the expression of a given marker to a stage of differentiation or activation of the cell is far from being established. However, a consensus conference published in 2006 criteria to more precisely defining the MSC and to discriminating them from more mature cell types, including fibroblasts, chondrocytes, osteoblasts, adipocytes and others (Table 1) [10]. This rather crude definition of MSC is a great tool for practical applications in the laboratory. But is has undisputedly obvious shortcomings. In attempts to define MSC more precisely, flow cytometry and fluorescence-activated cell sorting (FACS) yielded a plethora of data [11,16,17,25,42,58,63]. For instance, the expression of SSEA-4 identifies murine bmMSC [42]. This antigen was found on human pMSC as well, but not on human bmMSC [41]. Some cell surface antigens including CD10 (CALLA; neutral endopeptidase), CD29 (integrin β1chain), CD49 (integrin α-chain), CD56 (NCAM), CD63 (tetraspanin, LIMP), CD73 (ecto-5′ nuclease, SH3/SH4), CD90 (Thy-1), CD105 (TGFß receptor, endoglin, SH2 antigen) or CD166 (ALCAM) were described on MSC from different sources [9,11,14,17,22,33,63]. The expression of some of these antigens such as CD10, CD73, CD166, or CD318 seems to be linked to the proliferative activity of MSC in vitro. The nuclease CD73 and the peptidase CD10 facilitate the nutrition of the cells in cell cycle, and receptors for growth factors (e.g. CD105) are important for regulation of self-renewal, proliferation and differentiation. Antibodies to the individual antigens mentioned above or the combination of antibodies in two-tier separation protocols allow the direct enrichment of MSC from crude extracts. Alternatively, unwanted cells such as HSC, HSPC or endothelial cells can be removed from bmMSC with anti-CD34 mAB, monocytes or macrophages by antiCD11b or anti-CD68, respectively. In any case, the specificity of the antibody employed for selection or depletion has to be explored for each type of MSC and each protocol of MSC preparation, as some antibodies may deliver variable results [64]. The bmMSC differ in their expression of CD10, CD49, and CD318 from pMSC [41,65]. The expression of cell surface markers may even differ when isolated from the same tissue, albeit from individual donors (e.g. CD106 and CD146) [58]. In addition, the expression of cell surface markers may change during culture of the cells in vitro. A robust expression of the phosphatase TNAP (also referred to as MSCA1 antigen, detected by monoclonal antibody W8B2) was detected only on bmMSC ex vivo [11], but was reduced in primary culture bmMSC [66] or on primary culture pMSC [15]. MSC derived from adipose tissue change their immunophenotype in vitro considerably and the expression of CD40, a receptor involved in cell–cell interactions, increased whereas CD86, a type I membrane protein and member of the immunoglobulin superfamily, decreased significantly within three passages of in vitro culture [33]. Anyhow, flow cytometry and antibody-based cell sorting techniques (FACS, MACS or alike) are powerful tools not only to describe MSC more precisely, but also to enrich specific subsets of MSC from bulk populations. But care has to be taken to selecting the time point (MSC ex vivo/in primary culture/ passaged cells) and combination of antibodies and isolation techniques for the desired diagnostic or preparative purpose. For antibody-aided selection of MSC, two general strategies can be employed. On the one hand, the direct staining and – if required direct sorting – of MSC ex vivo can be used followed by expansion of a defined and purified subset of cells. Such a strategy can be applied when working with bmMSC, because isolation and purification of bmMSC does not require a time-consuming preparation of the tissue and omits proteolytic digestion. Moreover, the cell surface antigens on the cells are not degraded by proteolysis and such MSC can be stained ex vivo with specific antibodies. On the other hand, the expansion of bulk MSC from defined source (adipose tissue, placenta, etc.) may allow to generate large quantities of cells and the expression of autocrine growth factors may help in initiation, maintenance and expansion of the MSC cultures. Antibody-aided enrichment or

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selection allows them to pick the cells wanted, even by antigens found on MSC only in vitro (e.g. CD166 and CD318). A typical example for the ex vivo selection strategy is the isolation of a proliferative and chondrogenic subset of bmMSC. Expression of the receptor for platelet-derived growth factor CD140 defined a highly proliferative set of MSC [11]. It may well be that these CD140-positive MSC contain those MSC that were described about a decade ago and termed rapidly-cycling stem (RS) cells [19]. Using a whole panel of mAb to known or orphan antigens, several functional subsets of bmMSC were defined recently (Fig. 2). Colony forming units and rapidly growing bmMSC were found in the population that expressed ex vivo the nerve growth factor receptor CD271 and at the same time the adhesion molecule CD56 [16]. The CD56+, CD271+ MSC proliferated about thirty-times faster compared to the unsorted bulk MSC, and about three to four-times faster compared to the CD56-, CD271+ population. When the CD56+ CD271+ bmMSC were further separated according to their expression of TNAP, followed by expansion and differentiation in vitro, the triple positive subset (CD56+, CD271+, TNAP+) displayed a pronounced chondrogenic potential but a drastically reduced adipogenic differentiation potential (Fig. 2) [16]. The osteogenic, myogenic, and neurogenic potential did not differ between these subsets. This study indicated that (i) within the MSC population functionally different subsets can be found ex vivo, and (ii) that these subsets can be discriminated and separated by appropriate monoclonal antibodies or combinations thereof. It also suggested that (iii) in such subsets the proliferation and differentiation capacity are not necessarily linked together, as triple positive cells preferably generated one type of progeny (i.e. chondrocytes), failed to generate another type of cell (i.e. adipocytes), but were not different from bulk cultures with respect to additional differentiation pathways (i.e. osteoblasts and others).

Fig. 2. Selection of functional subsets of MSC by fluorescence-activated cell sorting of cells ex vivo. In a simplified scheme the basic steps of enrichment of subsets of MSC are shown. MSC are aspirated from bone marrow, purified by gradient centrifugation and immediately selected by monoclonal antibodies, exemplified by antibodies to CD56, CD271, and TNAP. The specifically enriched subsets of MSC are then expanded in vitro for further use. The different diameters of the circles indicating numbers, loss, or gain of MSC during this procedure are not drawn to scale.

At present a rule for the differentiation capacities of such MSC subsets cannot be delineated. However, during embryonic development the cartilagous anlagen of bone start to mineralize when the tissue is vascularized. Therefore the chondrogenic and osteogenic differentiation pathways of MSC might be connected or even linked. Comparably, myogenic precursor cells, termed satellite cells and the pericytes surrounded by the basement membrane of blood vessels may also belong to a family of related MSCs. But up to now this is far from being established. A typical example for the in vitro selection strategy was emerging recently (Fig. 3). When isolating bmMSC and pMSC under identical conditions, it was noted that the osteogenic differentiation of pMSC was not as efficient compared to the osteogenic differentiation achieved from bmMSC. Interestingly, the elevated osteogenic potential seemed to correlate with the expression of CD146 (Fig. 4) [15]. Furthermore, when separating the CD146+ from CD146- MSC by aid of magnetic sorting, efficient osteogenic differentiation was observed only in the CD146+ fraction, whereas in the CD146- population mineralization was not observed after osteogenic differentiation [67]. The expression of CD146 marked a population of stromal cells which were part of the hematopoietic niche in bone marrow, whereas the CD146 negative stromal cells failed to support growth of HSC [68]. A variable expression of CD146 (roughly between 40 and 90% of all MSC) was reported on MSC isolated from different donors after expansion in vitro [58,66]. MSC isolated from human umbilical cord express CD146 as well [64]. Furthermore, expression of CD146 was confirmed on bmMSC and pMSC when expanded under GMP-conform conditions indicating that CD146 is expressed on bmMSC or pMSC independent of cell culture conditions (unpublished observation). The expression of CD146 may still depend on cytokines or growth factors but this is not yet investigated in great detail. In contrast to MSC, CD146 is not found on synovial fibroblasts or mature osteoblasts [15]. By selection of CD146+ cells unwanted osteoblasts could be removed from bmMSC preparations. Additional selection strategies based on antibodies to known or orphan cell surface markers on MSC will open novel avenues for improved regeneration of cartilage, adipose tissue, smooth muscle regeneration and other. At present the complex selection based on

Fig. 3. Selection of functional subsets of MSC by magnet-activated cell sorting of cells after in vitro expansion. In a simplified scheme the basic steps of expansion of bulk MSC, their characterization [10] followed by enrichment or depletion according to the desired application. The different diameters of the circles indicating numbers, loss, or gain of MSC during this procedure are not drawn to scale.

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347

Fig. 4. Expression of CD146 on MSC correlates with their osteogenic differentiation capacity. Expression of CD146 on CD73+, CD90+ and CD105+ bmMSC (A) and pMSC (B) was investigated by flow cytometry, and the osteogenic differentiation was explored in vitro. After 4 week of incubation in an osteogenic differentiation medium, bmMSC displayed mineralization as depicted by von Kossa staining indicating osteogenesis (C), whereas pMSC did not generate a mineralized matrix (D).

three cell surface markers CD56, CD271, plus TNAP is complex but successful and can therefore be applied for further proof-of-principle studies in the laboratories (Fig. 2). But such a complex selection procedure may not reach clinical application. A strategy based on a single antibody, as used for selection of the osteogenic MSC (CD146), will facilitate the translation of this novel technique to the clinic (Fig. 3). Today bmMSC are often applied to escort HSC in bone marrow transplantation after irradiation and chemotherapy of cancer patients [69,70]. As bone marrow is utilized as a source for the HSPC, the MSC are taken from the same source for obvious reasons. One can imagine that distinct antigens will possibly allow enrichment of immunosuppressive bmMSC for improved therapy of GVHD. The same may apply for enrichment of myogenic MSC for regeneration of smooth muscles (e.g. M. sphincter) or myocardial infarction [71,72]. In this review we certainly cannot discuss strategies, outcome, risks, or chances of cellbased therapy of muscular tissue at length. But according to recent studies it is likely that not only CD133+ endothelial precursor cells [73], but also CD90+ MSC support and augment the regeneration of the tissue after myocardial infarct [74]. However, in some cases mineralization was observed after injection of bmMSC in the infarcted area of an affected heart. The mineralization process was associated with hypoxia and the inflammatory reactions during muscular repair [75]. Yet hypoxia promotes chondrogenesis of MSC but does not facilitate osteogenesis or adipogenesis [76] (and unpublished observation). Therefore the observed osteogenesis could be caused by the fact that osteogenic cells are enriched in preparations of bmMSC from

bone marrow [15]. Here a selection of such bmMSC, which promote vascularization and muscular regeneration, or the depletion of osteogenic subsets of bmMSC, may improve regeneration of the muscle after infarction. It is certainly possible that bmMSC do not contain all the subsets required for such a variety of clinical needs including muscular regeneration at sufficient amounts. Therefore the exploration of all clinically promising stem cell niches including human term placenta or adipose tissue is an important task nowadays. A completely different route to select MSC employs short random sequences of nucleic acids (DNA, RNA) termed aptamers [77]. So far, however, specific reactivity of aptamers with human MSC is not published, but experimental data from other species are promising [78]. Others investigate the attachment of MSC to proteins or peptides derived from the extracellular matrix, basement membranes, or specific tissues and hope to develop tools for propagation or enrichment of distinct MSC as well. Proteins derived from the different niches of MSC, including bone marrow, dental pulp, placenta, vasculature in adipose or muscular tissues as well as peptides derived from these proteins, are promising candidates for coating MSC culture vessels, biomaterials, and alike. 6. The choice for cell-based regimen: selected or bulk MSC? The selection of MSC for defined experimental or clinical purposes by antibodies may yield many advantages over the use of bulk MSC populations. However, several disadvantages cannot be ignored. For

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clones of bmMSC a high osteogenic differentiation capacity was reported. All clones generated osteoblast eventually, the adipogenic (80%) and especially the chondrogenic differentiation capacity (30%) of the bmMSC clones were considerably lower [9]. Moreover, is has been known for a long time that the differentiation potential of bmMSC changes over time of in vitro culture. Less chondrogenic clones were observed in MSC after long-term culture compared to the same MSC in early passages. In addition, more osteogenic clones were recorded in passaged MSC compared to the same cells in primary cultures [79]. Therefore cloning or selection of MSC may not be necessary for regeneration of bone, but it is of course a challenge when mineralization or osteogenesis must be avoided. For adipogenesis and especially for chondrogenesis selection of MSC may be therefore advantageous. But depending on the numbers of cells required for the investigation or therapy planned, selection of MSC subsets will require extended expansion of cells. But telomer lengths shorten during expansion of MSC considerably, and a loss of 80 bp to 90 bp per cell cycle were measured in MSC from older and young donors, respectively [80]. This certainly restricts an extended expansion of MSC even from young donors to the so called Hayflick limit [81]. Aging effects were observed in MSC isolated from the elderly and will influence the capacity for expansion and regeneration when autologous MSC are needed [82,83]. Here – at least for women – autologous MSC derived from the endometrium could be advantageous [15]. The benefit of stocks of umbilical cord stem cells generated individually right after birth for autologous clinical application must await careful evaluation, as the demand for such cells may come on average not after a few years, but likely after a few decades of the individuals' life. For heterologous use umbilical cord-derived stem cells could be applied for a variety of selection and differentiation procedures. This would even allow to develop of-the-shelf heterologous MSCs selected, expanded and bottled for a specific regenerative therapy (e.g. myocardial infarction), when time is a concern for success of the therapy. Although a specific selection of functional subsets of MSC seems to bring about several advantages, in may not be the best therapeutical option in all conditions. This is a lesson learned from treatment of infarcted myocard with purified CD133+ endothelial progenitor cells [84] or bone-marrow transplantation by CD34+ HSC [85]. It is known that highly purified allogenic CD34+ HSC restore the blood system completely after its erasure in a patient. Sometimes, however, graftversus-host-disease (GVHD) evolves. Under these circumstances the allogenic HSC generate immune cells which recognize the host tissue as “foreign” and mount an autoimmune attack. Here the combined application of highly purified CD34+ HSC together with MSC derived from the same donor was shown to ameliorate the GVHD and to be superior to any other therapy. The MSC not only modulate T-cell responses [86], but also improve the homing and seeding of the injected HSC to their niche in the bone marrow [85]. Today the risk of tumorigenesis by transplanted adult stem cells is considered to be rather low, but after extended in vitro culture (4– 5 months), transformation of MSC was observed [87]. The MSC entered a phase of senescence after about two months of culture. In 30% of the cells surpassing this stage of senescence, an ill karyotype, over expression of the proto-oncogen c-myc, or dysregulation of cyclin-dependent tumor suppressor p16 were observed [87]. This process, however, was accompanied by changes in the expression of CD90 and CD105, indicating that a careful analysis of the expression of cell surface markers may allow diagnosis of MSC even on a single cell level for a normal phenotype. To summarize, the advantages of selection of MSC according to the experimental or clinical needs bear great hope and bold perspectives for improved applications, but must furnish proof of superior efficacy in both, short-term and long-term follow-up studies. The disadvantages of enrichment or selection of MSC, including a more elaborate

preparation of the cells, extended culture, and other difficulties must be carefully balanced with the advantages of elementary application of bulk MSC. 7. Cell–matrix interactions modulate the differentiation of mesenchymal stromal cells As outlined above, soluble factors are key regulators for induction and continuation of osteogenic differentiation of MSC, both in vitro and in vivo. However, MSC are anchorage-dependent cells, which means that they grow in vivo and in vitro attached to or in contact with components of the extracellular matrix and neighboring cells. Therefore they rely not only on soluble factors to regulate their metabolism. True stem cells reside in specific places, called niches [13,88,89]. In the niche, the extracellular matrix, cells and growth factors provide the environment to maintain stem cells in the niche, and at the same time to generate other stem cells to refill the various pools of hematopoietic, mesenchymal and other progenitor cells needed for regeneration of blood and tissues, for wound repair, reproduction and more. At present the exact nature of the MSC niche in the bone marrow or in other tissues is not explored in great detail [13]. But some principles or components described for the HSC niche [89] may apply for the niche of MSC in bone marrow as well [90]. For MSC residing in other organs or tissues, the MSC niche is most likely different at least with respect to their cellular components. The osteoblasts (or even osteoclasts) contributing are residing typically in bone and other cells such as fibroblasts (or comparably macrophages or alike) have to substitute there. The differences in organization and composition of the MSC niches in different tissues as exemplified above may influence the regenerative capacity of MSC in vivo, and may even imprint the phenotypical characteristics observed with MSC isolated from different individuals or tissues in vitro. For instance, the biological signals regulating further proliferation, differentiation, metabolic responses, cellular survival and migration are provided by the pericellular microenvironment, again consisting of the extracellular matrix, cells and soluble factors. Under physiological conditions and in situ, attachment of MSC to the extracellular matrix (ECM) is mainly mediated by integrins [52] and expression of integrins on MSC or pericytes may vary on cells isolated from different sources [44]. The integrins are a family of cell surface receptors which occur in different stages of activation. In an inactive conformation the integrins bind to the extracellular matrix with low affinity. Upon activation of the cell, the integrins turn to an active conformation and promote the binding of the cell to the extracellular matrix with high affinity [91,92]. This may play a role for regulation of migration of stem cells including MSC in and out of their niche and in determining the amounts of MSC circulating in the peripheral blood [93]. Moreover, the extracellular matrix affects differentiation of MSC via integrin signaling and the ERK signaling pathway plays an important role for osteogenic differentiation of MSC [50]. In addition to the “chemical” composition of the MSC habitat (signaling by proteins, growth factors), the “physical” parameters of the neighborhood influence MSC considerably. It has been shown that osteogenic differentiation of MSC is facilitated when the cells are seeded on rather stiff substrates with an elasticity modulus E of 25– 40 kPa. In contrast, myogenic differentiation was promoted on substrates with higher elasticity (E approx. 12 kPa) [94]. There is experimental evidence that even individual stages of osteogenic differentiation from a MSC residing on a basal lamina of a vessel (Evessel probably below 15 kPa), to become a pre-osteoblast binding to type I collagen of the osteoid (Eosteoid around 30 kPa), to mature to an osteocyte binding to the mineralized cancellous bone (Ebone about 100 kPa) is accompanied by an increasing stiffness of the pericellular environment [94]. This difference in biomechanical predetermination by the MSC niche in vivo may prime bmMSC to preferably undergo osteogenic differentiation in vitro. In contrast, MSC or pericytes

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prepared from softer tissues such as muscle or placenta, seem to generate osteoblasts in vitro with lower efficacy [15]. At present it is not investigated conclusively, if this mechanism applies for other differentiation pathways of MSC such as the chondrogenic or adipogenic differentiation as well. The bmMSC in soft substrates prefer the adipogenic differentiation pathway [94]. MSC derived from the synovial membrane generated cartilagous tissue in vitro [30], but when injected in muscle tissue failed to form cartilage. Instead, a myogenic differentiation was reported [29]. This clearly indicates that MSC respond to the physical parameters of their pericellular environment. 8. Clinical application of mesenchymal stromal cells for regeneration of bone and cartilage For potential clinical use of MSC in humans three major areas of application are actively explored: (i) Local injection or local application (if necessary on a scaffold) to treat local defects. (ii) Systemic application to modulate for instance the activities of the immune system. Note that this type of application may include homing of MSC to the bone marrow or other sites in the recipient. (iii) Tissue engineering utilizing MSC or combinations of MSC with other cells. (iv) Application of recombinant gene technology and MSC represents an additional area of active research. Many cell-based clinical therapies involving MSC were performed in animal studies, and some included human patients. At present (November 2010) more than 140 clinical trials involving MSC are found in the official US registries (http://clinicaltrials.gov/ct2/results? term=mesenchymal+stem+cells&pg). Others are found in individual national registries. Most of the clinical studies with MSC focus on inflammation and autoimmune disorders. Most of them still recruiting patients. About a tenth of the US-registered trials deal with regeneration of cartilage and bone. Four of the 142 US registered studied were completed to date, one was withdrawn. It is therefore evident, that clinical application of MSC is a young but rapidly expanding field of research. But MSC are not yet a routine therapy in general for any case and everybody. The potential of bmMSC for repair of a meniscus was explored in a clinical phase I/II study and completed in 2003. The MSC were resuspended in a scaffold prepared from hyaluronic acid. Upon injection of 107 expanded bmMSC in the affected knee joint, regeneration of the meniscus was observed and the degerenative process in cartilage was retarded [95]. However, a translation of this approach to human patients seems to be hampered by the amount of MSC required to treating meniscal degeneration of a considerably larger joint in men. The use for MSC for cartilage regeneration was also explored. In one study suspensions of autologous bmMSC were expanded in vitro, harvested and mixed with collagen solutions and blotted on collagenous sheets. This technique allows to fix the cells in situ [96]. In addition, the defect was covered with a periosteal membrane to protect it and to provide nutrition as progenitor cells residing in the periost act like feeder cells. Recently, an extension of this study was presented covering 45 MSC transplantations to 41 patients and a mean of follow-up of more than 6 years (5–137 months post surgery) [97]. Application of MSC for cartilage repair seems to be a safe and effective procedure. Furthermore, in a direct comparison patients presenting with cartilagous defects were treated with either chondrocytes or MSC [98]. The autologous cells were expanded and 1.5 × 107 cells were injected in the defect covered with a periostal flap [99]. There was no gross difference in clinical outcome between the cohorts treated with chondrocytes derived from healthy appearing sites of the affected cartilage compared to patients treated with bmMSC. However, the contribution of the periostal cells should not be neglected as others have shown that a periosteal patch covering the defect may heal a cartilage defect by itself. Expansion of cells for clinical application in presence of animal sera is not accepted. Therefore this protocol has to be developed further to reach

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acceptance worldwide. But from these studies it is clear that bmMSC do have a great potential for cartilage repair, especially when number or quality of chondrocytes available in the affected joint are limited. Proof of principle for bone repair by MSC in humans was provided as well and several clinical phases studied were completed. One study investigated the potential of in vitro expanded MSC to heal large defects in long bone in three patients. All patients presented with good results one to two years after treatment [100]. In another study, amelioration of osteogenesis imperfecta was in focus [101]. Clinical outcome reported from a small number of cases (n = 5) confirmed the great potential for MSC for bone regeneration, even in the context of genetic bone disorders [101]. Recently, MSC were also applied to treat avascular necrosis after irradiation of bone. Again, clinical improvement – and most of all in this case less pain after treatment with MSC – were reported [18]. Tissue engineering to generate functional cell-augmented implants in vitro may require complex combinations of cell culture and bioreactors, strategies not only to define and differentiate MSC to the desired type of cell, but also may include concepts to provide either stimuli for vascularisation or provide structures for vessels in the scaffold. Furthermore tissue engineering with MSC also may require a geographically and temporally defined addition of different types of cells, and their in vitro conditioning to the chemical and physical needs in situ. This can be achieved probably best in bioreactor systems. Comparably, strategies combining MSC and recombinant gene technology require completely different sets of methods to generate the genetically altered cells, MSC treated with small interfering RNAs, or cells containing synthetic peptides or proteins. Furthermore, quality management of such additionally manipulated MSC is a special task as well. Finally, the combination of such technologies and stem cell therapy will probably be limited to a very small number of human patients or disorders treated ever. We therefore will not cover tissue engineering and strategies involving gene therapy in this review.

9. Outlook At present we entertain different strategies by utilizing antibodies, proteins, and peptides to define functionally different subsets of MSC from human bone marrow and human term placenta. This includes but is not limited antibodies to CD56, CD146, or CD271 and is mainly focusing on regeneration of bone, cartilage, and intervertebral disks. Other cell surface antigens on MSC may open new avenues for enrichment or even purification of distinct subsets of MSC, which differentiate towards other mature phenotypes, such as chondrocytes, smooth muscle cells, or adipocytes. Such tools will expand our possibilities to deplete unfavorable subsets from MSC preparations as well. The antigens on MSC indicating their pre-disposition towards a certain line of differentiation are not necessarily found on the mature cells. CD146 for instance is a marker for osteogenic MSC but not expressed on mature osteoblasts. Therefore in-depth characterization of the individual pathways of differentiation in connection with the phenotypic changes in regard to the expression of surface markers on MSC [102–106] will allow to better link selection or depletion antigens on MSC with the prospective regenerative potential or risk.

Acknowledgements The authors acknowledge the support of their research by grants from the BMBF, BMWiT, DFG, DGUV, and the Baden-WürttembergStiftung. They thank midwifes, obstetrics, and surgeons at both hospitals in Tübingen, UKT and BG, for providing biopsies to prepare MSC from different tissues, Christine Ulrich (MS) for providing artwork in preparation of figures, and Tanja Abruzzese for her excellent technical assistance.

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