In early 1970’s, Friedenstein and colleagues were the first to report the presence of fibroblastoid cells that could be flushed out from the adult bone marrow, form colonies on plastic and, when transplanted subcutaneously with appropriate carriers, could make bone and reconstitute a hematopoietic microenvironment. Friedenstein provided the first evidence of the existence in the bone marrow of what later would be called a “mesenchymal stem cell” or MSC. Over the years, it has become progressively clear that such cells, which can differentiate in vitro into a variety of mesenchymal lineages such as osteoblasts, chondrocytes and adipocytes, are not an exclusive feature of the bone marrow, but can also be isolated from other organs and tissues. A large number of studies have provided evidence in support of their plasticity, their potential use for tissue engineering purposes, their extraordinary immunomodulatory properties, as well as their ability to be recruited to sites of injury where they might contribute a “natural in vivo system for tissue repair”. Other studies have attempted the characterization of their cell-surface specific antigens and of their anatomical locations in vivo. However, despite this impressive body of investigations, numerous questions related to the developmental origin of these cells, their proposed pluripotency, and their participation to the physiological processes of bone modeling and remodeling and tissue repair in vivo, are still largely unanswered. Moreover, both a systematic phenotypic in vivo characterization of the MSC population and the development of a reproducible and faithful in vivo model that would test the ability of MSCs to self-renew, proliferate and differentiate in vivo are still just beginning. Lastly, a detailed analysis of the complex network of signaling pathways that very likely regulates their proliferative and differentiation potential has also just begun.
This brief review summarizes the current knowledge and the outstanding questions in the field of the study of mesenchymal stem cells.
1. Introduction
Any adult tissue with repair and/or regenerative capabilities is likely to harbor tissue-specific stem cells defined as cells that self-renew and retain sufficient proliferative and differentiation potential to be able to repair and/or reconstitute a specific tissue. It is well known that adult bone has an impressive ability to repair; therefore, it is not surprising that the identification and characterization of the stem cells responsible for this process is an active field of investigation (Bianco et al. 2008, Caplan, 2007, Kolf et al. 2007, Prockop, 1997).
The presence of non-hematopoietic stem cells in the bone marrow was first suggested by the German pathologist Cohneim about 130 years ago, who proposed that bone marrow can be the source of fibroblasts contributing to wound healing in numerous peripheral tissues (Prockop, 1997). In the early 1970’s, the pioneering work of Friedenstein and colleagues demonstrated that the rodent bone marrow had fibroblastoid cells with clonogenic potential in vitro (Friedenstein et al. 1970, Friedenstein, 1980). Friedenstein flushed out the whole bone marrow into plastic culture dishes, and, after discarding a the non-adherent cells a few hours later, isolated spindle-like cells adherent to the plastic, which were heterogeneous in appearance but capable of forming colonies (Colony-forming unit fibroblastic, CFU-F). These cells could also make bone and reconstitute a hematopoietic microenvironment in subcutaneous transplants. Moreover, Friedenstein demonstrated that they could regenerate heterotopic bone tissue in serial transplants, thus providing evidence in support of their self-renewal potential. Over the years, numerous laboratories have confirmed and expanded these findings (see Figure 1) by showing that cells isolated according to Friedenstein's protocol were also present in the human bone marrow, and by demonstrating that these cells could be sub-passaged and differentiated in vitro into a variety of cells of the mesenchymal lineages such as osteoblasts, chondrocytes, adipocytes and myoblasts (Bianco et al. 2008, Caplan, 2007, Kolf et al. 2007, Pittenger et al. 1999, Prockop, 1997). Friedenstein had thus isolated from the bone marrow what later on would have been renamed by Caplan and colleagues “mesenchymal stem cell” or MSC (Caplan, 2007).
Modified from Kuznetsov et al. 2004.
MSCs or MSC-like cells are not a unique feature of the bone marrow, as they also are found in tissues such as fat, umbilical cord blood, amniotic fluid, placenta, dental pulp, tendons, synovial membrane and skeletal muscle, though the complete equivalency of such populations has not been formally demonstrated using robust scientific methods (Rogers and Casper, 2004, Bieback and Kluter, 2007, Xu et al. 2005, Shi and Gronthos, 2003, Tsai et al. 2004, Bi et al. 2007, Igura et al. 2004, De Bari et al. 2001, Crisan et al. 2008). Much effort has been invested both in expanding and phenotypically characterizing these cells in vitro and in identifying factors that might keep them in undifferentiated state, in order to then transplant them back in vivo for the purpose of repairing specific tissues such as bone and cartilage (Tsutsumi, 2001, Kulterer et al. 2007, Pochampally et al. 2004, Hishikawa et al. 2004, Kratchmarova et al. 2005, Song et al. 2006). Therefore, our knowledge of MSCs is virtually entirely based on the characterization of cultured cells, and our definition of MSCs is indeed an “operational” definition based on the potential to self-renew and differentiate in vitro. Interestingly, no evidence of asymmetric cell division, which is considered a property of self-renewing cells in some settings (Wu et al. 2008), has been yet provided for MSCs.
Little is known about the phenotypic characteristics of MSCs in vivo, their developmental origin, their contribution to organogenesis and postnatal tissue homeostasis normally, and their anatomical localization. Moreover, a faithful assay that would rigorously test for their ability to self-renew in vivo, and would thus prove their “true” “stemness”, is still missing.
An in vivo characterization of MSCs could allow for either pharmacological or genetic manipulations of this cellular pool in vivo, and it could make possible the isolation of a more enriched population for tissue engineering applications with potentially better capabilities of self-renew and proliferate upon in vitro expansion and in in vivo transplants with appropriate scaffolds.
This brief review summarizes the current knowledge and the outstanding questions in the field of study of mesenchymal stem cells.
2. Are mesenchymal stem cells distinct from hematopoietic stem cells?
In vitro studies over many years established the essential role of bone marrow stromal cells for the development and differentiation of hematopoietic cells in vitro. The distinct lineage origin of such stromal cells was elegantly demonstrated by Simmons and colleagues, who showed that these cells isolated from patients with functioning sex-mismatched but HLA-identical allografts were exclusively of host genotype (Simmons et al. 1987). This finding clearly indicated that stromal cells supporting hematopoiesis are a population distinct from hematopoietic cells, and thus confirmed in vivo previous observations from Friedenstein's laboratory, which showed that in sex-matched transplants cells capable of forming heterotopic osseous tissue were physically different from HSCs (Friedenstein, 1980). An important implication of this discovery was that mesenchymal stem cells, which according to the work of Friedenstein and colleagues also give rise to stromal cells supporting hematopoiesis, were thus likely to be distinct from the hematopoietic stem cells.
The current model indeed is that there are at least two types of stem cells in the bone marrow, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). In this formulation, HSCs would give rise to hematopoietic cell types and to cells that resorb bone (osteoclasts), whereas MSCs would generate CFU-Fs and differentiate into a variety of mesenchymal lineages such as chondrocytes, adipocytes and osteoblasts in vitro.
Recent studies have challenged this clear distinction. For example, it has been reported that tissue fibroblasts/myofibroblasts, which are thought to play an important role in secretion of growth factors and accumulation of matrix in many organs and tissues, may derive from HSCs (Ogawa et al. 2006); others have failed to confirm these findings (Koide et al. 2007).
Numerous technical factors (possible cell contamination, differing culture conditions, possible cell fusion in vivo) may explain the investigators’ varying results, but the varying results also make clear the need for better phenotypic characterization.
3. Have mesenchymal stem cells specific cell-surface antigens?
If mesenchymal stem cells represent a population distinct from hematopoietic stem cells, they probably have a specific repertoire of cell surface antigens that would allow for a phenotypic separation from hematopoietic stem cells. For this and other reasons, the identification of cell-specific cell surface markers on MSCs would be very useful. Whereas numerous cells-surface antigens expressed by MSCs that have been cultured and sub-passaged in vitro have been identified over the years, only a few laboratories have attempted a phenotypic characterization of MSCs in vivo (Ratajczak et al. 2007).
In the early 1980’s, another elegant study from Simmons and colleagues led to the isolation of an antibody known as STRO1, which recognizes a cell surface antigen present in human bone marrow stromal cells. The STRO-1-positive population was considerably enriched in clonogenic cells that were able to both generate CFU-Fs and differentiate into multiple mesenchymal lineages in vitro (Simmons, and Torok-Storb, 1991). The same group reported that the degree of homogeneity of the STRO-1-positive population could be further enhanced by positive selection for VCAM/CD106 (Gronthos et al. 2003). More recently, an important study by Bianco and colleagues has demonstrated that MCAM/CD146 (+) cells isolated from the human bone marrow stroma adhere to the plastic in vitro and are clonogenic; moreover, they self-renew, at least in vitro, and they can generate bone and a hematopoietic supportive microenvironment in subcutaneous transplants in mice (Sacchetti et al. 2007). The relationship, if any, between these cells and the STRO-1/VCAM positive cells previously reported by Simmons’ group remains to be established.
Considerably less progress has been made in the characterization of the cell surface antigens that are expressed by murine MSCs in vivo. Van Vlasselaer and colleagues reported the purification of cells with osteogenic potential from murine bone marrow by two-color cell sorting using anti-Sca1 monoclonal antibody and wheat germ agglutinin (Van Vlasselaer et al. 1994. More recently, Simmons’ laboratory has identified a bone marrow pool of Sca1 (+) CD45 (-) CD31 (-) cells that appears to be enriched in MSCs/progenitors (Lundberg et al. 2007, Short et al. 2003). CD45, a pan-hematopoietic cell marker, and CD31 or PECAM, a classical marker for endothelial cells, were used in the study to negatively select for hematopoietic cells and endothelial cells, respectively.
Of note, all these markers have also been defined operationally, i.e used to enrich cells that are clonogenic/multipotent in vitro or that form heterotopic bone tissue in subcutaneous transplants, and none of them appears to participate directly in the molecular processes regulating self-renewal versus differentiation.
Interestingly, it has also been shown that a subset of CD45 (+) Lin (-) bone marrow cells was able to differentiate in vitro into a variety of cell types, including endothelial cells, osteoblasts, muscle cells, and neural cells (Rogers et al. 2007). The finding challenges the specificity of CD45 as a marker of hematopoietic cells. Alternatively, it may suggest that the boundary between hematopoietic stem cells and mesenchymal stem cells may not be as definite as thought in the past. Lastly, it raises the question about the existence in the bone marrow of pluripotent stem cells, which could give origin to tissues that embryologically derive from all the three embryonic germ layers, ectoderm, mesoderm and endoderm. This is an appealing and exciting possibility for which, however, only a few pieces of experimental evidence have been provided so far (see next paragraph).
4. Are pluripotent stem cells present in the bone marrow?
It has been proposed that a heterogeneous population of non-hematopoietic stem cells exists in the bone marrow of both humans and rodents, with MSCs being just a subset of this complex cellular make-up. The heterogeneity is exemplified by the different cell pools investigators over the years have isolated from the bone marrow using different methodology, and by the different names that have been assigned to these diverse populations, such as “endothelial progenitor cells” (EPCs), “multipotent adult progenitor cells” (MAPCs) or “marrow-isolated adult mutilineage inducible cells” (MIAMI), “mesenchymal stem cells” (MSCs), and “ very small embryonic-like stem cells” (VSELs; Asahara et al. 1999, Jiang et al. 2002, Reyes et al. 2001, D’ippolito et al. 2004, Ratajczak et al. 2007).
EPCs have been isolated in both mice and humans, and are considered endothelial precursors that reside in the bone marrow and are released into the bloodstream to contribute to vasculogenesis in injured organs (Asahara et al. 1999). MAPCs were isolated from both human and murine bone marrow mononuclear cells and exhibit a fibroblastic morphology similar to MSCs. To this end, they are the only population of bone marrow-derived cells that, after expansion in vitro, can contribute to all the three germ layers upon injection into blastocysts, at least in rodents (Jiang et al. 2002, Reyes et al. 2001). MIAMI cells were isolated from human bone marrow and cultured in vitro on fibronectin-coated plates in 5% oxygen, which is likely to be the physiological level of oxygen tension in the bone marrow. Upon expansion in vitro, MIAMI cells were shown to express markers for cells derived from all three germ layers (D’ippolito et al. 2004). Differently from MAPCS and MIAMI cells, which have been phenotypically characterized only after culture and expansion in vitro, VSEL cells express in vivo markers characteristic of pluripotent stem cells, and are found during embryogenesis in the epiblast of the cylinder-stage embryo (Ratajczak et al. 2007). More recently, the prospective isolation of stage-specific embryonic antigen-1 (SSEA-1) positive cells from the adult murine bone marrow, which are capable of differentiating in vitro into astrocyte-, endothelial-, and hepatocyte-like cells, has been reported (Anjos-Afonso and Bonnet, 2007).
How all these different populations are related to each other and their relevance in vivo are still open questions. Given the apparent heterogeneity of the non-hematopoietic stem cells existing in the bone marrow, standardization of methodologies for the isolation of these cells so that can be compared, and in vivo phenotypic characterization of this heterogeneous population are important next steps.
5. What is the developmental origin of mesenchymal stem cells?
Our knowledge of the developmental origin of MSCs is still quite limited. It is widely believed that MSCs derive from mesoderm; notably, however, a recent study showed that the earliest lineage providing MSC-like cells during embryonic trunk development is indeed generated from Sox1(+) neuroepithelium rather than from mesoderm, at least in part through a neural crest intermediate stage (Takashima et al. 2007). These early MSCs are then replaced, later in development, by MSCs from other origins. Perhaps relevant to these observations, it has been recently demonstrated that neural crest-derived cells migrate to the bone marrow through the bloodstream (Nagoshi et al. 2008). These cells are still present in the adult bone marrow, and can differentiate in vitro into neurons, glial cells and myofibroblasts. The potential link, if any, between these cells, the cells identified by Takashima et al. (2007) and the MSCs isolated according to Friedenstein's protocol remains to be established.
Notably, it has been reported that endothelial progenitors and mural cells may derive from a common vascular progenitor (Yamashita et al. 2000). In addition, lineage tracing experiments have shown that cells originated from the primary vascular plexus give also rise to mural cells with the capability to differentiate into osteoblasts and adipocytes (Brachvogel et al. 2005). Since vascular pericytes could be MSCs (see below), these findings would suggest that MSCs, HSCs, and endothelium progenitor cells (EPCs) could arise from a common progenitor.
Expanding our understanding of the developmental origin of MSCs will definitively broaden our knowledge about their role in organogenesis; moreover, it will provide further tools for a phenotypic characterization of these cells and, at the same time, important cues about their multi- or pluri-potentiality.
6. Is there a “niche” for mesenchymal stem cells?
In absence of specific and unique markers that would allow for a proper identification of MSCs in vivo, a histological localization of these cells is virtually impossible to identify and is clearly lacking.
An extensive literature has pointed to pericytes as a potential source of MSCs (Crisan et al. 2008, Dennis and Charbord, 2002, Jones and Mcgonagle, 2008, Da Silva Meirelles et al. 2008, Modder and Khosla, 2008). As already mentioned above, Bianco and colleagues have recently reported that MCAM/CD146 (+) subendothelial cells in the human bone marrow are the only cell population that is both clonogenic in vitro and capable of transferring a hematopoietic microenvironment in subcutaneous transplants (Sacchetti et al. 2007). These cells, which reside in the wall of the sinusoidal blood vessels of the bone marrow, are also positive for angiopoietin-1, a critical regulator of vascular remodeling. The findings by Bianco and colleagues represent the first rigorous attempt to histologically localize and phenotypically define MSC-like cells, or at least a subpool of this population. Notably, a recent paper by Crisan and colleagues suggests that multipotent MSCs with perivascular localization exist in numerous human organs (Crisan et al. 2008).
Whether the vascular setting provides a true niche for pericytic MSC-like cells and is the main source of MSCs in vivo, remains to be established. In this regard, however, it is important to note that an increasing amount of evidence has recently linked angiogenesis to osteoblastogenesis, suggesting that blood vessels could be a source of osteoprogenitors or of MSCs with osteogenic potential (Wang et al. 2007). A possible implication of these exciting findings is that MSCs could be the skeletal stem cells that contribute to the physiological processes of bone modeling and remodeling in vivo (Modder and Khosla, 2008).
Another extremely important but yet unanswered question in regard to MSCs and their site of origin is whether the bone periosteal compartment, which is critical for fracture repair, is also as a source of MSCs and whether this periosteal population shares significant similarities with the MSCs isolated from bone marrow (Modder and Khosla, 2008).
7. Why studying MSCs?
7.1. Organogenesis versus tissue engineering
A greater understanding of the biology of MSCs, particularly in the in vivo setting, will probably provide novel and critically important insights into the cellular mechanisms of bone development, hematopoiesis, vasculogenesis and angiogenesis. Surprisingly, despite a variety of very elegant studies that correlate the number of CFU-Fs to bone mass (Bonyadi et al. 2003, Hilton et al. 2008), rigorous evidence that MSCs are the skeletal stem cells has not been provided so far. For this purpose, it could be useful to inject MSCs in vivo, after appropriate isolation and expansion, and to study whether they contribute to the physiological process of bone remodeling and to fracture healing. Similarly, when appropriate gene markers have been identified, the tracing of the fates of MSCs in vivo will determine when these cells act physiologically as precursors of osteoblasts, chondrocytes and adipocytes.
Already, the abundance of in vitro studies has generated important information concerning the use of MSCs for tissue engineering applications. As we mentioned above, investigators have identified efficient modalities of expanding MSCs isolated from bone marrow or adipose tissue aspirates, while maintaining their multipotency (Caplan, 2007, Prockop, 2007). These cells have been used with appropriate scaffolds to form tissues such as bone and cartilage, upon transplantion at specific sites in experimental animal models. However, no human MSC-based technology is currently available.
7.2. MSCs as a “natural system for tissue repair”
Over the years, it has also become progressively clear that MSCs could be the basis for an extremely powerful “natural system of tissue repair” (Phinney and Prockop, 2007). MSCs have been demonstrated, upon exogenous administration, to serve as effective therapeutic agents in a variety of experimental models of tissue injuries (Ortiz et al. 2007, Kunter et al. 2006, Minguell and Erices, 2006, Lee et al. 2006, Phinney and Isakova, 2005). Curiously, in the vast majority of these studies the therapeutic efficacy did not correlate with the efficiency of engraftment, which was in general low (Prockop, 2007). This finding suggests that the ability to repair was very likely secondary not to transdifferentiation of MSCs into the appropriate cell phenotype or to cell fusion, but rather to the secretion by MSCs of soluble factors that altered the tissue microenvironment (Prockop, 2007). In other terms, MSCs may thus provide what Caplan and colleagues define as “trophic activity”(Caplan, 2007). In this regard, extensive proteomic analyses have indeed revealed that MSCs in vitro produce a variety of factors that influence a broad range of biological functions, including angiogenesis, and secrete neuroregulatory peptides and cytokines with critical roles in inflammation and repair (Caplan, 2007).
If MSCs do constitute a natural system for tissue repair, then the next important questions are how MSCs are mobilized and how they get to the site of injury. Chemokines such as SDF1 and its cognate receptor CXCR4 may have an essential role (Chamberlain et al. 2007) in directing MSCs to sites of injury. More recently, it has been reported that the cytokine receptor CCR2 and its intracellular adaptor molecule FROUNT are necessary for homing of bone marrow-derived mesenchymal stem cells to sites of injury (Belema-Bedada et al. 2008). Little is known about how MSCs are mobilized from the bone marrow. It has been reported that MSCs can be observed in the circulating blood and that this circulating pool is dramatically increased by exposure to chronic hypoxia (Rochefort et al. 2006). The molecular mechanism and the biological relevance of this interesting phenomenon remain to be elucidated.
7.3. Gene therapy and immunomodulatory properties
Over the years, investigators have attempted to use MSCs as cellular vehicles for gene therapy in experimental models, or to treat conditions such as osteogenesis imperfecta in humans upon their systemic administration (Horwitz et al. 2002, Nixon et al. 2007). Much still needs to be done in order to be able in the future to employ MSCs as therapeutic agents.
Probably the most surprising feature of MSCs is their extraordinary and still partially unexplained immunomodulatory properties. Adult human MSCs have been reported to express intermediate levels of major histocompatibility complex (MHC) class I proteins, but not MHC class II proteins (Le Blanc and Ringden, 2005). This phenotype has been regarded as non-immunogenic, which implies that transplant into an allogenic host would not require any immunosuppression. Even more surprising, numerous studies have shown that MSCs have immunosuppressive properties by modulating specific T-cell functions in vitro (Beyth et al. 2005), and some of these observations have even been translated into in vivo settings such as experimental models of graft versus host disease (GVHD; Dazzi and Marelli-Berg, 2008).
8. A future perspective
As we have emphasized in this brief summary, a systematic, phenotypic in vivo characterization of the MSC population is needed for numerous reasons, including to learn about the role of these cells in organogenesis, to be able to appropriately expand them in vivo, and thus modulate in vivo repair and regeneration processes without the need for in vitro expansion.
A necessary precondition for an adequate characterization of the MSC population is the development of a reproducible and faithful in vivo model that would test the ability of MSCs to self-renew, proliferate and differentiate in vivo. The subcutaneous transplants first introduced by Friedenstein and colleagues are undoubtedly very useful and informative, but are not the equivalent of the primary and secondary bone marrow transplants that are classically used in the hematopoietic field to test stemness. Murine intratibial injection of MSCs and characterization of their contribution to bone homeostasis and fracture repair is becoming progressively more popular, even if to this end it is not a fully standardized test (Wang et al. 2003).
An important goal, in addition to a proper in vivo characterization, would be the identification of pharmacological tools that could be used to expand in vivo and/or in vitro the MSC pool. Some attempts in this direction have been already pursued: for example the proteosome inhibitor Velcade has been reported to be able to expand the MSC pool in a murine model in vivo (Mukherjee et al. 2008).
A successful identification of useful pharmacological tools requires a detailed and systematic analysis of the complex network of signaling pathways and cells that regulates the ability of MSCs to self-renew, proliferate and eventually differentiate. The definition of a possible niche for MSCs and its regulation is a clear priority. The identification of this network is critically important, in order to reach a deep understanding of the rules that govern the size of the MSC pool in vivo, which would then ultimately allow for the appropriate pharmacological interventions. But, how to unveil this network? It is likely that a global gene expression profiling approach could be extremely helpful to gain insights into the molecular mechanisms that regulate both the size and the differentiation potential of MSCs (Tsutsumi et al. 2001, Kulterer et al. 2007, Pochampally et al. 2004, Hishikawa et al. 2004, Kratchmarova et al. 2005, Song et al. 2006). Alternatively, or better in parallel, the in vivo analysis of genetically modified experimental models could be an invaluable source of information. Lastly, human diseases could open new windows on our understanding of mesenchymal stem cells. In this regard, analysis of the Gsalpha signaling pathway in human fibrous dysplasia, a disease involving early cells of osteoblast lineage in mosaic individuals, is likely to be a very promising model (Riminucci et al. 2006).
Acknowledgments
This work was supported by NIH Grants RO1 AR048191–06 to ES and U54 HL081030 to HMK