There is a growing interest in rheumatology in the role of mesenchymal progenitor cells (MPCs) in the pathogenesis of the rheumatic diseases (1–4). These primitive progenitors possess high proliferative potential and can differentiate into several mesenchymal lineages, including bone and cartilage (5–10). It is thought that MPCs may participate in joint repair, and their possible role in regeneration of damaged joints has been suggested (1, 4). Despite recent progress in the field of MPC biology, the in vivo characteristics of these cells remain largely unknown (7–9), and their presence in various tissues, including synovium, is established retrospectively by lengthy expansion/differentiation assays (3). As a consequence, their precise numbers, anatomic distribution within the joint, possible routes of entering the synovium, and relationship to other types of progenitor and stem cells remain poorly defined (3).
The phenotype of MPCs expanded in culture has been previously described (6). Cultured MPCs derived from the bone marrow (BM) (5, 6) and synovium (3), as well as from peripheral (11), cord (12, 13), and fetal (14) blood, all appear as monolayers of fibroblastic cells capable of multilineage mesenchymal differentiation. These cultured MPCs are known to derive from individual clonogenic cells, termed colony-forming units–fibroblastic (CFU-Fs) (6, 11). In the BM, CFU-Fs were documented to have a fibroblastic appearance, characteristic prominent nucleoli (5, 15, 16), and an average frequency of 1 cell per 104–105 mononuclear cells (MNCs) (15, 17). In human peripheral blood, they are even less frequent (11). The extreme rarity of these cells remains the main reason for the current lack of data on their phenotype, and the inability to purify CFU-Fs still leads to ambiguity regarding their precise relationship to hematopoietic and other stem cells.
To date, only a few surface antigenic markers have been described that were used in the purification of MPCs/CFU-Fs from the BM (18–20). The molecules commonly seen as specific for cultured MPCs, such as CD44 and CD29 (13), have in fact broad cell reactivity and, hence, are unsuitable for the detection of MPCs in vivo. STRO-1, the first antibody used to partially enrich CFU-Fs from human BM, also cross-reacts with other cells (erythroblasts) (18). Another potential candidate MPC molecule, bone morphogenetic protein receptor type IA (BMPRIA), was recently used to detect putative mesenchymal precursors in the synovium (2). However, the proliferation and differentiation potentials of BMPRIA+ cells have not been reported, and the BMPRIA status of BM MPCs is still unknown. Therefore, there is a clear need for novel markers and methods of detection, enumeration, and isolation of MPCs from the BM and other tissues as a prerequisite for establishing their roles in joint homeostasis and arthritis.
In the present study, we isolated and characterized MPCs in the BM and established their phenotype and relationship to hematopoietic progenitors. We purified BM MPCs and showed that these cells were adherent, fibroblast-like, and capable of proliferation and osteogenic, adipogenic, and chondrogenic lineage progression. We demonstrated that BM MPCs expressed several unique antigens that distinguished them from dermal fibroblasts, cells lacking multilineage differentiation potentials. These findings should provide a basis for identification of MPCs in the joints and further our understanding of their roles in joint physiology and in diseases such as rheumatoid arthritis, osteoarthritis, and osteoporosis.
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- MATERIALS AND METHODS
MPCs may be of major pathogenic and therapeutic importance in the rheumatic diseases, but to date, they have only been indirectly characterized following expansion in culture (1–4, 6). The phenotype and topography of MPCs in vivo and their relationship to other progenitor and stem cells are poorly defined (3, 7–10). The purpose of this study was to isolate and characterize MPCs resident in human BM. We showed that all of the BM MPC activity was confined to a rare, phenotypically distinct, and homogeneous population of D7-FIB+,CD45low cells that are different from both skin fibroblasts and hematopoietic progenitors. Representing only 0.01% of BM MNCs, D7-FIB+,CD45low cells were purified here using a combination of magnetic selection and FACS, resulting in levels of enrichment that had not been reported before.
Using multiparameter flow cytometry, we obtained a detailed phenotype profile of BM MPCs. Published data on the MPC phenotype in vivo are very limited (18–20, 24). Our findings confirmed that BM MPCs expressed STRO-1 and CD105 antigens (18–20). Both markers, however, are known to be present on other types of BM cells and therefore are not very selective for MPCs. STRO-1 is expressed on erythroblasts (17–19), and CD105/SH2/endoglin (25, 26) is expressed on endothelial cells (27) and on pre–B cells (28). In comparison, the D7-FIB molecule showed minimal coexpression on other BM cells. It therefore appears superior to STRO-1 or CD105 as a marker of MPCs in the BM.
Apart from the D7-FIB antigen, which is a fibroblast-specific molecule of yet-unknown function, BM MPCs were found to express other antigens present on human fibroblasts, the peptidases CD10 and CD13, and CD90 (Thy-1) (29, 30). The molecules strongly expressed on BM MPCs and absent on uncultured skin fibroblasts were LNGFR, STRO-1, and HLA–DR. LNGFR expression was previously reported on adventitial reticular cells (ARCs), an interconnected network of nonhematopoietic star-like cells in the BM (31, 32). This indicated that ARCs were the likely in vivo equivalents of MPCs in the BM. The function of LNGFR on MPCs is unclear, but it may have a morphogenic role in the development of the BM cavity, kidney, and other organs (31–33).
The expression of HLA–DR on BM MPCs was unexpected. It is possible that HLA–DR may play a role in hematopoietic progenitor cell maturation, since HLA–DR was previously shown on stromal cells in the developing thymus (34). Interestingly, thymic stroma was also shown to express TGFβ receptors (35), a large superfamily of molecules that includes BMPRs (36). BMPs are known to regulate the growth, differentiation, and apoptosis of various cell types, including osteoblasts, chondroblasts, and neural and epithelial cells (36). In this study, one member of this family, BMPRIA, was found to be expressed on BM MPCs and not on skin fibroblasts. This strengthens the idea that BMPRIA+ cells described in inflamed synovial tissue are related to MPCs (2). The expression of other members of the BMPR family on BM MPCs is currently under investigation.
To date, the relationship between MPCs and hematopoietic progenitor cells has not been fully established. Our study clearly demonstrated that BM MPCs did not express CD34 or CD133, the most commonly used markers of hematopoietic progenitors. Morphologically, fibroblast-like MPCs also differed markedly from hematopoietic progenitors, which are known to be small, blast-like cells. This indicated that hematopoietic and mesenchymal progenitors in the BM are morphologically and phenotypically different cell types.
There is a great deal of debate in the literature as to what constitutes a stem or progenitor cell as well as the degree of plasticity of various progenitor cells. Stem cells are capable of self-renewal and can differentiate into all cell types (pluripotential) or to more than one differentiated cell type (multipotential) (37). Progenitor cells are the intermediate step between stem cells and fully differentiated cells (38). Early progenitors can still be multipotential cells, but unlike stem cells, they are not capable of self-renewal (38). Because MPCs have been described as multipotential cells, many investigators have adopted the term mesenchymal stem cells, in one view somewhat prematurely, since no suitable assay to assess their self-renewal has yet been developed (39).
A broad range of alternative terms (such as “marrow stromal stem cells” or “osteogenic stem cells”) historically used to define cells with MPC properties tends to add even more confusion to the contentious nature of this field (6–10, 40). In addition, recent findings suggest that BM may contain putative stem cells more primitive than MPCs (40) as well as pluripotent cells capable of producing progeny with characteristics of mesoderm, neuroectoderm, and endoderm (41). Such cells can be copurified with MPCs from the CD45−,GPA− cell fraction of human BM, but their more detailed phenotype has not yet been reported. It may be possible in the future to isolate these primitive cells using the D7-FIB–based strategy described in this report. With regard to the MPCs themselves, further investigation is required to determine whether D7-FIB+,CD45low cells are a self-renewing cell population or whether they are derived from more primitive progenitors with a yet-unknown phenotype.
It has recently been shown that MPCs are present in normal synovium, and their role in the development of arthritis has been postulated (1–4). However, in the absence of a “phenotypic fingerprint,” the nature and roles of MPCs in bone and joint homeostasis have remained speculative. In this study, we identified and described the phenotype of these exceptionally rare cells in human BM. On the basis of this research, precise quantification of BM MPCs in such diseases as osteoporosis and osteoarthritis could be performed. The phenotype data from the present study could allow identification of MPCs in the joint, based on the D7-FIB+,CD45low,LNGFR+ phenotype. This will lead to further progress in the understanding of the distribution and plasticity of MPCs in vivo and the roles they may play in the joint physiology in health and disease.