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Cell Journal، جلد ۱۶، شماره Suppl ۱، صفحات ۱۷-۱۷
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عنوان انگلیسی Is-17: Isolation and Characterization of Primary Human Bone Marrow Mesenchymal Stem Cells
چکیده انگلیسی مقاله Objective: Human bone marrow (BM) contains a rare population of non-hematopoietic mesenchymal stem cells (MSC) that are of central importance for the hematopoietic microenvironment. We and others have shown that primary BM-MSC were enriched in lin-/CD45-/CD271+ cells but not in the CD271- cell fraction. Furthermore, we have shown that CD146 expression on primary BM-MSC allowed to discriminate endosteally- from perivascularly-located BM-MSC in human bone marrow in-situ (Tormin et al. Blood 2011, 117, 5067-5077). Although CFU-F frequencies in lin-/ CD45-/CD271+ cells are as high as up to ca. 1 in 20, a more precise phenotypical definition of these rare cells is required to be able to study the exact cellular properties of this putative stem/progenitor cell population. We therefore aimed to identify novel and potentially better markers for the isolation and characterization of primary human MSC by utilizing comparative gene expression profiling on human lin-/CD45- BM cells sorted based on CD271 expression. Materials and Methods: BM aspirates were from healthy volunteer donors. Primary MSC were pre-enriched by RosetteSep lineage depletion and then isolated by multi-color FACS sorting. Illumina gene arrays were used for comparative gene expression analysis. MSC properties were evaluated by standard in vitro and in vivo assays. Results: In total, 219 genes were significantly upregulated in the CD271+ subset, including typical MSC genes as well as genes encoding for cytokines, growth factors and extracellular matrix protein. Twenty-eight of the upregulated genes related to surface-expressed molecules. Four of the genes were cell surface markers that had been previously used to isolate human MSC from different tissues, while the remaining 24 genes had not been reported in the context of MSC isolation. FACS analysis of the expression of the potential novel markers on lin-/CD45-/CD271+ cells revealed two staining patterns, i.e. marker expression was either directly correlated with CD271 expression and did thus not enable to further enrich for CFU-F (e.g. CD151), or the marker was only expressed on a fraction of the lin-/CD45-/CD271+ cells, thus potentially allowing to identify a CFU-F and a non-CFU-F containing population within the CD271+ cells. In fact, sorting based on CD140a (PDGFR alpha) expression allowed to sort a population of lin-/CD45-/CD271+/CD140alow/- cells with a CFU-F frequency of 1 in 5. These cells furthermore demonstrated typical in vitro and in vivo stroma formation and differentiation capacities and exhibited high levels of genes associated with mesenchymal lineages as well as HSC supportive function. Moreover, lin-/ CD45-/CD271+/CD140alow/- cells effectively mediated the ex vivo expansion of transplantable CD34+ hematopoietic stem cells. Conclusion: Taken together, these data indicate that PDGFRα is a key marker for adult human BM MSC which enables to prospectively isolate a close to pure population of candidate stroma stem/progenitor cells. These results will enable to better characterize this important cellular component of the hematopoietic microenvironment.
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نشانی اینترنتی http://celljournal.org/journal/article/abstract/311
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