Recently, we developed a new protocol, called the subfractionation culturing method (SCM), to generate single-cell-derived clonal MSC lines from whole bone marrow aspirate without any centrifugation step for mononuclear cells or enzyme treatment process. Since a mononuclear cell fraction produced by gradient centrifugation or a culture-expanded population of MSCs was the usual source for the limiting dilution method, there is still a possibility of losing some “real” MSCs in the isolation of mononuclear cells and culture expansion of the cells. The limiting dilution method has been the only option to generate single-cell-derived clonal MSC lines. However, although significant improvements have been made in purification techniques, these isolation methods have not yet been sufficient to isolate completely homogeneous populations of MSCs. In order to obtain more homogeneous MSC populations, fluorescence-activated cell sorting (FACS) isolation and specific cell-surface antibody selection have been applied, improving the purity of the final MSC population. Experimental and clinical data over the last decade have indicated that cultured adherent MSCs are heterogeneous and exhibit variations in terms of their differentiation potentials and clinical outcomes. The conventional isolation method of MSCs relies on the fractionation of mononuclear cells by gradient centrifugation and selection of fibroblast-like cells adhering to the culture plate surface by removing nonadherent floating cells. In addition, more experimental data have become available about the immune modulation and hypoimmunogenic property of MSCs and there has been a surge of interest in both the basic biology and the potential clinical applications of these cells. Therefore, these cells are now also called multipotent stem/stromal cells since they are capable of differentiating into mesodermal, ectodermal and/or endodermal origin cells. Recently, there have been reports regarding additional potential areas for lineage differentiation, such as neurogenic, cardiogenic, myogenic and hepatogenic differentiation. The culture-expanded MSCs appear to have multilineage differentiation potential, including at least adipogenic, chondrogenic and osteogenic differentiation under appropriate induction conditions. MSCs can be readily obtained from humans and animals, isolated by means of their ability to adhere to plastic culture plates and can be expanded for many generations in culture. Mesenchymal stem cells (MSCs) were first isolated from the bone marrow by Friedenstein et al in the 1960s and they are now known to be present in a variety of tissues, including adipose tissue, umbilical cord blood and muscle. T-cell suppression analysis showed that 75% of the lines exhibited T-cell suppression capability.ĬONCLUSION: mcMSC lines have similar cell morphology and cell growth rate but exhibit variations in their cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability. Differentiation assays showed that 90% of the mcMSC lines were capable of differentiating into adipogenic and/or chondrogenic lineages, but only 20% showed osteogenic lineage differentiation. However, some lines exhibited different expression levels in a few epitopes, such as CD44 and CD105. Cell surface epitope profiles of these mcMSC lines were similar to those of nonclonal MSCs. Lines differed in optimal growth density requirement. RESULTS: All mcMSC lines showed typical nonclonal MSC-like spindle shape morphology. Nonclonal MSCs isolated by the conventional gradient centrifugation method were used as controls. These lines were characterized by measuring cell growth, cell surface epitopes, differentiation potential, lineage-specific gene expression and T-cell suppression capability. METHODS: We established mcMSC lines using subfractionation culturing method from bone marrow samples obtained from long bones. AIM: To characterize single-cell-derived mouse clonal mesenchymal stem cells (mcMSCs) established with bone marrow samples from three different mouse strains.
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