Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells

Figure 1

In vitro characteristics of NOD-derived MSCs with cell culture expansion. (a) FACS analysis and enrichment of NOD-derived MSCs. Following culture for two passages, NOD bone marrow stromal cells were stained with nil antibody (unstained), CD45 mAb conjugated to fluorochrome APC (CD45-APC), Ly6 MAb conjugated to fluorochrome PE (Ly6-PE), and both mAbs (CD45-APC/Ly6-PE). Fluorescence dot plots of CD45-APC (-axis) and Ly6-PE (-axis) were used to identify the MSC (CD45^-/Ly6⁺; orange) and double-positive (CD45⁺/Ly6⁺; red) cell subpopulations ready for cell sorting using the BD FACSAria™ II instrument. Representative of three individual FACS experiments. (b) Plastic adherence, fibroblast-like morphology, and self-renewal without differentiation into other cell types. MSCs maintained fibroblast-like morphology as assessed using light microscopy (Leica DM microscope; 10x magnification; scale bar = 100 μM). (c) Improved cell proliferation with culture expansion. Data are presented as mean viable cells ± SDs (). A two-way ANOVA with Tukey’s posttests was performed, . (d) Improved fibroblastic colony formation following methylene blue staining. Data are presented as mean number of colonies ± SEMs (). A one-way ANOVA and Tukey’s posttests were performed, . (e) Semiquantitative analysis of adipogenic differentiation under defined conditions. NOD-derived MSCs maintained fat formation following Oil Red O staining, albeit at reduced levels, with increasing passage number. Data are presented as mean cell count/cm² ± SEM (). A two-way ANOVA and Tukey’s posttests were performed, . (f) Semiquantitative analysis of osteogenic differentiation under defined conditions. NOD-derived MSC maintained bone formation following Alizarin Red staining, albeit at lower levels, with increasing passage number. Data are presented as mean cell count/cm² SEM (). A two-way ANOVA and Tukey’s posttests were performed, . (g) Pancreatic transcription factor and hormone and protein expression levels were determined by RT-PCR. MSCs did not express any transcription factors, hormones, or protein found in the pancreas. Positive control mouse pancreas (lane 1), plastic-adherent MSCs (lane 2), plastic-adherent hematopoietic cells (lane 3), adherent bone marrow cells (lane 4), and negative control mouse liver (lane 5).

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