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

Figure 4

Analysis of INS-FUR-, Neurod1-, and Pdx1-transduced MSC. (a) Fluorescence imaging of MSCs transduced with empty vector (EV), Neurod1, and INS-FUR. Transduced cells were returned to culture after sorting for GFP positivity and imaged for GFP expression (day 7 posttransduction) using a Leica DM fluorescence microscope (Leica Microsystems), 10x magnification with bright field and GFP fluorescence filter sets; scale bar = 100 μm. (b) Fluorescence imaging of MSCs and MSC-INS-FUR transduced with Pdx1. Transduced cells were returned to culture post sorting and imaged for GFP and mCherry expression (day 7 posttransduction) using a Leica DM microscope, 10x magnification under bright field, GFP and Texas Red fluorescence filter sets; scale bar = 100 μm. (c) Morphological characterization of MSCs posttransduction. Bright-field images were acquired at 28 days posttransduction on a Leica DM microscope (Leica Microsystems) at 10x magnification under bright-field setting; scale bar = 100 μm. (d) Gene expression profiling of INS-FUR and Neurod1-expressing MSC. Lane 1: untransduced MSC; lane 2: MSC-EV; lane 3: MSC-INS-FUR; lane 4: MSC-Neurod1; lane 5: MSC-INS-FUR/Neurod1, and lane 6: mouse pancreas (positive control). (e) Gene expression profiling of Pdx1-expressing MSCs. Lane 1: MSC-Pdx1; lane 2: MSC-INS-FUR/Pdx1; and lane 3: mouse pancreas (positive control). (f) Chronic insulin secretion from transduced MSCs. Human insulin was quantified using the ARCHITECT™ i4000SR Immunoassay Analyser (Abbott Diagnostics^©). Data are represented as means ± SDs (). A one-way ANOVA with Sidak’s posttests were performed, . (g) Acute glucose-stimulated insulin secretion from INS-FUR- and Neurod1-transduced MSCs. Human insulin was quantified using the ARCHITECT™ i4000SR Immunoassay Analyser (Abbott Diagnostics^©). Data were presented as means ± SDs (). Two-way ANOVA with Tukey’s posttests were performed, .

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