Figure 4.. SIMR iPSCs Produce MSCs with…
Figure 4.. SIMR iPSCs Produce MSCs with Trilineage Differentiation Potential
(A) Flow cytometry of MSC…
Figure 4.. SIMR iPSCs Produce MSCs with Trilineage Differentiation Potential(A) Flow cytometry of MSC biomarkers CD73, CD90, and CD105, and a cocktail of negative MSC biomarkers. Immunostained samples are indicated in color, with isotype negative controls in gray. Data for BJ-MSC control are the same data as in Figure S5A. Representative clones for native BJ-MSCs and BJ ρ0+PBMC1-MSCs are indicated. (B) Bright-field microscopy showing unmanipulated BJ-MSC and BJ ρ0+PBMC1-MSC clones 1, 2, and 11 adhering to plastic at 203 magnification (scale bars, 100 mm). Data for BJ-MSC control are the same data as in Figure S5B. (C) OCR measurements for ~1.5 × 104 native BJ-MSCs and BJ ρ0+PBMC1-MSC clones 1, 2, and 11. Data for BJ-MSC control are the same as in Figure S5C. Data are the means ± SD of three technical replicates. Statistical significance by unpaired, two-tailed Student’s t test. *p ≨ 0.05; **p ≨ 0.01. (D) Quantitative phase microscopy of native BJ-MSCs and a 1:1:1 mix of BJ ρ0+PBMC1-MSC clones 1, 2, and 11. Data for BJ-MSC control are the same as in Figure S5D. Shown are box-and-whisker Tukey plots with outliers identified. Data were averaged from 77 and 172 cells for native BJ-MSCs and BJ ρ0+PBMC1- MSCs, respectively. Statistical significance by Welch’s t test. (E) T cells were added into native BJ-MSC or BJ ρ0+PBMC1-MSC clone 1, 2, or 11 cultures at 1:2, 1:1, 5:1, and 10:1 T cell:MSC ratios. After 5 days of co-culture, T cell proliferation was measured using a CFSE dye dilution assay by flow cytometry. The data labeled “NS” (no stimulus) denote T cells without CD3/CD28 bead activation. The data labeled “-ve” (negative) denote no addition of MSCs to stimulated T cells. The data labeled “+ve” (positive) denote a 1:1 addition of myeloid-derived suppressor cells to T cells. Data are the means ± SD of three technical replicates. (F) Trilineage differentiation of native BJ-MSCs and BJ ρ0+PBMC1-MSC clones 1, 2, and 11. Representative sections were fixed and stained with 1 μM Bodipy 493/503, 1% alizarin red S, and 0.1% Safranin O, respectively. Shown are adipocytes (first row, 20×; scale bars, 100 μm), osteocytes (second row, 20×; scale bars, 200 μm), and chondrocytes (fourth row, 5×; scale bars, 500 μm). See also Figure S1.
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