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. 2015 May 15;4(7):731–742. doi: 10.5966/sctm.2014-0270

Figure 3.

Figure 3.

Expansion and characterization of hESCs on synthetic microcarriers in stirred culture conditions. (A): Representative images from cell-viability assays (fluorescein diacetate, live cells in green; propidium iodide, dead cells in red) of hESC-C and hESC-M cells on microcarriers at the last day of culture. Scale bars = 100 μm. (B): Growth curves of hESC-C and hESC-M cells in spinner vessels on SP microcarriers. (C): Percentage of cell recovery after the harvesting process, using TrypLE Select. (D): Immunofluorescence images of Oct-4 and TRA-1-60 labeling of hESC-M cells on SH microcarriers, and phase contrast images of alkaline phosphatase activity staining of hESC-M cells on SP microcarriers. Scale bars = 100 μm. (E): Flow cytometry analysis: percentages of TRA-1-60-, SSEA-4-, hES-Cellect-, and SSEA-1-positive cells at day 0 and at the last day of culture on SP microcarriers. Error bar denotes SD of 2 measurements. (F): Gene expression of stemness markers Oct-4 and Nanog relative to day 0 of both hESC-C and hESC-M cells cultured on SP microcarriers and 2D culture. Gene expression was quantified using the ΔΔCt method (housekeeping:RPL22; as described elsewhere [29]). The primers' sequences are listed in supplemental online Table 2. Error bar denotes SD of 3 replicates. (G): Images of embryoid bodies of both hESCs (after expansion on SP microcarriers) and the differentiated cultures labeled for α-SMA (mesoderm), FOXA2 (endoderm), and β-III tubulin (ectoderm). Nuclei were stained with DAPI (blue). Scale bars = 100 μm. (H): Hierarchical clustering given by Pearson correlation method implemented in Spotfire of initial and final samples for both hESC lines in the 2D culture system (feeder layer for hESC-C and Coat-1 for hESC-M) and on the Synthemax synthetic surface. ∗∗∗, p < .001 determined by two-way analysis of variance. Abbreviations: 2D, two-dimensional culture; α-SMA, α-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; FOXA2, forkhead box A2; hESC, human embryonic stem cell; SH, Synthemax II hydrogel; SP, Synthemax II polystyrene.