101 Massive Expansion and Cryopreservation of Beating Human iPSC-derived Cardiomyocytes 4 64. Wash the cells with 2.5 ml of flow cytometry buffer and centrifuge at 200xg for 5 min at 4°C; discard the supernatant and repeat the wash two more times. 65. Resuspend in 50 μl of flow cytometry buffer containing the secondary-antibody Goatanti-mouse (1:300 dilution). 66. Analyse the cells with a flow cytometer, adjusting the gates according to the standard gating strategy as shown in Figure 4. CELL LINE CTR. 1 N = 9 CTR. 2 N = 4 CTR. 3 N = 4 AVERAGE N = 25 ΑACTININ-POSITIVE CELLS P0 (%) 84 ± 12 88 ± 14 93 ± 8 89 ± 12 ΑACTININ-POSITIVE CELLS P5 (%) 88 ± 10 92 ± 1 91 ± 5 91 ± 5 %-INCREASE OF CMS CELL NUMBER 2229 ± 1070 1129 ± 203 1303 ± 49 1343 ± 374 Alpha-actinin SSC Pure sample Unpure sample Isotype control Negative control A B CELL LINE CTR. 1 N = 9 CTR. 2 N = 4 CTR. 3 N = 4 AVERAGE N = 25 ΑACTININ-POSITIVE CELLS P0 (%) 84 ± 12 88 ± 14 93 ± 8 89 ± 12 ΑACTININ-POSITIVE CELLS P5 (%) 88 ± 10 92 ± 1 91 ± 5 91 ± 5 %-INCREASE OF CMS CELL NUMBER 2229 ± 1070 1129 ± 203 1303 ± 49 1343 ± 374 P1 CTR. N = 9 0 (%) 84 ± 1 5 (%) 88 ± 1 2229 ± 1 FOLD INCREASE CHIR (µM) 0 2 4 6 0 20 40 60 CHIR990021 (M) 0 2 4 6 0 20 40 60 %ki67+ % Ki67 + Ki-67 Ki-67 + Alpha-actinin C D 0.11% 0.07% 45.6% 92.2% Figure 4. Differentiation efficiency and expansion capacity in different control hiPSC lines. (A) Quantitative analysis of small molecule-based Wnt-modulated directed cardiac differentiation at the passage (P) number P1 and P5. Replicate numbers refer to the number of performed expansion experiments from individual differentiation batches. The percentages of increase in hiPSC-CMs are relative to the input of day 11 hiPSC-CMs. Mean values ± s.d. are given. (B) Representative gating strategy for α-actinin positive hiPSC-CMs in a pure population of hiPSC-CMs versus negative control, isotype control, and an impure or un-purified hiPSC-CM culture. The number of α-actinin positive analyzed cells is 25 × 105. SSC – side scatter. (C) Representative immunofluorescence for proliferation assessed by Ki67 expression after 48 hours of CHIR treatment. Immunofluorescence: Hoechst (blue), Ki67 (red), and α-actinin (green). Scale bar: 200 μm. (D) Quantitative graph indicates high proliferation (37%) of cardiomyocytes stimulated with the optimal dose of 2 μM CHIR.
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