Figure 2
A top-down chemical screening to find the most efficient combination of signaling factors for self-renewal of CMCs under adherent culture conditions. (a–d) The expansion fold and percentage of MESP1+ cells were evaluated as two preliminary criteria to find the minimal essential pool of signaling molecules. In each run, the effect of different combinations of n-1 signaling factors in culture media was assessed according to these criteria. Serial removal of factors resulted in the selection of 7-IV (5), 5-N (4), 4-P (3) and 3-A (2). “–”: Indicates withdrawal of signaling factors. A: A83-01; B: bFGF; C: CHIR; I: IGF1; N: NRG1 agonist; P: Pioglitazone; V: Vitamin C; P1: passage 1; P2: passage 2. (e) The doubling time of CMCs in culture media that contained selected combinations of factors. The 3 and 4 combinations of signaling chemicals had the least doubling time of the CMCs. (f) The total cell count of CMCs after three passages in media that contained the selected factors. As presented, CMCs had the highest cell count in media that contained the 3 and 4 combinations of chemicals. All data: mean ± SD and statistically analyzed by one-way ANOVA followed by Tukey’s post-hoc test. *P ≤ 0.05; **P ≤ 0.01; #Number.
