Figure 4. Optimized culture conditions are required to generate and maintain MP cells.
(A) Human ES cells were treated with CHIR98014 (CHR) for 24 hr. After 48 hr, cells were cultured on either Matrigel or the optimal matrix (C1 C3 C4 FN VN) in the absence (no factor) or in the presence of the optimal GF/SM combination (CHR + FGF). Only cells cultured with CHR + FGF could be serially passaged. (B) QPCR analysis for mesodermal markers MESP1, MIXL1, and LHX1. Conditions containing no factor did not grow beyond passage 1, while the CHIR + FGF samples represent expression at passage 3. NF = no factor; C + F = CHR + FGF. Statistical comparisons are made to C1 C3 C4 FN VN with CHR + FGF condition. *p < 0.05, **p < 0.005. (C) MP cells were expanded to p6 on the optimal ECMP (C1 C3 C4 FN VN) and GF/SM combination (CHR + FGF). MP cells were then either transitioned to Matrigel or maintained on C1 C3 C4 FN VN in the absence or presence of CHR + FGF. (D) QPCR analysis for mesodermal markers MESP1, MIXL1, and LHX1. Conditions containing no factor did not grow past p7, while the CHR + FGF sample represents expression at p9. All statistical comparisons are made to the C1 C3 C4 FN VN with CHR + FGF condition. *p < 0.05, **p < 0.005.