Fig. 3: Balancing pro-self-renewal and pro-differentiation signals enables propagation of MIXL1-GFP/SSEA-3 cells.
From: Generation and trapping of a mesoderm biased state of human pluripotency
Flow cytometry density plots of SSEA-3 versus MIXL1-GFP for HES3 MIXL1-GFP cells growing in E8 (a) and E8 with 3 µM CHIRON for 24 hours and 72 h (b). In E8 SSEA-3 is highly expressed but MIXL1-GFP is not detected. The addition of CHIRON drives MIXL1 expression and eventual loss of SSEA-3. c Flow cytometry density plots displaying SSEA-3 expression versus MIXL1-GFP expression for HES3 MIXL1-GFP cells grown in E8 medium with 3 µM CHIRON and increasing levels of LPA, higher levels of LPA prevent MIXL1-GFP expression. d Flow cytometry histograms of SSEA-3 expression for cells grown in 3 µM CHIRON + 0.48 µM LPA with IWP-2 (blue) or without IWP-2 (orange). Histograms are from 3 days in culture and 3 days post first passage. SSEA-3 was maintained post passage in samples cultured with IWP-2 present only. e A schematic diagram of the development of PRIMO medium. Cells grown under GSK3B inhibition supplemented with LPA and with or without IWP2 induce expression of MIXL1-GFP(+)/SSEA-3(+) cells after 3 days. Post passage without IWP-2 addition cells differentiate, losing SSEA-3, but with IWP-2 cultures maintained SSEA-3 and some MIXL1 expression. f After optimisation, the new formulation consisted of 3 µM CHIRON, 1 µM IWP-2 and 0.48 µM LPA. Density plots display the MIXL1/SSEA-3 expression under PRIMO revealing high double expression.
