Fig. 3.

Media supplements added to hiPSC colonies to promote differentiation of mesodermal lineage. a In comparison to culture in basal conditions, RT-PCR demonstrated higher mRNA levels for brachyury (T) at day 2 (D2), along with similarly high mRNA levels for the mesoderm markers MIXL1 and CDX2 (ANOVA, *p < 0.05). Higher levels in mRNA for the node/notochord markers FOXA2, SHH, and Noggin observed at 3–5 days (D3–D5) after induction of differentiation as compared to culture in basal conditions (*p < 0.05). b Notochord marker NOTO was not upregulated at any time following induction of differentiation with FGF2 and BMP4; however, addition of Wnt-3a and Activin A promoted an early (D2) and sustained elevation (D3–D5) in mRNA for NOTO (*p < 0.05). This observation was key in our choosing to supplement colony cultures with Wnt-3a and Activin A at the earliest time points, days 1–3 (D1–D3). bFGF2 basic fibroblast growth factor, BMP bone morphogenetic protein 4, MIXL1 paired-type homeobox transcription factor identified in human, CDX2 member of the caudal-related homeobox transcription factor family, FOXA forkhead box protein A2, SHH sonic hedgehog, NOG noggin, NOTO notochord homeobox