Fig. 7: Cells in PRIMO medium can be maintained in a mesoderm biased state and revert back to an unbiased state.

Flow Cytometry analysis of cells grown in PRIMO Plus for three passages. a Density plot of MIXL1-GFP vs SSEA-3 for HES3 MIXL1 b Density plot of T-Venus vs SSEA-3 for H9 T-Venus c. Histogram of SSEA-3 expression for MIFF1. d Algorithm score for EBs generated from HES3-MIXL1, H9 T-Venus and MIFF1 (iPS line) grown in PRIMO medium for three passages. All lines exhibited strong mesoderm signatures under neutral conditions (n = 1 biological repeat from three separate lines). e Principal component analysis of cells grown in PRIMO for 3 days (purple stars) or ten passages (purple triangles) compared to the differentiation time course (grey squares). Also shown are cells, which had been grown in PRIMO for nine passages and then transitioned back into E8 conditions (red and orange triangles). Cells maintained in PRIMO cultures are close to 18–24 h of differentiation and reverted cultures close to 0 h (E8 only) cultures (white circles). f Heatmap analysis of key genes related to Pluripotency and mesendoderm differentiation generated with Log2 counts that have been mean centred per gene with a colour scale from −2 to 2. g Lineage proportion graphs from neutral EBS generated from HES3-MIXL1 and H9 T-Venus after three passages in PRIMO Plus and subsequent reversion into E8 medium. The strong mesoderm bias was alleviated upon transition back into standard conditions. b, d, e Source data are provided as a Source Data file.