Extended Data Figure 9. Characterization of alternative culture conditions for ESCs.

(a) Representative images of XY ESCs cultured in the indicated conditions.

(b) Heatmap and dendrogram of global gene expression levels in male ESCs cultured in the indicated conditions.

(c) Principal component analysis of ESCs cultured in S/L, 2i/L, a2i/L or PKCi/L using a previously published set of known pluripotency and differentiation genes²². Two male ESC lines maintained in S/L for 4 passages were switched to each condition and cultured for additional 6 passages (final passage 10).

(d) Heatmap and dendrogram showing expression levels of pluripotency and developmental genes²² in male ESC lines cultured in the indicated conditions.

(e) Relative expression levels of transcripts associated with naïve pluripotency in (d).

(f) Violin plots of global methylation levels in the indicated ESC lines. White dots, median value.

(g) Heatmap and dendrogram of ICR methylation levels in the indicated ESCs lines.

(h) Flow-cytometric analysis of GFP and TdTomato after culturing X^GX^T ESCs in S/L, S/L+PD and a2i/L for the indicated passages.

(i) Agouti germline offspring (red arrows), obtained from across between an all-ESC male generated from p10 a2i/L-cultured ESCs and a C57B6 female.

(j–k) Karyotype analysis of male ESCs cultured in a2i/L for 6 or 16 passages (j), or PKCi/L for 6 or 16 passages (k). Red boxes indicate abnormal chromosomes detected.