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. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: Cell Stem Cell. 2019 May 16;25(2):210–224.e6. doi: 10.1016/j.stem.2019.04.015

Figure 6. Combination of Calcitonin and GSK3i (CTCH) in N2B27 Media Supports Self-Renewal of Wild-Type ESCs.

Figure 6.

(A) DIC images displaying the morphology and density of wild-type and Atp2b1−/− Tcf7l1−/− double-mutant ESCs in 2iL, N2B27 alone, or CTCH media for indicated number of passages (P) and days (d). Scale bar represents 200 mm. See also Figures S6F and S6G.

(B) (Left) DIC and epifluorescence images (from Rex1-GFPd2) of wild-type cells in CTCH media for 26 passages. (Right) Flow cytometry analysis of the naive state with Rex1-GFPd2 of wild-type cells in CTCH media (green) or 2iL (red) for 26 passages. n = 2 biological replicates of >10,000 live-cell, singlet events were acquired for each sample.

(C) Intracellular free Ca2+ levels are localized in wild-type or Atp2b1−/− cells transfected with the genetically encoded calcium indicator, GCaMP5G. Transfected cells are treated with 2iL, GSK3i only, calcitonin (CT) only, or combinations thereof. Scale bar represents 14 μm.

(D) The relative mRNA expression measured by qRT-PCR (ΔΔCt-method) of naive, formative, and primed state marker genes in wild-type cells maintained in CTCH media. Data represent mean ± SD for 3 biological replicates. Gapdh expression is used as a loading control. * represents FDR < 5% for indicated comparisons.

(E) The relative mRNA expression measured by qRT-PCR (ΔΔCt-method) of pluripotency genes and germ layer (ectoderm, mesoderm, and endoderm) marker genes during embryoid body differentiation from wild-type cells previously maintained in either 2iL or CTCH media. Data represent individual measurements from biological duplicates. See also Figure S7.