Human ES Cell Culture Conditions Fail to Preserve the Mouse Epiblast State

Figure 4

Continuous exposure to the hESC/EpiSC condition draws the PSCs towards posterior mesoderm and endoderm lineages. (a) qRT-PCR analysis of the indicated lineage-specific genes in the indicated samples. Gapdh and Pmm2: housekeeping genes. The endoderm (Gata6 and Sox17) and posterior mesoderm (Tbx6 and Msgn1) markers were induced by 6 days. The neuroectoderm (Pax6 and Sox1), lateral mesoderm (Foxf1), and intermediate mesoderm (Osr1) genes were not expressed. (b) Western blot analysis of the mesoderm (Msgn1 and Tbx6) and endoderm (Sox17) proteins in the cells treated with AA/F2 for the indicated durations. The posterior mesoderm (Msgn1 and Tbx6) and endoderm (Sox17) proteins were expressed by 6 days of the treatment. (c) Flow-cytometric analysis of the fraction of Sox2+ve and T+ve cells after 3 and 6 days of AA/F2 treatment. Error bars: (); and ( and ) (Student’s -test). By 6 days of treatment, the count of Sox2-expressing cells was very low, and almost half the proportion of the cells expressed the PS marker, T. (d) Analysis of the indicated genes in the FACS-sorted T+ve cells from the AA/F2-6D-treated cells. The T-expressing cells expressed early markers of PS, mesoderm, and endoderm. (e) A model that summarizes the major finding of this study. The mESCs were converted to naïve state of pluripotency (represent the pluripotent embryonic cells in the E3.5-E4.5 developmental stages) using the 2i medium (LIF/2i). AA/F2 treatment for 2 to 3 days led these cells to a primed state of pluripotency (may represent the pluripotent epiblast cells in the E5.5-E7.5 developmental stages). This study shows that the continued treatment of AA/F2 for 6 days led to the enrichment of cells expressing the genes of the primitive streak, mesoderm (primarily, posterior mesoderm), and endoderm (may represent the pluripotent epiblast cells and PS/mesendoderm cells in the E6.5-E7.5 developmental stages).

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