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. 2022 May 19;17(6):1303–1317. doi: 10.1016/j.stemcr.2022.04.013

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© 2022 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Characterization of hPSC-derived TSCs

(A) Principal-component analysis of hPSCs (undifferentiated/day 0), H9 hESCs treated with BMP4/IWP2 for 1–4 days, hPSC-derived TSCs after 6–8 passages in the modified Okae TSC medium (TSC), and primary (placenta-derived) TSCs (lines 1048 and 1049). Each dot on the principal-component analysis (PCA) represents a sample from an independent experiment (n = 2 for H1 and iPSCs, day 0 and TSC; n = 3 for all other samples). Sample clusters from k-means clustering, marked by circles, show that hPSC-derived TSCs cluster together with primary (placenta-derived) hTSCs.

(B) Heatmaps of undifferentiated hPSCs and primary and hPSC-derived TSCs showing genes that are either upregulated (839 genes) or downregulated (779 genes) in primary TSCs compared with undifferentiated hPSCs. GSEA shows that hPSC-derived TSCs were enriched in primary TSC-associated genes (NES 2.44, padj < 0.004) but not in (undifferentiated) hPSC-associated genes. A few TSC-associated genes are noted in the heatmap.

(C) qPCR of the indicated CTB markers in undifferentiated primary TSC (1049) compared with hPSC (H9)-TSCs. Data were normalized to L19 and shown as fold change over undifferentiated 1049 (D0 = day 0) and represent mean ± SD for n = 3 independent experiments. ^∗p < 0.05; ^∗∗p < 0.01 by Student’s t test.

(D) DNA methylation surrounding the ELF5 promoter in undifferentiated (U) H9 ESCs, TSCs derived from both H9 and a human dermal fibroblast (HDF)-derived iPSCs, an umbilical-cord-derived mesenchymal stem cell line (1754), and a primary (placenta-derived) TSC line (1049). Each line represents a distinct sequenced clone (n = 3 to 9).

(E) Flow-cytometric analysis of primary (1049) and hPSC (H9)-derived TSCs for HLA-A2 and HLA-Bw6 with (gray) and without (purple) valproic acid (VPA) in the culture medium. Data are representative of 2 independent experiments.

(F) Heatmap of amnion-specific markers (based on a more stringent analysis by Seetheram et al. in this issue of Stem Cell Reports of the Roost et al., 2015 dataset) in undifferentiated hPSCs and primary (1048 and 1049 TSC) and hPSC-derived TSCs, as well as amnion (AM). GSEA showed that neither primary nor hPSC-derived TSCs were enriched in amnion-specific genes (NES = -0.94 with padj = 0.561 and NES = -1.06 with padj = 0.354, respectively).

See also Figure S2.