Fig. 5: Derivation of highly proliferative bipotent hTSL cells from C19MC-reactivated primed hES cells.

a Phase-contrast images of hTSL cells obtained from C19MC-reactivated primed hES cells (hTSL^C19MC cells). We also analyzed two clones isolated from these bulk hTSL^C19MC cells. hTSL^empty cells were derived from hES cells transfected with an empty gRNA expression vector and used for comparison. The scale bar indicates 300 μm. Representative data obtained from two independent experiments are shown for hTSL^empty (bulk) and hTSL^C19MC (bulk) cells. Two hTSL^C19MC clones were analyzed. See Supplementary Fig. 5a, b for detailed experimental design. b RNA-FISH of C19MC in hTSL^C19MC and hTSL^empty cells. C19MC expression was labeled with a Cy3-labeled oligonucleotide probe (shown in yellow). Nuclei were stained with Hoechst 33258 (shown in gray). The scale bar indicates 100 μm. c Percentage of the number of C19MC FISH signals per nucleus in hTSL^C19MC and hTSL^empty cells. Bulk hTSL^empty and hTSL^C19MC cells and two hTSL^C19MC clones were analyzed. 500–1000 nuclei were examined for each cell type. d Relative expression levels of C19MC miRNAs in hTSL^C19MC cells. Expression levels in hTS cells are set as 1. Two hTSL^C19MC clones were analyzed. Bar charts are shown as mean. e DNA methylation analysis of the C19MC DMR by bisulfite sequencing. Black and white circles indicate methylated and unmethylated CpGs, respectively. The methylation levels are shown on the right. f Growth curve of hTSL^C19MC clones and hTSL^empty cells. Genetically unmodified hTS cells were also analyzed for comparison. Cells were cultured for 12 days. g Phase contrast and immunofluorescence images of EVT-like cells derived from hTSL^C19MC and hTSL^empty cells. Cells were stained for HLA-G. Nuclei were stained with Hoechst 33,258. The scale bar indicates 100 μm. Representative data obtained from two independent experiments are shown. Similar results were obtained for hTSL^C19MC clones 1 and 2. h Flow cytometry analysis of HLA-G expression on EVT-like cells derived from hTSL^C19MC clones. EVT-like cells derived from genetically unmodified hTS cells were analyzed as a positive control. i Phase contrast and immunofluorescence images of ST-like cells derived from hTSL^C19MC and hTSL^empty cells. Cells were stained for SDC1 and CGB. Nuclei were stained with Hoechst 33,258. The scale bar indicates 100 μm. Representative data obtained from two independent experiments are shown. Similar results were obtained for hTSL^C19MC clones 1 and 2. j Fusion efficiency of ST-like cells derived from hTSL^C19MC clones and hTSL^empty cells. k Levels of hCG secreted by ST-like cells derived from hTSL^C19MC clones and hTSL^empty cells.