Figure 5.

XCI reacquisition restores the differentiation ability of female hPSCs

(A) Experimental schemes for evaluating the differentiation potential by XIST repression. XIST re-expressing hPSC lines and their parental lines without XIST expression were used. Immuno-FISH (for MN) or immunofluorescence (for cortical organoid) analysis was used for the evaluation.

(B and C) The efficiency of MN differentiation from XIST re-expressing hPSC lines. The representative images (B) and bar graphs are quantification results for ISL1+ cells (C). Each dot and n.c. indicate the percentage in the observed area and number of cells analyzed in total, respectively. Scale bar represents 50 μm. Error bars show the SD. p values were calculated using Student’s t test.

(D and E) The efficiency of neuronal differentiation from Rett-iPSC:XIST^re-on. The representative images (D) and bar graphs are quantification results of TUJ-1 or ISL1+ cells (E). Each dot and n.c. indicate the percentage in the observed area and number of cells analyzed in total, respectively. Scale bar represents 50 μm. Error bars show the SD. p values were calculated using Student’s t test.

(F) Summary of female hPSC erosion and de-erosion. XIST is irreversibly repressed by DNMT3A/3B (Fukuda et al., 2021), leading to XCI erosion. XCI erosion affects various phenotypes, not only X-linked gene dysregulation but also the autosomal gene functional assay. XIST is reactivated by the editing of XIST promoter regions, resulting in the generation of de-eroded hPSCs by natural selection under normal culture conditions.