Single-Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells

Abstract

The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs), as well as during the extensive programming that occurs in human preimplantation development, is not well-understood. Indeed, studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here, we examine expression of the long noncoding RNA, XIST, in single cells of human embryos through the oocyte-to-embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4-cell stage, coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally, we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however, expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells.

Keywords: Human; Human induced pluripotent stem cells; Preimplantation embryo development; X chromosome inactivation; XIST RNA.

Figure 1

XIST expression in whole embryos and single cells during human preimplantation development. (A): Schematic of XIST and housekeeping gene, CTNNB1, qRT‐PCR on whole and single or grouped blastomeres of preimplantation human embryos. (B): Log2Ex values for XIST (red) and CTNNB1 (black) from single preimplantation human embryos from the 1‐cell to blastocyst stage. (C): Table of XIST and CTNNB1 expression in single or grouped blastomeres from five stages of preimplantation development (2–3 cell, 4–5 cell, 6–7 cell, 8–10 cell, and morula–blastocyst stages). Schematic demonstrates XIST+ blastomeres (red), XIST− blastomeres (light grey), and nonviable blastomeres, CTNNB1− (dark grey). Abbreviation: qRT‐PCR, quantitative real‐time polymerase chain reaction.

Figure 2

mRNA reprogramming produces fully reprogrammed colonies as early as 11 days post‐reprogramming initiation. (A): mRNA reprogramming protocol used to reprogram fetal and adult fibroblasts into iPSCs. The reprogramming process was only successful when supplemented with miRNAs on days 1 and 5. Images show progression of cell morphology and the appearance of colonies. Magnification 5×. (B): Normalized single cell expression values for housekeeping and pluripotency markers compared to an ESC control, showing that both iPSC lines are indeed pluripotent. Abbreviation: iPSC, induced pluripotent stem cell.

Figure 3

XIST expression in single cell newly reprogrammed female iPSCs. (A): Schematic for isolating pluripotent cells from a heterogeneous reprogramming well. Following successful reprogramming, some cells were manually passaged and analyzed at later passages, while the remainder were fluorescence‐activated cell‐sorted and analyzed by quantitative real‐time polymerase chain reaction for single‐cell gene expression. (B): Normalized log2Ex values for newly reprogrammed fetal and adult iPSCs. (C): Percentage of single cells expressing XIST. Loss of XIST is first significant, relative to the parental fibroblasts, at passage 20 in fetal cells and at passage 3 in adult cells (only in 5% O₂) using Fisher's test: **, p < .01; ***, p < .001. (D): RNA fluorescence in situ hybridization for XIST in female fetal iPSCs. Abbreviation: iPSCs, induced pluripotent stem cells.

Figure 4

Fibroblasts maintain XIST expression in various passage and culture conditions. (A): Schematic for isolating fibroblasts after continuous culture. Some cells were passaged and analyzed at later passages. All cells were fluorescence‐activated cell‐sorted for double negative populations and analyzed using quantitative real‐time polymerase chain reaction for single‐cell gene expression. (B): Normalized log2Ex values for single female fibroblasts. All fibroblasts maintain XIST expression regardless of length of time in culture. (C): Percentage of single fibroblasts expressing XIST. No significant difference in percentage of XIST positive cells was detected in fibroblasts. Fisher test was used to determine whether groups were statistically different. (D): RNA fluorescence in situ hybridization for XIST in female fetal fibroblasts. Abbreviation: DAPI, 4′,6‐diamidino‐2‐phenylindole.

Figure 5

X‐linked gene expression varies with XIST expression. Density plot for each of four X‐linked genes from fetal and adult iPSCs were plotted with XIST+ populations (light red) and XIST‐ populations (black) separated. In adult iPSCs, there is an increase in the overall mean of silenced genes in the XIST− population (A) while the difference in means of pseudoautosomal genes is almost zero (B). However, in fetal iPSCs, ATRX does not behave as expected (A) and there is a significant difference in one of the pseudoautosomal genes, CD99 (B). p values for the differences in means was calculated using a t test (C). Abbreviation: iPSCs, induced pluripotent stem cells.