Modeling X chromosome inactivation using t5iLA naive human pluripotent stem cells

Abstract

Background: X chromosome inactivation (XCI) is a critical epigenetic event for dosage compensation of X-linked genes in female mammals, ensuring developmental stability. A robust in vitro model is required for mimicking XCI during the early stages of embryonic development. This methodology article introduces an advanced framework for the in-depth study of XCI using human pluripotent stem cells (hPSCs). By focusing on the transition between naive and primed pluripotent states, we highlight the role of long non-coding RNA X-inactive specific transcript (XIST) and epigenetic alterations in mediating XCI.

Results: Our methodology enables the distinction between naive and primed hESCs based on XIST expression and the activity of X-linked reporters, facilitating the investigation of XCI initiation and maintenance. Through detailed experimental procedures, we demonstrate the utility of our hESC lines in modeling the process of human XCI, including the establishment of conditions for random XCI induction and the analysis of X chromosome reactivation.

Methods: The study outlines a comprehensive approach for characterizing the X chromosome status in hPSCs, employing dual fluorescent reporter hESC lines. These reporter lines enable real-time tracking of XCI dynamics through differentiation processes. We detailed protocols for the induction of X chromosome reactivation and inactivation, as well as the X status characterization methods including cultivation of hESCs, flow cytometric analysis, RNA fluorescence in situ hybridization (FISH), and transcriptome sequencing, providing a step-by-step guide for researchers to investigate XCI mechanisms in vitro.

Conclusions: This article provides a detailed, reproducible methodology for studying XCI mechanisms in vitro, employing hPSCs as a model system. It presents a significant advance in our ability to investigate XCI, offering potential applications in developmental biology, disease modeling, and regenerative medicine. By facilitating the study of XCI dynamics, this methodological framework paves the way for deeper understanding and manipulation of this fundamental biological process.

Keywords: Early embryogenesis; Human embryonic stem cells; X chromosome inactivation.

Fig. 1

Characterization of X chromosome status in initial primed hESCs. A strategy for generating WIBR3^MGT and WIBR2^MGT primed hESCs carrying a dual X-reporter system using TALEN-mediated gene editing. B Phase contrast and fluorescence images of WIBR3 WT (top panels) and post- (middle panels) and pre-XCI (bottom panels) WIBR3^MGT primed hESCs. Scale bar indicates 100 μm. C Flow cytometric analysis of the GFP and tdTomato expression in WIBR3^MGT primed hESCs. D Representative images of WIBR3^MGT primed hESCs maintained in E8 medium, analyzed by RNA-FISH to detect XIST (red) and ATRX (yellow) transcripts and by immunofluorescence co-staining of H3K27Me3 (green). Scale bar indicates 10 μm. E Upper panel: position of 4 predefined X-linked SNPs in WIBR3 [40]. Different colors represent different bases. Lower panel: sequencing peaks of 4 random SNPs in cDNA extracted from WIBR3^MGT cells. Panels A and E of this figure is adapted, with permission, from Ref.30© (2020) Cell Press

Fig. 2

Characterization of X Status of t5iLA naive hESCs. A Phase contrast and fluorescence images of WIBR3^MGT HT and LT t5iLA and WT naive hESCs. B flow cytometric analysis of the GFP and tdTomato expression at P10 of WIBR3^MGT t5iLA resetting. C Representive images of WIBR3 HT and LT t5iLA naive hESCs. Scale bar indicates 100 μm. D Left panel: representative RNA-FISH images of WIBR3^MGT t5iLA HT and LT naive hESCs, detecting XACT (green), XIST (red), and ATRX (yellow) transcripts. Right panel: cartoons of the HT and LT naive hESCs having different RNA FISH patterns. E PCA analysis of RNA-seq data from WIBR3^MGT primed hESCs, LT and HT naive hESCs derived from WIBR3^MGT or WIBR3. Every single dot represents one sample in each cell line. F Heatmap of allelic expression of X-linked genes in WIBR3 and WIBR3^MGT naive and primed hESCs, based on reads covering X-linked SNPs

Fig. 3

Inducing XCI in t5iLA naive hESCs through teratoma formation in vivo. A Upper panel: representative image of the mouse carring terotoma (red circled). Lower panel: representative images of WIBR3^MGT HT naive hESCs teratoma shows differentiation of three germ layers by immunohistochemistry. Red arrows point respiratory epithelial tissue in endoderm, cartilage tissue in mesoderm and neuroepithelial tissue in ectoderm. B Histogram showing the single GFP (SG, in green), single tdTomato (ST, in red) and double positive (TG, in yellow) populations of teratoma cells differentiated from WIBR3^MGT HT naive hESCs. C Representative images of the teratoma cells, analyzed by RNA-FISH to detect XIST (red) and ATRX (yellow) expression with immunofluorescence co-staining of H3K27Me3 (green). Scale bar indicates 30 μm

Fig. 4

Inducing XCI in t5iLA naive hESCs through re-priming in vitro. A Phase contrast and fluorescence images of repriming WIBR3^MGT HT naive hESCs at day 5. B Histograms showing the distribution of single GFP (SG, in green), single tdTomato (ST, in red), and double positive (TG, in yellow) hESCs re-primed from WIBR3^MGT 5iLA HT naive hESCs, by flow cytometric analysis. C Examples of WIBR3^MGT re-primed hESCs analyzed by RNA-FISH to detect XIST (red), XACT (green), and ATRX (yellow) expression. Scale bar indicates 10 μm. D Heatmap of allelic expressing X-linked SNPs in above samples, based on reads covering indicated SNPs

Fig. 5

Random XCI through embryoid body differentiation into fibroblasts. A Representative images of EBs (left panel) and fibroblasts (right panel) derived from pre-XCI WIBR3^MGT re-primed hESCs. Scale bar indicates 100 μm. B Flow cytometric analysis showing the distribution of GFP and tdTomato-positive fibroblasts derived from pre-XCI WIBR3^MGT re-primed hESCs through EB differentiation. Histograms reporting the proportions of single GFP (SG, in green), single tdTomato (ST, in red) and double positive (TG, in orange) cells. C Using differentiated fibroblasts in B, expression of XIST (red) and H3K27Me3 enrichment (green) are analyzed by RNA-FISH and immunofluorescence co-staining. D Heatmap of allelic expressing X-linked SNPs in SG and ST fibroblasts, based on reads covering indicated SNPs

Fig. 6

Workflow of human XCI research in vitro