Inhibition of PRC2 enables self-renewal of blastoid-competent naive pluripotent stem cells from chimpanzee

Abstract

Naive pluripotent stem cells (PSCs) are counterparts of early epiblast in the mammalian embryo. Mouse and human naive PSCs differ in self-renewal requirements and extraembryonic lineage potency. Here, we investigated the generation of chimpanzee naive PSCs. Colonies generated by resetting or reprogramming failed to propagate. We discovered that self-renewal is enabled by inhibition of Polycomb repressive complex 2 (PRC2). Expanded cells show global transcriptome proximity to human naive PSCs and embryo pre-implantation epiblast, with shared expression of a subset of pluripotency transcription factors. Chimpanzee naive PSCs can transition to multilineage competence or can differentiate into trophectoderm and hypoblast, forming tri-lineage blastoids. They thus provide a higher primate comparative model for studying pluripotency and early embryogenesis. Genetic deletions confirm that PRC2 mediates growth arrest. Further, inhibition of PRC2 overcomes a roadblock to feeder-free propagation of human naive PSCs. Therefore, excess deposition of chromatin modification H3K27me3 is an unexpected barrier to naive PSC self-renewal.

Keywords: Polycomb; developmental drift; epiblast; higher primate; mammalian early embryo; naive pluripotency; pluripotent stem cells; self-renewal; single-cell transcriptomics; stem cell-based embryo model.

Graphical abstract

Figure 1. Generation and self-renewal of chimpanzee naive PSCs

(A) Morphology and SUSD2 live staining of resetting chimpanzee PSCs in PXGL and PXGL-A6E after exposure to VPA. Plot shows SUSD2-PE flow cytometry at P2. (B) Live-cell staining of primed and stabilized reset PSCs for SUSD2. (C) qRT-PCR analysis of pluripotent state markers in reset and primed PSC lines. SD from three biological replicates. (D) Immunostaining of primed PSCs and reset PSCs at P18. (E) SUSD2 staining of feeder-free reset PSCs at P8. (F) Phase image and SUSD2 live staining during capacitation. (G) Phase contrast images and flow cytometry analyses for SUSD2 and CD90 of primed, reset, and capacitated PSCs. (H) Somatic lineage differentiation of capacitated chimpanzee PSCs. (I) Immunostaining of H3K27me3 in primed and reset PSCs. Scale bar: 20 μm. (J) Immunostaining of H3K27me3 during capacitation scored for nuclei with an intense focus. (K) Genome-wide methylation distribution for CpGs with ≥5× coverage. (L) Promotor methylation comparison between naive and primed cells. Red indicates promoters with significantly higher methylation in naive cells. (M) Top 10 GO biological process terms for genes associated with promoters hypermethylated in naive cells. (N) Comparison between chimpanzee and human of genes with increased promoter methylation in naive PSCs. Human genes from Guo et al. Scale bars: 278.5 μm unless otherwise indicated. See also Figure S1.

Figure 2. Transcriptome identity and trophectoderm differentiation of chimpanzee naive PSCs

(A) Mapping of bulk RNA-seq samples for chimpanzee reset and primed PSCs onto UMAP embedding of human embryo scRNA-seq datasets. Left, embryo UMAP; center, projection of PSC samples; right, expanded ICM, hypoblast, and epiblast region. (B) Heatmap of correlation distance metrics between chimpanzee cell samples and human embryo cell types. Trophectoderm samples are from differentiation time points as in (G) below. (C) Heatmap of top 200 differentially expressed genes between naive and primed PSC samples in both chimpanzee and human. (D) Cluster map of pluripotency factor expression in human and chimpanzee naive or primed PSCs. (E) Immunostaining after treatment of naive PSCs with PD+A83 for 5 days. Scale bar: 278.5 μm. (F) qRT-PCR analysis at days 0, 3, and 5 of naive or primed PSCs differentiating in PD+A83 with or without BMP signal inhibition by LDN. SD from two biological replicates. (G) Projection onto the human embryo UMAP embedding of bulk RNA-seq samples from differentiation of two chimpanzee naive PSC lines in PD+A83 (four samples of each cell line). See also Figure S1 and Table S1.

Figure 3. Chimpanzee naive PSCs form blastoids and can be generated by direct reprogramming

(A) Phase images of day 4 blastoids from indicated lines. Scale bar: 139 μm. (B) Confocal images of blastoids immunostained for indicated lineage markers. Scale bar: 50 μm. (C) Numbers of hypoblast (FOXA2)- or epiblast (NANOG)-positive inner cells in individual day 4 blastoids. Double-negative cells may be trophoblast or nascent hypoblast. (D) UMAP with Leiden clustering of day 4 blastoid scRNA-seq data. (E) Projection of blastoid scRNA-seq samples on human embryo UMAP. (F) Comparison of blastoids and human embryo datasets by correlation distance metrics. Sample colors as in (D). (G) Morphology of naive iPSC colonies emerging during direct reprogramming and expansion after passage in indicated media. (H) SUSD2 flow cytometry of directly reprogrammed cells after 2 passages in indicated medium. (I) Immunostaining of KLF17 and SUSD2 in directly reprogrammed naive iPSCs at P10. Scale bar: 144 μm. (J) Projection onto the human embryo UMAP embedding of scRNA-seq data from chimpanzee naive PSCs cultured on MEF or feeder free. See also Figure S2.

Figure 4. Reduced activity of PRC2 sustains self-renewal of chimpanzee and human naive PSCs

(A) SUSD2 flow cytometry and images after transfer to indicated conditions for 2 or 3 passages (P). Scale bar: 278.5 μm. (B) Immunostaining for H3K27me3 and immunoblotting after culture in indicated concentrations of EPZ for 7 days. Scale bar: 278.5 μm. (C) Schematic of PRC2 knockouts and images of knockout colonies. (D) Immunoblotting analysis of knockout cells. (E) Immunostaining of H3K27me3 and EZH2 in EZH2-KO cells. Scale bar: 278.5 μm. (F) Schematic of cell competition experiment and images of co-cultures showing SUSD2 staining and GFP expression at P1, P3, and P5. (G) Flow cytometry detection of GFP in P1, P3, and P5 mixed cultures for each knockout. (H) Immunoblot of H3K27me3 in human naive PSC lines after three passages in indicated conditions. EPZ 0.2 μM. (I) Cell counts over sequential passages for feeder-free human naive iPSCs in PXGL with or without EPZ. SD from triplicate cultures. See also Figures S3 and S4.