Laminin111-based defined culture promoting self-renewing human pluripotent stem cells with properties of the early post-implantation epiblast

Abstract

In the mammalian embryo, a formative pluripotent phase is proposed to exist at the early post-implantation period, during the transition from the pre-implantation naive-to the post-implantation primed-epiblast. By recapitulating a laminin component of the extracellular matrix niche during embryonic formative transition, and defined culture conditions, we generated cultures highly enriched for self-renewing human pluripotent stem cells (hPSCs), exhibiting properties of early post-implantation epiblast cells. These hPSCs display post-implantation-epiblast gene expression profiles. FGF and TGF-β signaling maintain their self-renewal for multiple passages. They have inactive canonical Wnt signaling, do not express primitive streak markers, and are competent to initiate differentiation toward germline and somatic fates. hPSCs exhibiting early post-implantation epiblast properties may shed light on human embryonic PSCs development and may serve for initiating somatic and germ cell specification.

Keywords: canonical Wnt signaling; early post-implantation epiblast; formative pluripotency; germ cell specification; human pluripotency states; human pluripotent stem cells; laminin-111.

Figure 1

Laminin111-based defined culture conditions support prolonged self-renewal of genetically stable hPSCs, retaining their pluripotent potential (A) Phase-contrast image of HES-1-LN hPSC colony with a magnified section. Scale bar, 200 μm. (B) Karyotype analysis of HES-1-LN-hPSCs cultured for 10 passages. (C) Immunostaining of HES-1-LN-hPSCs differentiated in vitro into ectodermal (β-tubulin-III), mesodermal (muscle actin), and endodermal (FOXA2) cells. (D) Histological analysis of teratomas generated from HES-1-LN-hPSCs. Scale bars, 200 μm. (E) Immunostaining and FACS analyses of POU5F1, NANOG, SOX2, and OTX2 in HES-1-LN-hPSCs. Immunostaining- POU5F1, SOX2, OTX2 (n = 3), NANOG (n = 5); FACS- POU5F1, NANOG (n = 5), SOX2, OTX2 (n = 4). (F) FACS analysis of the expression of TRA-1-60 (n = 10) and TRA-1-81 (n = 7) in HES-1-LN-hPSCs. (G) Image of alkaline phosphatase expression in HES-1-LN-hPSC colonies. Scale bar, 1 mm. (H–J) FACS and immunostaining analyses of PSA-NCAM and A2B5 at day 7 of neural induction (H), TBXT at day 3 of mesodermal induction (I), and SOX17 at day10 of endodermal induction (J), in differentiated HES-1-LN- and primed hPSCs. n = 3 for all experiments. (K and L) Images (K) and quantification (L) of crystal violet staining of colonies formed from single-cell suspensions of HES-1 hPSCs cultured under naive, LN, and primed conditions, ± Y27632 (RI). Data are mean ± SEM of (n) wells –RI hPSCs n ≥ 4, +RI-naive hPSCs n ≥ 7. p values were calculated using two-tailed Student’s t test for naive and primed hPSCs versus LN-hPSCs, seeded with RI. ^∗p < 0.05, ^∗∗∗p < 0.001. (E, F, and H–J) Data are mean ± SEM of (n) independent experiments. (C, E, and I–J) Nuclei are counterstained with DAPI (blue). Scale bars, 100 μm.

Figure 2

The gene expression profile of LN-hPSCs resembles a post-implantation pluripotency state (A) Heatmap of the Human Stem Cell Pluripotency Gene Expression Array, of LN-hPSCs (HES-1, HADC100, and HADC102). Gene expression is presented as Delta-Ct value. HES-1 sample data is the average of two independent experiments. (B) PCA plot of RNA-seq data of HES-1, HES-2, and H7 hPSCs cultured under LN and primed conditions, and HES-1 and HES-2 hPSCs cultured under naive conditions. PCA was computed using the top variable 3,800 genes. (C–E) RNA-seq data of selected core pluripotency (C), naive pluripotency (D), and post-implantation epiblast (E) associated genes, in naive, LN and primed HES-1 and HES-2 hPSCs. p values depicted in black show the significant statistical differences between the naive samples and LN and primed samples. p values depicted in red show the significant statistical differences between the LN and primed samples. (F–G) RNA-seq data of selected lineage-specific genes (F) and CDH2 (G) in LN and primed HES-1 and HES-2 hPSCs. LN, LN-hPSCs; P, primed hPSCs; N, naive hPSCs. p values were calculated using two-tailed Student’s t test when two groups were compared, and ANOVA test when three groups were compared. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (C–G) Data are mean ± SEM of two independent samples (except for HES-1-LN- three samples). LN-hPSC samples were at passage 3.

Figure 3

LN-hPSCs display a methylation profile characteristic of the post-implantation epiblast (A) RRBS analysis showing average global CpG methylation levels of the autosomal chromosomes of naive, LN, and primed HES-1 and HES-2 hPSCs. Data are mean ± SEM of (n) independent samples; naive hPSCs (n = 2), LN and primed hPSCs (n = 3). (B) Unsupervised hierarchical clustering of RRBS data of LN, primed, and naive hPSCs. (C) Heatmaps of the methylation levels of DMRs that were hypermethylated (left panel), or hypomethylated (right panel), in LN compared with primed hPSCs. (D) Top 20 enriched pathways related to hypermethylated DMRs in LN compared with primed hPSCs. Analysis was performed using Metascape (Zhou et al., 2019b). (E and F) Methylation profiles (E) and RNA-seq data (F) of selected genes in LN compared with primed hPSCs. RNA-seq data are mean ± SEM of two independent samples (except for HES-1-LN, three samples). LN, LN-hPSCs; P, primed hPSCs; N, naive hPSCs. p values were calculated using two-tailed Student’s t test when two groups were compared, and ANOVA test when three groups were compared. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 4

The FGF and TGF-β/activin dependent signaling pathways are required for the self-renewal of LN-hPSCs (A and E) RNA-seq data of components of the FGF (A) and TGF-β/activin (E) signaling pathways in LN-hPSCs (HES-1, HES-2, and H7). Data are mean ± SEM of two independent samples (except for HES-1-LN, three samples). (B, D, F, and G) Phase-contrast images of HES-1-LN-hPSCs cultured in the basic medium containing FGF2, and in the absence of FGF2 (−F) (B), –F + PD173, −F + PD032, and –F + LY (D), SB, and −F + SB (F), and combinations of the various inhibitors (G). Scale bars, 200 μm. (C) FACS analysis of TRA-1-60 expression in HES-1-LN-hPSCs cultured ± FGF2. Data are mean ± SEM of three independent experiments. (H) qRT-PCR analysis of the relative expression levels of POU5F1 and NANOG, in HES-1-LN-hPSCs cultured in the basic medium, and in the presence of the various inhibitors. Expression levels are normalized to the expression of β-actin. Data are mean ± SEM of (n ≥ 3) independent experiments except for −F + PD032 + LY, −F + SB + LY, −F + SB + PD032 + LY (n = 2). (I) Immunostaining for NANOG (red) in HES-1-LN-hPSCs cultured in the presence of the various inhibitors. Nuclei are counterstained with DAPI (blue). Scale bars, 100 μm. Data are mean ± SEM; n (fields) = 3 for all experiments. F, FGF2; PD032, PD0325901; PD173, PD17304; LY, LY294002; SB, SB431542. p values were calculated using two-tailed Student’s t test for all samples compared with the basic medium sample. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 5

The canonical Wnt signaling is inactive in LN-hPSCs, and its induction upregulates primitive streak markers (A) Immunostaining for β-catenin (green) in HES-1-LN-hPSCs. (B–D) RNA-seq data of various components of the canonical Wnt signaling in LN-hPSCs (HES-1, HES2, and H7). Data are mean ± SEM of two independent samples (except for HES-1-LN, three samples). (E) Phase-contrast images of HES-1-LN-hPSCs cultured in the basic medium, or in the presence of XAV939. Scale bars, 200 μm. (F) FACS analysis of the expression of TRA-1-60 in HES-1-LN-hPSCs cultured as in (E). Data are mean ± SEM of five independent experiments. (G) Phase-contrast image of HES-1-LN-hPSCs cultured in the basic medium, in the presence of CHIR. Scale bar, 200 μm. (H and I) qRT-PCR analyses of the relative expression levels of pluripotency-associated markers (H), and lineage-specific markers (I), in HES-1-LN-hPSCs cultured in the basic medium, and in the presence of CHIR. Expression levels are normalized to the expression of GusB. Data are mean ± SEM of (n) independent experiments; NANOG, TBXT (n = 5), POU5F1, SOX17, GSC, PAX6 (n = 4), SOX2, OTX2 (n = 3). (J) Immunostaining for POU5F1, NANOG, OTX2, and TBXT in HES-1-LN-hPSCs cultured as in (H). XAV, XAV939; CHIR, CHIR99021. p values were calculated using two-tailed Student’s t test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (A and J) Nuclei are counterstained with DAPI (blue). Scale bars, 100 μm.

Figure 6

LN-hPSCs are competent to initiate germ cell specification (A) qRT-PCR analyses of the relative expression levels of pluripotency-associated markers and the early PGC marker PRDM1 in HES-1-LN-hPSCs cultured in the basic medium or in the basic medium without FGF2, and in the presence of CHIR. POU5F1 (n = 4), NANOG (n = 5), SOX2, OTX2, PRDM1 (n = 3). (B and C) Immunostaining images (B) and quantification (C) for the expression of PRDM1 and SOX17 in HES-1-LN-hPSCs cultured as in (A). n = 3. (D) qRT-PCR analyses of the relative expression levels of early PGC markers in HES-1-LN-hPSCs cultured in the basic medium, or as EBs, in the absence of FGF2, and in the presence of BMP4, LIF, SCF, and EGF. Basic medium, 4 days, BMP4-EBs (n = 4), 7 days, BMP4-EBs (n = 3). (E–H) Immunostaining images (E and F) and quantification (G) for the co-expression of AP2γ and SOX17 (E) and AP2γ and NANOG (F), and Nanos3 expression (H), in HES-1-LN-hPSCs cultured as in (D). AP2γ and SOX17, basic medium (n = 3); BMP4-EBs (n = 5); AP2γ and NANOG, basic medium (n = 2), BMP4-EBs (n = 3). (I) qRT-PCR analyses of the relative expression levels of POU5F1, NANOG, SOX2, and OTX2, in HES-1-LN-hPSCs cultured as in (D). Basic medium, 4 days-BMP4-EBs (n = 5), 7 days-BMP4-EBs (n = 3). (J and K) Immunostaining images (J) and quantification (K) for the expression of POU5F1, NANOG, SOX2, and OTX2, in HES-1-LN-hPSCs cultured as in (D). n ≥ 3. (L and M) Quantification of immunostaining for the co-expression of AP2γ and SOX17 (L), and AP2γ and NANOG (M) in BMP4-induced EBs generated from HES-1-LN and primed hPSCs. n ≥ 3. F, FGF2; CHIR, CHIR99021; EBs, embryoid bodies. p values were calculated using two-tailed Student’s t test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. For all experiments, data are mean ± SEM of (n) independent experiments. (A, D, and I) expression levels were normalized to the expression of GusB. (B, E, F, H, and I) Nuclei are counterstained with DAPI. Scale bars, 100 μm.