Three Dimensional Matrigel Endows Human Formative Pluripotent Stem Cells with Pre-gastrula Stage Epiblast Features [bulk RNA-seq]

Human embryos undergo pivotal morphogenetic remodeling shortly after implantation into the uterus. However, our understanding of this crucial stage is severely impeded by the scarcity of embryonic samples and ethical constraints. Pluripotent stem cells with the competence for somatic and germline differentiation serve as in vitro simulants of epiblast pluripotency continuum. Here, we establish human formative pluripotent stem cells (hfPSCs) from naïve human embryonic stem cells (hESCs), conventional hESCs of different backgrounds, human induced pluripotent stem cells (hiPSCs) as well as human blastocysts in three-dimensional (3D) Matrigel. Similar to pre-gastrula stage epiblast, hfPSCs self-organize into renewal clones with apical lumen and exhibit unique features of formative pluripotency. Functionally, hfPSCs correspond to pre-amnion epiblast cells and exhibit the enhanced tri-lineage differentiation capacity and superior germline competence. Mechanistically, Matrigel-based three-dimensional culture conditions ensure the formative pluripotency through dual ways: biomechanical support and biochemical priming. Thus, hfPSCs open new avenues for creating faithful stem cell-based embryo models and developing the relevant differentiation protocols for regenerative medicine. Overall design: To decipher the multi-omics landscape of hfPSCs cultured in three-dimensional Matrigel, we conducted bulk RNA-seq, single-cell RNA-seq (scRNA-seq), ATAC-seq, and whole-genome bisulfite sequencing (WGBS). The primary mfPSCs (mfPSCs_P0) were transformed from mouse embryonic stem cells (CMTI-1) in N2B27 medium supplemented with 1% KSR, 20 ng/ml Activin A, 12 ng/ml bFgf2, and 5 µM XAV939 in Matrigel. In RNA-seq samples formatted as mfPSCs_P0_24-72h_1/2_R1/2.fq.gz, '24-72h' denotes the time interval of 24 to 72 hours post-passage; ?'1/2'? indicates two biological replicates; ?'R1/R2'? represents the paired-end sequencing reads. Conventional embryonic stem cells (hESCs-H9/MGT3) were maintained in commercial E8 medium (Cat# A15169) on Matrigel (Cat# 356230/354230/356231) coated dishes at 37 °C and 5% CO2. The hESCs were passaged mechanically every 5–7 days by a brief PBS (HUANKE, Cat #HK2109.21) wash followed by dissociation using EDTA (Cat# 15575-038). hESCs_H9_R1/R2.fq.gz and? hESCs_MGT3_R1/R2.fq.gz represent conventional embryonic stem cells. The hfPSCs/hifPSCs were cultured in medium (50 ml) consisted of: 50 ml E8 complete medium, 2 µM IWP2 (Merck, Cat# 681671), 1% HSA (Sigma, Cat# A1653) and 10 µM Rho-associated kinase inhibitor Y27632 (TOCRIS, Cat# 1254) or 10% CloneR (STEM CELL, Cat# 05888). hfPSCs were established from H9 and MGT3 cell line. hifPSCs were obtained from human induced pluripotent stem cells (hiPSCs). The RNA-seq samples associated with this culture method are labeled hfPSCs_H9_R1/R2.fq.gz, hfPSCs_MGT3_R1/R2.fq.gz, hifPSCs_R1/R2.fq.gz Human amnion-like stem cell (hALSCs) were established from hfPSCs based on the following procedures. Firstly, hfPSCs were dissociated into single cells using 50% TrypLE Express and seeded in Matrigel. Then they were cultured in amnion-like stem cell medium. Human amnion-like stem cell medium (50 ml) including: 50 ml N2B27 basal medium, 1 mM PD0325901 (LC Laboratories, Cat# P-9688) and 1 mM A83-01 (Selleck, Cat# S7692). For ?human amnion-like stem cells (hALSCs)? cultured using the above method, the corresponding RNA-seq samples are ? hALSCs_1_R1/R2.fq.gz and hALSCs_2_R1/R2.fq.gz hESCs_sus were hESCs cultured in a suspension manner in E8 medium supplemented with 5uM Y27632. Suspension-cultured hESCs are labeled as hESCs_sus_2_R1/R2.fq.gz and hESCs_sus_1_R1/R2.fq.gz. Following stable 3D culture, mfPSCs were adapted to Matrigel-coated 2D plates for 1-2 passages (3D to 2D_P1/P2) in mfPSCs medium. In the filenames: 3D_to_2D_P1_1/2_R1/R2.fq.gz denote the first generation of cells cultured under this protocol. And 3D_to_2D_P2_1/2_R1/R2.fq.gz ? denote the second generation of cells cultured under this protocol. ?'1/2'? indicates two biological replicates.