Derivation of Human Primordial Germ Cell-Like Cells in an Embryonic-Like Culture

Introduction:: Primordial germ cells (PGCs) are the embryonic precursors of sperm and eggs. Germline defects underlie several human diseases, most notably infertility. Development of human PGC-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) is promising for studying human germline development. Existing protocols for generating hPGCLCs require three-dimensional embryoid body culture and conversion of primed hPSCs into either a naïve-like or mesoderm-like state, posing challenges for high-throughput screening. Herein, we report the induction of hPGCLCs in a bioengineered hPSC culture that mimics peri-implantation human development. In this culture, amniotic ectoderm-like cells (AMLCs) derived from hPSCs induce hPGCLC specification through paracrine signaling downstream of ISL1. hPGCLCs are successfully derived from eight non-obstructive azoospermia (NOA) participant-derived induced hPSCs using this biomimetic platform, demonstrating its promise for screening applications.

Materials and Methods:: Singly dissociated hPSCs were suspended in mTeSR containing 10 μM Y27632, and seeded in a 24-well plate pre-coated with 1% Geltrex at 30,000 cells/sq cm. To generate an embryonic-like environment (Fig. 1A), 2 hours after initial cell seeding, the culture medium was changed to mTeSR containing 4% (v/v) Geltrex and was replenished daily. Y27632 was removed from medium at 24 hours. Immunostaining of cell lineage markers was performed at different time points. Single-cell RNA-sequencing (scRNA-seq) was conducted at Day 3 and Day 8 using 10X Genomics Chromium system. Analysis of scRNA-seq data and integration of scRNA-seq datasets were performed using Seurat R package. To validate cell identities, scRNA-seq datasets were integrated with the scRNA-seq datasets of in vivo human embryos. ISL1-knockout hPSC line was generated using CRISPR/Cas9. Disease-specific hiPSC lines were generated from research participants diagnosed with nonobstructive azoospermia (NOA, no sperm in the ejaculate).

Results, Conclusions, and Discussions:: In the culture, hPSCs spontaneously form lumenal structures with distinct morphologies, containing amniotic ectoderm-like cells (AMLCs), mesoderm-like cells (MeLCs), and hPGCLCs (Fig. 1A). hPGCLCs become evident by Day 3 (TFAP2C+SOX17+BLIMP1+) and continue to develop up to Day 8 (KLF4+TFCP2L1+)(Fig. 1B). By Day 8, flow cytometry analysis shows the percentage of hPGCLCs of around 20%, comparable with the efficiency from other protocols (Fig. 1C). scRNA-seq data reveal three distinct cell clusters, including hPGCLC (Fig. 1D,E). The lineage annotations were validated through the transcriptomic comparison with in vivo human embryos (Fig. 1F). Experimental results generated from ISL-knockout hPSCs suggest that paracrine induction from AMLCs, downstream of ISL1, is critical in hPGCLC specification (Fig. 1G,H). Using our protocol, we successfully generated hPGCLCs from hiPSCs derived from eight NOA participants, suggesting the possibility of deriving sperms for NOA participants (Fig. 1I,J).

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