Scalable and Efficient Generation of Mouse Primordial Germ Cell-like Cells

Abstract

Primordial germ cells (PGCs) are the founder cells of the germline. The ability to generate PGC-like cells (PGCLCs) from pluripotent stem cells has advanced our knowledge of gametogenesis and holds promise for developing infertility treatments. However, generating an ample supply of PGCLCs for demanding applications such as high-throughput genetic screens has been a limitation. Here, we demonstrated that simultaneous overexpressing 4 transcriptional factors - Nanog and three PGC master regulators Prdm1, Prdm14 and Tfap2c - in suspended mouse epiblast like cells (EpiLCs) and formative embryonic stem cells (ESCs) results in efficient and cost-effective production of PGCLCs. The overexpression of Nanog enhances the PGC regulatory network and suppresses differentiation of somatic lineages, enabling a significant improvement in the efficiency of PGCLC production. Transcriptomic analysis reveals that differentiated PGCLCs exhibit similarities to in vivo PGCs and are more advanced compared to cytokine-induced PGCLCs. These differentiated PGCLCs could be sustained over prolonged periods of culture and could differentiate into spermatogonia-like cells in vitro. Importantly, the ability to produce PGCLCs at scale, without using costly cytokines, enables biochemical and functional genomic screens to dissect mechanisms of germ cell development and infertility.

A high throughput CRISPR perturbation screen identifies epigenetic regulators impacting primordial germ cell development

“…1B and C; Table S4). We then prioritized genes within the set of candidate pro-PGCLC differentiation factors using 2 criteria: 1) beta score and 2) increased mRNA levels during PGCLC differentiation compared to the ESC and EpiLC stages 31 . Amongst the 25 prioritized candidates (Fig.…”

“…(D) Gene expression heatmap of 25 candidate genes with β scores and exhibited increased expression during PGCLC differentiation compared to ESC and EpiLC stages. RNA-seq data is from Ding et al 31 .…”

“…To generate sufficient PGCLCs necessary for screening the library, we used a highly scalable, cytokine-free culture system for differentiation of ESCs into PGCLCs 31 . This entails the use of ESCs engineered with four doxycycline-inducible transcription factors (TFs; Prdm1 , Prdm14 , Tfap2c , and Nanog ) that together drive efficient PGCLC differentiation in 8 days (Fig.…”

“…Mouse ESCs with the Stella-eGFP reporter were a kind gift of Azim Surani 56 . As we’ve described elsewhere 31 , these ESCs were then stably transfected with plasmid constructs encoding doxycycline-inducible Prdm1 , Prdm14 , Nanog , and Tfap2c transcription factors. Culture plates were first coated by poly-L-ornithine (0.01%) at room temperature for at least 1 hour, and then coated with laminin (10ng/mL) at 37°C for at least 1 hour.…”

“…The development of methods for in vitro differentiation of embryonic stem cells (ESCs) into primordial germ cell like cells (PGCLCs) has proven to be valuable for studying the regulatory mechanisms of germline development 28–30 . Here, we report the results of a CRISPR inhibition (CRISPRi) screen to identify essential epigenetic regulators of PGC development, using a recently-developed scalable culture system for differentiation of ESCs into PGCLCs 31 . The screen identified several epigenetic factors that impact the efficiency of PGCLC specification and proliferation, including Ncor2 , a nuclear receptor co-repressor facilitating histone deacetylation.…”