Sorting and Manipulation of Human PGC-LC Using PDPN and Hanging Drop Cultures

Abstract

The generation of oocytes from induced pluripotent stem cells (iPSCs) was proven efficient with mouse cells. However, no human iPSCs have yet been reported to generate cells able to complete oogenesis. Additionally, efficient sorting of human Primordial Germ Cell-Like Cells (hPGC-LCs) without genomic integration of fluorescent reporter for their downstream manipulation is still lacking. Here, we aimed to develop a model that allows human germ cell differentiation in vitro in order to study the developing human germline. The hPGC-LCs specified from two iPS cell lines were sorted and manipulated using the PDPN surface marker without genetic modification. hPGC-LCs obtained remain arrested at early stages of maturation and no further differentiation nor meiotic onset occurred when these were cultured with human or mouse fetal ovarian somatic cells. However, when cultured independently of somatic ovarian cells, using BMP4 and the hanging drop-transferred EBs system, early hPGC-LCs further differentiate efficiently and express late PGC (DDX4) and meiotic gene markers, although no SYCP3 protein was detected. Altogether, we characterized a tool to sort hPGC-LCs and an efficient in vitro differentiation system to obtain pre-meiotic germ cell-like cells without using a gonadal niche.

Evidence of the ability of endocrine disrupting compounds to induce testicular germ cell cancer in humans

“…Total RNA was extracted from B2M cells using the RNeasy kit (QIAGEN), reverse-transcribed using High Capacity Reverse Transcription Kit (Thermofisher Scientific), and processed for qRT-PCR with PowerUp SYBR Green Master Mix (Applied Biosystems) and a StepOne system (Thermofisher Scientific) with gene-specific probes (B2M: 5'-CACAGCCCAAGATAGTTAAGT & 5'-CCAGCCCTCCTAGAGC, the other probes have been already described in (Arkoun et al, 2022;Frydman et al, 2017;Le Bouffant et al, 2010) supplier recommendations (Twist Bioscience). Then a capture of the transcriptome coding regions is performed and the resulting library is suitable for subsequent cluster generation and sequencing.…”