Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells

Abstract

Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for pluripotent stem cells (iPSCs) culture in the common marmoset (Callithrix jacchus, cj), which stably propagate in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes culminating in the establishment of a primate specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.

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

“…Although rare hPGC-LC surface markers were identified such as CD38 [ 14 ], difficulties to sort germ cells are still encountered and alternative surface markers are lacking. It was previously reported that PDPN distinguishes early PGC from gonadal somatic cells in humans and marmosets [ 26 , 27 , 28 ]. ScRNA-seq indicates that PDPN was strongly expressed and CD38 was very weakly detected in PGC-LC ( Figure 2 E).…”

Efficient derivation of embryonic stem cells and primordial germ cell-like cells in cattle

“…These results indicate that the concentration of BMP4 should be refined for individual ES cell clones, at least in cattle and rhinoceroses. bPGCLCs expressed transcription factors important for PGC specification, such as BLIMP1, TFAP2C, SOX17, and NANOS3 [2,[4][5][6][7]10]. In contrast, SOX2 was not expressed in the bPGCLCs (Fig.…”

“…Recent reports have demonstrated that primordial germ cells (PGCs), the precursor cell population of gametes, are induced from ES/iPS cells in mammalian species including humans, monkeys, rabbits, rats, and rhinoceroses [2][3][4][5][6][7][8][9][10]. However, in vitro gametogenesis remains underdeveloped for use in livestock.…”

mRNA-based generation of marmoset PGCLCs capable of differentiation into gonocyte-like cells

“…Species difference or different methods used likely account for the discrepancy of the effect of BLIMP1 . Recently, another group reported marmoset PGCLC induction from PSCs (Seita et al, 2022). Their induction efficiency was 40% at the highest using a similar method reported in Cynomolgus monkeys and rabbits (Kobayashi et al, 2021; Sakai et al ., 2020).…”