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 cell (iPSC) culture in the common marmoset (Callithrix jacchus, cj), allowing stable propagation 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.

Generation of marmoset primordial germ cell–like cells under chemically defined conditions

“…To this end, we differentiated cjiPSCs in cjPSCM with or without ActA into pre-ME, which were then induced into cjPGCLCs. CjPGCLCs were isolated by FACS from d4 EBs after staining for the cell surface markers INTEGRINα6 (INTα6) and CXCR4 ( Fig 6A–C ), which are routinely used to sort primate PGCLCs ( Sasaki et al, 2015 ; Kojima et al, 2017 ; Mitsunaga et al, 2017 ; Sakai et al, 2020 ; Seita et al, 2023 ).…”

“…The developmental pathway of mammalian germ cells can be reconstituted in vitro. In particular, pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) of rodents, primates, and other mammals can be differentiated into PGC-like cells (PGCLCs), which represent early PGCs in vivo ( Hayashi et al, 2011 ; Irie et al, 2015 ; Sasaki et al, 2015 ; Sugawa et al, 2015 ; Sosa et al, 2018 ; Overeem et al, 2023 ; Seita et al, 2023 ; Shono et al, 2023 ). Mouse PGCLCs (mPGCLCs) can even give rise to functional mature oocytes or spermatocytes in vitro when aggregated with gonadal somatic cells to generate reconstituted ovaries or testes ( Hayashi et al, 2012 ; Hikabe et al, 2016 ; Ishikura et al, 2016 , 2021 ; Yoshino et al, 2021 ).…”

“…Mouse PGCLCs (mPGCLCs) can even give rise to functional mature oocytes or spermatocytes in vitro when aggregated with gonadal somatic cells to generate reconstituted ovaries or testes ( Hayashi et al, 2012 ; Hikabe et al, 2016 ; Ishikura et al, 2016 , 2021 ; Yoshino et al, 2021 ). In addition, human or NHP PGCLCs aggregated with mouse gonadal cells or transplanted into the gonadal niche can further differentiate and give rise to late PGCLCs, pre-meiotic spermatogonia-/oogonia-like cells, or meiotic oocytes ( Yamashiro et al, 2018 ; Hwang et al, 2020 ; Gyobu-Motani et al, 2023 ; Seita et al, 2023 ; Shono et al, 2023 ).…”

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

“…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.…”

“…Its value can be further enhanced by its application in humans, livestock, and endangered species, where it could eventually contribute to the treatment of infertility, improvement of animal breeding, and conservation of vulnerable species [ 1 ]. 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.…”