Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells [bulk RNA-seq]
Ontology highlight
ABSTRACT: Reconstitution of germ cell fate from pluripotent stem cells provide the opportunities to understand the molecular underpinning of germ cell development. Here, we established a several culture methods of induced pluripotent stem cells (iPSCs) in common marmoset (Callithrix jacchus, cj), which stably propagate in an undifferentiated state. Notably, iPSCs cultured on feeder layer in the presence of a WNT signaling inhibitor upregulate genes related to ubiquitin-dependent protein catabolic process and acquire permissive state that readily differentiate into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs. Induction of cjPGCLCs accompanies transient upregulation of mesodermal genes and culminate in establishing primate-specific transcriptional network similar to humans and cynomolgus monkeys. Moreover, cjPGCLCs can be expanded in monolayer while retaining cellular state. Upon co-culture with mouse testicular somatic cells, these cells acquire DDX4+ early prospermatogonia-like state. Our findings provide a framework for understanding and reconstituting germ cell development in marmoset in vitro, which serve as an comparative tool and foundation for a preclinical model for human in vitro gametogenesis.
Project description:Reconstitution of germ cell fate from pluripotent stem cells provide the opportunities to understand the molecular underpinning of germ cell development. Here, we established a several culture methods of induced pluripotent stem cells (iPSCs) in common marmoset (Callithrix jacchus, cj), which stably propagate in an undifferentiated state. Notably, iPSCs cultured on feeder layer in the presence of a WNT signaling inhibitor upregulate genes related to ubiquitin-dependent protein catabolic process and acquire permissive state that readily differentiate into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs. Induction of cjPGCLCs accompanies transient upregulation of mesodermal genes and culminate in establishing primate-specific transcriptional network similar to humans and cynomolgus monkeys. Moreover, cjPGCLCs can be expanded in monolayer while retaining cellular state. Upon co-culture with mouse testicular somatic cells, these cells acquire DDX4+ early prospermatogonia-like state. Our findings provide a framework for understanding and reconstituting germ cell development in marmoset in vitro, which serve as an comparative tool and foundation for a preclinical model for human in vitro gametogenesis.
Project description:Reconstitution of germ cell fate from pluripotent stem cells provide the opportunities to understand the molecular underpinning of germ cell development. Here, we established a several culture methods of induced pluripotent stem cells (iPSCs) in common marmoset (Callithrix jacchus, cj), which stably propagate in an undifferentiated state. Notably, iPSCs cultured on feeder layer in the presence of a WNT signaling inhibitor upregulate genes related to ubiquitin-dependent protein catabolic process and acquire permissive state that readily differentiate into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs. Induction of cjPGCLCs accompanies transient upregulation of mesodermal genes and culminate in establishing primate-specific transcriptional network similar to humans and cynomolgus monkeys. Moreover, cjPGCLCs can be expanded in monolayer while retaining cellular state. Upon co-culture with mouse testicular somatic cells, these cells acquire DDX4+ early prospermatogonia-like state. Our findings provide a framework for understanding and reconstituting germ cell development in marmoset in vitro, which serve as an comparative tool and foundation for a preclinical model for human in vitro gametogenesis.
Project description:Reconstitution of germ cell fate from pluripotent stem cells provide the opportunities to understand the molecular underpinning of germ cell development. Here, we established a several culture methods of induced pluripotent stem cells (iPSCs) in common marmoset (Callithrix jacchus, cj), which stably propagate in an undifferentiated state. Notably, iPSCs cultured on feeder layer in the presence of a WNT signaling inhibitor upregulate genes related to ubiquitin-dependent protein catabolic process and acquire permissive state that readily differentiate into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs. Induction of cjPGCLCs accompanies transient upregulation of mesodermal genes and culminate in establishing primate-specific transcriptional network similar to humans and cynomolgus monkeys. Moreover, cjPGCLCs can be expanded in monolayer while retaining cellular state. Upon co-culture with mouse testicular somatic cells, these cells acquire DDX4+ early prospermatogonia-like state. Our findings provide a framework for understanding and reconstituting germ cell development in marmoset in vitro, which serve as an comparative tool and foundation for a preclinical model for human in vitro gametogenesis.
Project description:Primordial germ cells (PGCs) are the embryonic precursors of sperm and oocytes and are responsible for the transmission of genetic and epigenetic information across generations. The development of mouse gametes from PGCs can be reconstituted from pluripotent stem cells in vitro, opening up new avenues for the study of germ cell biology in biomedical research. However, the development of germ cells exhibits molecular and morphological differences between rodents and humans. Therefore, to establish an in vitro model system that is closely related to humans, we studied PGC development in vivo and in vitro in the common marmoset Callithrix jacchus (cj), a New World monkey. Post-migratory cjPGCs in gonads at embryonic day 74 express the germ cell-related genes SOX17, AP2Ɣ, BLIMP1 and the pluripotency markers NANOG and OCT4A (POU5F1), which is reminiscent of human PGCs. We established transgene-free induced pluripotent stem cell (cjiPSC) lines from foetal and postnatal fibroblasts. These cjiPSCs, cultured in a defined medium and in feeder-free conditions, can be differentiated into precursors of mesendoderm and subsequently into cjPGC-like cells (cjPGCLCs) with a transcriptome similar to human PGCs/PGCLCs. Our results not only pave the way to study PGC development in a non-human primate in vitro under experimentally controlled conditions without foetal calf serum and feeder cells, but also provide the opportunity to derive functional marmoset gametes in vitro in future studies.
Project description:Epstein-Barr virus (EBV)-based episomal vector system enables persistent transgene expression, which is advantageous for efficient derivation of transgene-free induced pluripotent stem cells (iPSCs) without viral transduction. Here, we report establishment of an iPSC line from somatic fibroblasts of a neonatal common marmoset monkey (marmoset; Callithrix jacchus) using an all-in-one episomal vector that we newly developed. The established iPSC line, named NM-iPS, showed standard characteristics of pluripotency such as pluripotency-related marker expression, three germ layer differentiation, and normal karyotype (2n = 46). The novel iPSC line would be a useful resource for stem cell research using non-human primates.
Project description:Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers as well as germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem-cell-based therapy, generation of gene-modified animals, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrated the derivation of iPSCs from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the somatic fibroblasts temporarily acquired a neural stem cell (NSC)-like state during the reprogramming procedure. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem-cell-based research using various animals in Mammalia.
Project description:Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers as well as germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem-cell-based therapy, generation of gene-modified animals, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrated the derivation of iPSCs from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the somatic fibroblasts temporarily acquired a neural stem cell (NSC)-like state during the reprogramming procedure. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem-cell-based research using various animals in Mammalia.
Project description:Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers as well as germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem-cell-based therapy, generation of gene-modified animals, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrated the derivation of iPSCs from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the somatic fibroblasts temporarily acquired a neural stem cell (NSC)-like state during the reprogramming procedure. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem-cell-based research using various animals in Mammalia.
Project description:Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers as well as germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem-cell-based therapy, generation of gene-modified animals, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrated the derivation of iPSCs from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the somatic fibroblasts temporarily acquired a neural stem cell (NSC)-like state during the reprogramming procedure. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem-cell-based research using various animals in Mammalia.
Project description:Induced pluripotent stem cells (iPSCs) are capable of providing an unlimited source of cells from all three germ layers as well as germ cells. The derivation and usage of iPSCs from various animal models may facilitate stem-cell-based therapy, generation of gene-modified animals, and evolutionary studies assessing interspecies differences. However, there is a lack of species-wide methods for deriving iPSCs, in particular by means of non-viral and non-transgene-integrating (NTI) approaches. Here, we demonstrated the derivation of iPSCs from somatic fibroblasts of multiple mammalian species from three different taxonomic orders, including the common marmoset (Callithrix jacchus) in Primates, the dog (Canis lupus familiaris) in Carnivora, and the pig (Sus scrofa) in Cetartiodactyla, by combinatorial usage of chemical compounds and NTI episomal vectors. Interestingly, the somatic fibroblasts temporarily acquired a neural stem cell (NSC)-like state during the reprogramming procedure. Collectively, our method, robustly applicable to various species, holds a great potential for facilitating stem-cell-based research using various animals in Mammalia.