Rat embryonic stem cells produce fertile offspring through tetraploid complementation
Ontology highlight
ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE97970 | GEO | 2017/10/11
SECONDARY ACCESSION(S): PRJNA383532
REPOSITORIES: GEO
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Project description:Pluripotency of embryonic stem cells (ESCs) can be functionally assessed according to the developmental potency. Tetraploid complementation, through which an entire organism is produced from the pluripotent donor cells, is taken as the most stringent test for pluripotency. It remains unclear whether ESCs of other species besides mice can pass this test. Here we show that the rat ESCs derived under 2i (two small molecule inhibitors) conditions at very early passages are able to produce fertile offspring by tetraploid complementation. However, they lose this capacity rapidly during culture due to a nearly complete loss of genomic imprinting. Our findings support that the naïve ground state pluripotency can be captured in rat ESCs but also point to the species-specific differences in its regulation and maintenance, which have implications for the derivation and application of naïve pluripotent stem cells in other species including human.
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Project description:Pluripotency of embryonic stem cells (ESCs) can be functionally assessed according to their developmental potency. Tetraploid complementation, through which an entire organism is produced from donor pluripotent cells, is taken as the most stringent test for pluripotency. It remains unclear whether ESCs from other species besides mice can pass this test. Here we show that the rat ESCs at very early passages are also capable to produce fertile offspring by tetraploid complementation, however, this capacity is rapidly lost during culture due to the loss of genomic imprinting. Our findings support that the naïve ground state pluripotency exists in rat and can be captured in rat ESCs, yet may be subjected to species-specific regulations, which have implications for the derivation and application of naïve pluripotent stem cells in other species including human.
2017-10-11 | GSE97968 | GEO
Project description:Pluripotency of embryonic stem cells (ESCs) can be functionally assessed according to their developmental potency. Tetraploid complementation, through which an entire organism is produced from donor pluripotent cells, is taken as the most stringent test for pluripotency. It remains unclear whether ESCs from other species besides mice can pass this test. Here we show that the rat ESCs at very early passages are also capable to produce fertile offspring by tetraploid complementation, however, this capacity is rapidly lost during culture due to the loss of genomic imprinting. Our findings support that the naïve ground state pluripotency exists in rat and can be captured in rat ESCs, yet may be subjected to species-specific regulations, which have implications for the derivation and application of naïve pluripotent stem cells in other species including human.
2017-10-11 | GSE97966 | GEO
Project description:Rat embryonic stem cells produce fertile offspring through tetraploid complementation
| PRJNA383532 | ENA
Project description:Genetic mutations could cause sperm deficiency, leading to male infertility. Without functional gametes in the testes, patients cannot produce progeny even with assisted reproduction technologies such as in vitro fertilization. It has been a major challenge to restore the fertility of gamete-deficient patients due to genetic mutations. In this study, using a Kit(w)/Kit(wv) mouse model, we investigated the feasibility of generating functional sperms from gamete-deficient mice by combining the reprogramming and gene correcting technologies. We derived embryonic stem cells from cloned embryos (ntESCs) that were created by nuclear transfer of Kit(w)/Kit(wv) somatic cells. Then we generated gene-corrected ntESCs using TALEN-mediated gene editing. The repaired ntESCs could further differentiate into primordial germ cell-like cells (PGCLCs) in vitro. RFP-labeled PGCLCs from the repaired ntESCs could produce functional sperms in mouse testes. In addition, by co-transplantation with EGFP-labeled testis somatic cells into the testes where spermatogenesis has been chemically damaged or by transplantation into Kit(w)/Kit(wv) infertile testes, non-labeled PGCLCs could also produce haploid gametes, supporting full-term mouse development. Our study explores a new path to rescue male infertility caused by genetic mutations.
| S-EPMC4493281 | biostudies-literature
Project description:Ectopic expression of four transcription factors including Oct4, Sox2, Klf4 and c-Myc in differentiated fibroblast cells could reset the cell fate of fibroblast cells to pluripotent state. Subsequently, fully pluripotency of these so-called induced pluripotent stem cells (iPSCs) has been demonstrated as viable mice could be generated autonomously from iPS cells through tetraploid blastocyst complementation. Moreover, the generation of human and patient-specific iPS cells have raised the possibility of utilizing iPS cells clinically. However, the utilization of c-Myc in iPS cells induction greatly increased the incidence of tumorigenecity in the iPS-chimeric mice and also might hinder the clinical application of human iPS cells in the future. Fortunately, c-Myc has been recently found dispensable for iPS induction even though the iPS induction efficiency is greatly reduced in the absence of c-Myc. However, it remains unknown if these three factors-induced iPS cells are fully pluripotent. In the present study, we have successfully demonstrated that 3-factor iPS cells could also be fully pluripotent as viable mice could be generated from 3-factor iPS cells autonomously via tetraploid complementation and moreover, our data indicated that the pluripotency regulatory mechanism in 3-factor iPS cells might be distinct from 4-factor iPS cells. We compared the gene expression profile of iPS cells with and without the tetraploid embryo complementation competence. Three biological repeats were included for each line.
2010-10-19 | E-GEOD-24046 | biostudies-arrayexpress
Project description:Ectopic expression of four transcription factors including Oct4, Sox2, Klf4 and c-Myc in differentiated fibroblast cells could reset the cell fate of fibroblast cells to pluripotent state. Subsequently, fully pluripotency of these so-called induced pluripotent stem cells (iPSCs) has been demonstrated as viable mice could be generated autonomously from iPS cells through tetraploid blastocyst complementation. Moreover, the generation of human and patient-specific iPS cells have raised the possibility of utilizing iPS cells clinically. However, the utilization of c-Myc in iPS cells induction greatly increased the incidence of tumorigenecity in the iPS-chimeric mice and also might hinder the clinical application of human iPS cells in the future. Fortunately, c-Myc has been recently found dispensable for iPS induction even though the iPS induction efficiency is greatly reduced in the absence of c-Myc. However, it remains unknown if these three factors-induced iPS cells are fully pluripotent. In the present study, we have successfully demonstrated that 3-factor iPS cells could also be fully pluripotent as viable mice could be generated from 3-factor iPS cells autonomously via tetraploid complementation and moreover, our data indicated that the pluripotency regulatory mechanism in 3-factor iPS cells might be distinct from 4-factor iPS cells.
2010-10-19 | GSE24046 | GEO
Project description:One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem (ES) cells, which is used to produce gene-targeted mice for wide applications in biomedicine. However, a major bottleneck in this approach is that the robustness of germline transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing, which impairs the efficiency and robustness of mouse model generation. Recently, we have established a new type of pluripotent cells termed extended pluripotent stem (EPS) cells, which have superior developmental potency and robust germline competence compared to conventional mouse ES cells. In this study, we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage. Based on gene targeting in mouse EPS cells, we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation, which could be accomplished in approximately 2 months. Importantly, using this approach, we successfully constructed mouse models in which the human interleukin 3 (IL3) or interleukin 6 (IL6) gene was knocked into its corresponding locus in the mouse genome. Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting, which have great application potential in biomedical research.
| S-EPMC6321812 | biostudies-literature
Project description:ObjectivesRecently, pluripotency of induced pluripotent stem (iPS) cells has been displayed after producing adult mice, in tetraploid complementation assays. These studies lead us to the last piece of the puzzle for reprogramming somatic cells into fully pluripotent cells which function as embryonic stem cells in most applications. However, in all of previous studies, skin fibroblasts were used as the starting population for reprogramming, raising questions as to whether the pluripotency of the iPS cells was dependent on the particular starting cell type.Materials and methodsOur iPS cell lines were prepared from murine adipose stem cells (ASCs). Their multi-potency was first tested by teratoma formation in nude mice. Then, tetraploid complementation was performed to generate progeny from them.ResultsWe succeeded to the birth of viable and fertile adult mice derived entirely from reprogrammed ASC, indicating cell types other than fibroblasts can also be restored to the embryonic level of pluripotency.ConclusionsWe also directed differentiation of iPS cells into chondrocytes, thus adipose-derived iPS cells can be used as models to study chondrogenic differentiation and cartilage regeneration.
| S-EPMC6496444 | biostudies-literature
Project description:Rat embryonic stem cells (ESCs) offer the potential for sophisticated genome engineering in this valuable biomedical model species. However, germline transmission has been rare following conventional homologous recombination and clonal selection. Here, we used the CRISPR/Cas9 system to target genomic mutations and insertions. We first evaluated utility for directed mutagenesis and recovered clones with biallelic deletions in Lef1. Mutant cells exhibited reduced sensitivity to glycogen synthase kinase 3 inhibition during self-renewal. We then generated a non-disruptive knockin of dsRed at the Sox10 locus. Two clones produced germline chimeras. Comparative expression of dsRed and SOX10 validated the fidelity of the reporter. To illustrate utility, live imaging of dsRed in neonatal brain slices was employed to visualize oligodendrocyte lineage cells for patch-clamp recording. Overall, these results show that CRISPR/Cas9 gene editing technology in germline-competent rat ESCs is enabling for in vitro studies and for generating genetically modified rats.
| S-EPMC5639479 | biostudies-literature