Direct induction of meiosis from human iPSCs under defined conditions
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1209977 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31983971_1.fastq.gz | Fastqsanger.gz | |||
| SRR31983971_2.fastq.gz | Fastqsanger.gz | |||
| SRR31983972_1.fastq.gz | Fastqsanger.gz | |||
| SRR31983972_2.fastq.gz | Fastqsanger.gz | |||
| SRR31983973_1.fastq.gz | Fastqsanger.gz |
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Project description:Meiotic failure is a significant cause of infertility, but the lack of an in vitro model of human meiosis is a barrier to understanding its mechanism. Here, we establish a method to initiate meiosis directly from male or female human induced pluripotent stem cells (iPSCs). DNMT1 inhibition, retinoid signaling activation, and overexpression of regulatory factors (anti-apoptotic BCL2, and pro-meiotic HOXB5, BOLL, or MEIOC) rapidly activates meiosis over a 15-day protocol. Our protocol bypasses the primordial germ cell stage and directly generates cells expressing genes similar to meiotic oogonia, including oogonia markers, all synaptonemal complex components, and meiotic recombination machinery. DNMT1 inhibition rapidly erases DNA methylation, including at imprinting control regions and promoters of meiotic genes. Microscopy shows key aspects of meiosis, including chromosome axis formation and synapsis in live human cells. Our model of human meiosis provides opportunities for studying this critical reproductive process under chemically defined conditions in vitro.
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Project description:Induced pluripotent stem cells (iPSCs) have been generated from various somatic cells under feeder-layer conditions. These feeder-derived iPSCs generated in different labs exhibit greater variability than between different traditional embryo derived hESC lines. For that reason, it is important to develop a standard and defined system for deriving autologous patient stem cells. We have generated iPSCs under feeder-free conditions using Matrigel coated vessels in chemically defined medium, mTeSR1. These feeder-free derived iPSCs are in many ways similar to feeder-derived iPSCs and also to hESCs, with respect to their pluripotent gene expression (OCT4, NANOG, SOX2), protein expression (OCT4, NANOG, SSEA4, TRA160) and differentiation capabilities. We conducted a whole genomic transcript analysis using Affymetrix Human Gene 1.0 ST arrays to elucidate the important differences between traditional feeder-derived iPSCs and feeder-free derived iPSCs. We reveal that feeder-free iPSCs have over-represented terms belonging to DNA replication and cell cycle genes which are lacking in feeder-derived iPSCs. Feeder-free iPSCs are in many aspects more similar to hESCs including; apoptosis, chromatin modification enzymes and mitochondrial energy metabolism. We have also identified potential biomarkers for fully reprogrammed iPSCs (FRZB) and partially reprogrammed iPSCs (POTEG, MX2) based on their expression trends across all cell types. In conclusion, feeder-free derived iPSCs is transcriptomically more similar to hESCs than feeder derived iPSCs, in many biological functions.
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Project description:Non-human primates (NHPs) are pivotal animal models for translating novel cell replacement therapies into clinical applications, including validating the safety and efficacy of induced pluripotent stem cell (iPSC)-derived products. Preclinical development and the testing of cell-based therapies ideally comprise xenogeneic (human stem cells into NHPs) and allogenic (NHP stem cells into NHPs) transplantation studies. For the allogeneic approach, it is necessary to generate NHP-iPSCs with generally equivalent quality to the human counterparts that will be used later on in patients. Here, we report the generation and characterization of transgene- and feeder-free cynomolgus monkey (Macaca fascicularis) iPSCs (Cyno-iPSCs). These novel cell lines have been generated according to a previously developed protocol for the generation of rhesus macaque, baboon, and human iPSC lines. Beyond their generation, we demonstrate the potential of the novel Cyno-iPSCs to differentiate into two clinically relevant cell types, i.e., cardiomyocytes and neurons. Overall, we provide a resource of novel iPSCs from the most frequently used NHP species in the regulatory testing of biologics and classical pharmaceutics to expand our panel of iPSC lines from NHP species with high relevance in preclinical testing and translational research.
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Project description:An in vitro model of human meiosis would accelerate research into this important reproductive process and development of therapies for infertility. We have developed a method to induce meiosis starting from male or female human pluripotent stem cells. We demonstrate that DNMT1 inhibition, retinoid signaling activation, and overexpression of regulatory factors (anti-apoptotic BCL2, and pro-meiotic HOXB5, BOLL, or MEIOC) rapidly activates meiosis, with leptonema beginning at 6 days, zygonema at 9 days, and pachynema at 12 days. Immunofluorescence microscopy shows key aspects of meiosis, including chromosome synapsis and sex body formation. The meiotic cells express genes similar to meiotic oogonia in vivo, including all synaptonemal complex components and machinery for meiotic recombination. These findings establish an accessible system for inducing human meiosis in vitro.
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Project description:We have developed a serum-free chemical defined medium, namely 6C, that can directly convert mouse embryonic fibroblast (MEFs), glia cells into neurons in vitro. Human cells such as human foreskin fibroblast(HHFs), Hela cells and born marrow derived mesenchymal stem cells (BM-hMSC) can also be converted into neuron-like cells by this medium with some modification such as including several other small molecules. To understand the possible mechanisms, the transdifferentiation of MEFs by 6C was chosen as a model system and gene profiling at different time point during the conversion was carried out by RNA sequencing using Illumina MiSeq. MEFs was maintained in MEF medium (DMEM containing 10% FBS, 1mM Glutamax and 100X NEAA), to start the induction, the culture medium was shift to 6C and marked as day 0, neurons could be generated in day 15. mRNA samples was collected at day 0, 2, 5, 10, 15 during the process, with cell lysed by Trizol and mRNA enriched by Illumina TruSeq RNA Sample Preparation v2 kit. We find that most cell cycle related genes were up regulated during the first few days of induction, while many Notch pathway genes up regulated during the later phase of this process. The RNA sequencing data provided important cues for further study on the mechanisms of the direct neuronal induction process. Gene profiling at different time point during the transdifferentiation mediated by 6C medium.
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Project description:An in vitro model of human meiosis would accelerate research into this important reproductive process and development of therapies for infertility. We have developed a method to induce meiosis starting from male or female human pluripotent stem cells. We demonstrate that DNMT1 inhibition, retinoid signaling activation, and overexpression of regulatory factors (anti-apoptotic BCL2, and pro-meiotic HOXB5, BOLL, or MEIOC) rapidly activates meiosis, with leptonema beginning at 6 days, zygonema at 9 days, and pachynema at 12 days. Immunofluorescence microscopy shows key aspects of meiosis, including chromosome synapsis and sex body formation. The meiotic cells express genes similar to meiotic oogonia in vivo, including all synaptonemal complex components and machinery for meiotic recombination. These findings establish an accessible system for inducing human meiosis in vitro.
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Project description:We have developed a serum-free chemical defined medium, namely 6C, that can directly convert mouse embryonic fibroblast (MEFs), glia cells into neurons in vitro. Human cells such as human foreskin fibroblast(HHFs), Hela cells and born marrow derived mesenchymal stem cells (BM-hMSC) can also be converted into neuron-like cells by this medium with some modification such as including several other small molecules. To understand the possible mechanisms, the transdifferentiation of MEFs by 6C was chosen as a model system and gene profiling at different time point during the conversion was carried out by RNA sequencing using Illumina MiSeq. MEFs was maintained in MEF medium (DMEM containing 10% FBS, 1mM Glutamax and 100X NEAA), to start the induction, the culture medium was shift to 6C and marked as day 0, neurons could be generated in day 15. mRNA samples was collected at day 0, 2, 5, 10, 15 during the process, with cell lysed by Trizol and mRNA enriched by Illumina TruSeq RNA Sample Preparation v2 kit. We find that most cell cycle related genes were up regulated during the first few days of induction, while many Notch pathway genes up regulated during the later phase of this process. The RNA sequencing data provided important cues for further study on the mechanisms of the direct neuronal induction process.
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