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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