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Analysis of multi-lineage gene expression dynamics during primordial germ cell induction from human induced pluripotent stem cells.

ABSTRACT: BACKGROUND:In mammals, specification of primordial germ cells (PGCs) is established in the early post-implantation embryo. The bone morphogenetic protein (BMP)-SMAD and WNT3-?-catenin signaling initiate the gene regulatory network for PGC specification. The activation of SOX17-BLIMP1 axis is critical for human PGC program. Moreover, EpCAM and INTEGRIN?6 were identified as surface markers of human PGC-like cells (PGCLCs) recently. However, the signaling mechanism for PGC specification in non-rodent mammals remains to be clarified. METHODS:We differentiated human induced pluripotent stem cells (hiPSCs) into PGCLCs in vitro in response to Activin A and BMP4. The percentage of EpCAM/INTEGRIN?6 double-positive cells (PGCLCs) was analyzed by flow cytometry. The expression of PGC genes was evaluated by qRT-PCR and immunofluorescence. The expression dynamic of multi-lineage genes during the differentiation process was evaluated by qRT-PCR. RESULTS:Under the stimulation for PGCLC induction, the embryoids derived from hiPSCs initiated significant upregulation of the early PGC genes (BLIMP1, TFAP2C, and NANOS3), but maintained low or no levels of DPPA3 and late PGC genes (DAZL and DDX4). The percentage of EpCAM/INTEGRIN?6 double-positive PGCLCs reached the highest at day 6 of induction. After pre-induction, the incipient mesoderm-like cells (iMeLCs) upregulated most of the mesoderm genes (EOMES, T, MSXI, RUNX2, and MIXL1). The differentiating embryoids showed high levels of key pluripotency genes, OCT4 and NANOG, but became negative for SOX2. In contrast to iMeLCs, the differentiating embryoids downregulated mesoderm genes RUNX2 and EOMES, and ectoderm gene PAX6, but increased the expression of endoderm gene SOX17. CONCLUSIONS:During PGCLC induction process in vitro, the differentiating embryoids not only activated the PGC-related genes, but also displayed complex regulation of pluripotency genes and multi-lineage genes. These results would be meaningful for future research investigating the regulation of human early germ line development.

SUBMITTER: Fang F

PROVIDER: S-EPMC7055065 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Analysis of multi-lineage gene expression dynamics during primordial germ cell induction from human induced pluripotent stem cells.

Fang Fang F Li Zili Z Zhao Qian Q Xiong Chengliang C Ni Ke K

Stem cell research & therapy 20200304 1

<h4>Background</h4>In mammals, specification of primordial germ cells (PGCs) is established in the early post-implantation embryo. The bone morphogenetic protein (BMP)-SMAD and WNT3-β-catenin signaling initiate the gene regulatory network for PGC specification. The activation of SOX17-BLIMP1 axis is critical for human PGC program. Moreover, EpCAM and INTEGRINα6 were identified as surface markers of human PGC-like cells (PGCLCs) recently. However, the signaling mechanism for PGC specification in ...[more]

PMID: 32127035

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Project description:BACKGROUND:Premature ovarian insufficiency (POI) is a common disease in reproductive women. The pathogenesis of POI is not clear, although it is known that it involves the disorder of oocyte differentiation and development. The introduction of reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) offers a unique opportunity to study many aspects of POI from cell differentiation in vitro that could ultimately lead to novel drug development and testing to help treat the disorder. METHODS:The fibroblasts from POI patients, including fragile X syndrome, abnormal karyotype (45, X; 45, X/46, XX; 45, XO and 47, XXX), and the gene mutation (FIGLA and GDF9) were reprogrammed to pluripotency status by retroviral transduction using defined factors. The morphology, growth characteristics, gene expression profiles, epigenetic status, and in vitro and in vivo differentiation potential of the POI-1-iPSCs (from fragile X syndrome) were analyzed. Then, POI-1-iPSCs were induced to differentiation into primordial germ cells (PGCs) with DNA methyltransferase inhibitors. RESULTS:The iPSCs were successfully generated from POI patients' fibroblasts. The formed iPS clones have the same characteristics of human ESCs. POI-1-iPSCs were successfully generated with germline competence. The POI-1-iPSCs, with genotypes of fragile X syndrome, can be induced to differentiation into PGCs with high efficiency under our culture system by DNA demethylation. This study proved that disease-specific iPSC lines derived from POI patients could be generated and successfully differentiated into PGCs. CONCLUSIONS:We established some novel, systemic cell models for the studying of the pathogenesis of POI patients. Second, DNA demethylation may accelerate the induction of human PGCs from iPSCs in vitro and the conclusion needs further exploration. This represents an important step in the novel approach for the study of the pathophysiology and potential egg resource for POI patients.

Dataset Information

Analysis of multi-lineage gene expression dynamics during primordial germ cell induction from human induced pluripotent stem cells.

Publications

Analysis of multi-lineage gene expression dynamics during primordial germ cell induction from human induced pluripotent stem cells.

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