Abstract
BackgroundAs the precursors of sperm and eggs, human primordial germ cells (hPGCs) emerge as early as weeks 2 to 3 of post-implantation development. Recently, robust hPGC induction models have been established in vitro with different protocols, but global 5mC/5hmC epigenetic reprogramming is not initiated in vitro. Previous studies found that vitamin C can enhance Tet (ten-eleven translocation) enzyme expression and improve 5hmC level in cells. But the effect of vitamin C supplementation on hPGC in vitro induction is still unknown.MethodsWe generated a gene-edited human embryonic stem cell (hESC) line carrying a BLIMP1-mkate2 reporter by CRISPR/Cas9 technology and used flow cytometry to optimize the PGC differentiation protocol; meanwhile, the expression of PGC genes (BLIMP1, TFAP2C, SOX17, OCT4) was evaluated by qRT-PCR. When different concentrations of vitamin C were added to the induction medium, the percentage of hPGCLCs (hPGC-like cells) was analyzed by flow cytometry; dot blot and ELISA were used to detect the levels of 5hmC and 5mC. The expression of TET enzymes was also evaluated by qRT-PCR.ResultsWe optimized the PGC differentiation protocol with the BLIMP1-mkate reporter hESCs, and the efficiency of PGC induction in vitro can be improved to 30~40%. When 50 μg/mL vitamin C was added, the derived hPGCLCs not only upregulated the expression of key genes involved in human early germ cell development such as NANOS3, TFAP2C, BLIMP1, and SOX17, but also increased the levels of 5hmC and TET enzymes.ConclusionsTaken together, supplementation of vitamin C can promote the in vitro induction of hPGCLCs from hESCs, which might be related to vitamin C-mediated epigenetic regulations during the differentiation process.
Highlights
The germ cell lineage originates from primordial germ cells (PGCs) and serves as the foundation for heredity and evolution
It has been demonstrated that SOX17 is a critical regulator of human primordial germ cells (hPGCs) fate and that WNT signals induce the expression of EOMES to activate SOX17, which works together with TFAP2C to instate the human primordial germ cell-like cells (hPGCLCs) transcriptional program, including the BLIMP1 expression [5, 7,8,9]
Establishment of human embryonic stem cell (hESC) bearing BLIMP1-mkate2 reporter BLIMP1 has been reported to be expressed in hPGCs and human fetal germ cells [12]
Summary
The germ cell lineage originates from primordial germ cells (PGCs) and serves as the foundation for heredity and evolution. The mechanism for germ cell development has been extensively studied in the mouse models and significant progress has been made recently [1]. Researchers have been working on the reconstitution of human germ cell development in vitro using human ESCs and iPSCs. Recently, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have. Li et al Stem Cell Research & Therapy (2019) 10:324 been induced into human primordial germ cell-like cells (hPGCLCs) in response to signals simulating the natural developing environment of hPGC in vivo, and several key questions during hPGC specification have been illuminated using this in vitro system. Recent studies have investigated the genomewide DNA demethylation dynamics in hPGCs. Consistent with the findings in mice, hPGCs undergo epigenetic reprogramming during early embryo development [10,11,12,13]. The effect of vitamin C supplementation on hPGC in vitro induction is still unknown
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