generation-human-primordial-germ-cell-like-cells-at-surface-embryoid

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent ...

long-term-culture-of-human-primordial-germ-cell-like-cells

Long-term Culture of Human Primordial Germ Cell-like Cells

Primordial germ cells (PGCs) are common precursors of all male and female germline cells. PGCs arise in peri-implantation mammalian embryos and become ...

Massachusetts General Hospital

Pluripotent stem cell-derived human primordial germ cell-like cells (hPGCLCs) provide important opportunities to study primordial germ cells (PGCs). We robustly produced CD38 hPGCLCs [∼43% of FACS-sorted embryoid body (EB) cells] from primed-state induced pluripotent stem cells (iPSCs) after a 72-hour transient incubation in the four chemical inhibitors (4i)-naïve reprogramming medium and showed transcriptional consistency of our hPGCLCs with hPGCLCs generated in previous studies using various and distinct protocols. Both CD38 hPGCLCs and CD38 EB cells significantly expressed  and , although PRDM1 mRNA in CD38 cells lacked the 3'-UTR harboring miRNA binding sites regulating mRNA stability. Genes up-regulated in hPGCLCs were enriched for cell migration genes, and their promoters were enriched for the binding motifs of TFAP2 (which was identified in promoters of , , and ) and the RREB-1 cell adhesion regulator. In EBs, hPGCLCs were identified exclusively in the outermost surface monolayer as dispersed cells or cell aggregates with strong and specific expression of POU5F1/OCT4 protein. Time-lapse live cell imaging revealed active migration of hPGCLCs on Matrigel. Whereas all hPGCLCs strongly expressed the CXCR4 chemotaxis receptor, its ligand CXCL12/SDF1 was not significantly expressed in the whole EBs. Exposure of hPGCLCs to CXCL12/SDF1 induced cell migration genes and antiapoptosis genes. Thus, our study shows that transcriptionally consistent hPGCLCs can be readily produced from hiPSCs after transition of their pluripotency from the primed state using various methods and that hPGCLCs resemble the early-stage PGCs randomly migrating in the midline region of human embryos before initiation of the CXCL12/SDF1-guided chemotaxis.

Relevance of iPSC-derived human PGC-like cells at the surface of embryoid bodies to prechemotaxis migrating PGCs.

Exposure of mammalian fetuses to endocrine disruptors can increase the risk of adult-onset diseases. We previously showed that exposure of mouse fetuses to bisphenol A (BPA) caused adult-onset obesity. To examine roles of epigenetic changes in this delayed toxicity, we determined the effects of fetal mouse exposure to BPA on genome-wide DNA methylation and messenger RNA (mRNA) expression in gonadal white adipose tissues (WATs) by deep sequencing, bisulfite pyrosequencing, and real-time quantitative polymerase chain reaction. Pregnant CD-1 mice (F0) were dosed daily with 0, 5, or 500 μg/kg/d BPA during gestational days 9 to 18, and the weaned F1 animals were fed ad libitum with standard chow until they were euthanized at 19 weeks old. In the vehicle-exposed F1 animals, fggy promoter showed a clear bimodal pattern of very strong (55% to 95%) or very weak (5% to 30%) DNA methylation occurring at nearly equal incidence with no intermediate strength. Promoter hypermethylation completely suppressed mRNA expression. BPA exposure eliminated this naturally occurring dichotomy, shifting fggy promoter toward the hypomethylation state to release transcriptional suppression. The strength of Fggy mRNA expression significantly correlated with increased whole body weight and gonadal fat weight of males but not females. Bioinformatics studies showed that expression of Fggy mRNA is stronger in mouse WATs than in brown adipose tissues and enhanced in gonadal fat by diet-induced obesity. These observations suggest that prenatal exposure to BPA may disrupt the physiological bimodal nature of epigenetic regulation of fggy in mouse WATs, possibly contributing to the adult-onset obesity phenotype.

Prenatal Exposure to Bisphenol A Disrupts Naturally Occurring Bimodal DNA Methylation at Proximal Promoter of fggy, an Obesity-Relevant Gene Encoding a Carbohydrate Kinase, in Gonadal White Adipose Tissues of CD-1 Mice.

Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.

Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells.

Emergence of antiestrogen-resistant cells in MCF-7 cells during suppression of estrogen signaling is a widely accepted model of acquired breast cancer resistance to endocrine therapy. To obtain insight into the genomic basis of endocrine therapy resistance, we characterized MCF-7 monoclonal sublines that survived 21-day exposure to tamoxifen (T-series sublines) or fulvestrant (F-series sublines) and sublines unselected by drugs (U-series). All T/F-sublines were resistant to the cytocidal effects of both tamoxifen and fulvestrant. However, their responses to the cytostatic effects of fulvestrant varied greatly, and their remarkably diversified morphology showed no correlation with drug resistance. mRNA expression profiles of the U-sublines differed significantly from those of the T/F-sublines, whose transcriptomal responsiveness to fulvestrant was largely lost. A set of genes strongly expressed in the U-sublines successfully predicted metastasis-free survival of breast cancer patients. Most T/F-sublines shared highly homogeneous genomic DNA aberration patterns that were distinct from those of the U-sublines. Genomic DNA of the U-sublines harbored many aberrations that were not found in the T/F-sublines. These results suggest that the T/F-sublines are derived from a common monoclonal progenitor that lost transcriptomal responsiveness to antiestrogens as a consequence of genetic abnormalities many population doublings ago, not from the antiestrogen-sensitive cells in the same culture during the exposure to antiestrogens. Thus, the apparent acquisition of antiestrogen resistance by MCF-7 cells reflects selection of preexisting drug-resistant subpopulations without involving changes in individual cells. Our results suggest the importance of clonal selection in endocrine therapy resistance of breast cancer.

Antiestrogen-resistant subclones of MCF-7 human breast cancer cells are derived from a common monoclonal drug-resistant progenitor.

Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.

Masculine epigenetic sex marks of the CYP19A1/aromatase promoter in genetically male chicken embryonic gonads are resistant to estrogen-induced phenotypic sex conversion.

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor α protein in MCF-7 cells require the CSK c-Src tyrosine kinase.

The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.

Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells.

The DOHaD (developmental origins of health and disease) hypothesis claims that fetal malnutrition or exposure to environmental pollutants may affect their lifelong health. Epigenetic changes may play significant roles in DOHaD; however, access to human fetuses for research has ethical and technical hurdles. Umbilical cord blood (CB) has been commonly used as an epigenetic surrogate of fetuses, but it does not provide direct evidence of fetal exposure to pollutants. Here, we propose umbilical cord tissue (UC), which accumulates substances delivered to fetuses during gestation, as an alternative surrogate for epigenetic studies on fetuses. To explore the feasibility to examine UC epigenome by deep sequencing, we determined CpG methylation profiles of human postnatal UC by reduced representation bisulfite sequencing. Principal component analysis clearly separated the DNA methylomes of UC and CB pairs isolated from the same newborn (n = 10). Although all UC chromosomes were modestly hypomethylated compared to CB chromosomes, GO analysis revealed strong enrichment of differentially methylated regions (DMRs) at promoter-associated CpG islands in the HOX gene clusters and other genes encoding transcription factors involved in determination of the body pattern. DNA methylomes of UC autosomes were largely comparable between males and females. Deficiency of folate during pregnancy has been suggested to affect fetal DNA methylation to cause congenital anomalies. Whereas DNA methylome of UC was not significantly affected by early-gestational (12 weeks) low levels of maternal plasma folate (< 8 ng/ml, n = 10) compared to controls (>19 ng/mL, n = 10), two specific loci of LTR12C endogenous retroviruses in chromosome 12 were significantly hypermethylated in the low-folate group. Our study suggests that UC is useful as an alternative surrogate for studying environmental effects on DNA methylation in human fetuses, compensating CB by providing additional information about epigenetic regulation of genes involved in developmental body patterning and endogenous retroviruses.

DNA methylome of human neonatal umbilical cord: Enrichment of differentially methylated regions compared to umbilical cord blood DNA at transcription factor genes involved in body patterning and effects of maternal folate deficiency or children's sex.

Primordial germ cells (PGCs) are common ancestors of all germline cells. In mammals, PGCs emerge in early-stage embryos around the timing of gastrulation at or near epiblast, and specification of PGCs from their precursor cells involves multiple growth factors secreted by adjacent cells. Recent advancements in germline stem cell biology have made it possible to generate PGC-like cell culture models (PGCLCs for PGC-like cells) from human and mouse pluripotent stem cells by mimicking the embryonic growth factor environment in vitro. Here we describe a method of producing human PGCLCs from primed-pluripotency induced pluripotent stem cells (iPSCs) via temporal conversion to naive pluripotency followed by formation of embryoid bodies (EBs) using the spin-EB method.

Production and Analysis of Human Primordial Germ Cell-Like Cells.

Whereas in ovo exposure of genetically male (ZZ) chicken embryos to exogenous estrogens temporarily feminizes gonads at the time of hatching, the morphologically ovarian ZZ-gonads (FemZZs for feminized ZZ gonads) are masculinized back to testes within 1 year. To identify the feminization-resistant "memory" of genetic male sex, FemZZs showing varying degrees of feminization were subjected to transcriptomic, DNA methylome, and immunofluorescence analyses. Protein-coding genes were classified based on their relative mRNA expression across normal ZZ-testes, genetically female (ZW) ovaries, and FemZZs. We identified a group of 25 genes that were strongly expressed in both ZZ-testes and FemZZs but dramatically suppressed in ZW-ovaries. Interestingly, 84% (21/25) of these feminization-resistant testicular marker genes, including the DMRT1 master masculinizing gene, were located in chromosome Z. Expression of representative marker genes of germline cells (eg, DAZL or DDX4/VASA) was stronger in FemZZs than normal ZZ-testes or ZW-ovaries. We also identified 231 repetitive sequences (RSs) that were strongly expressed in both ZZ-testes and FemZZs, but these RSs were not enriched in chromosome Z. Although 94% (165/176) of RSs exclusively expressed in ZW-ovaries were located in chromosome W, no feminization-inducible RS was detected in FemZZs. DNA methylome analysis distinguished FemZZs from normal ZZ- and ZW-gonads. Immunofluorescence analysis of FemZZ gonads revealed expression of DMRT1 protein in medullary SOX9+ somatic cells and apparent germline cell populations in both medulla and cortex. Taken together, our study provides evidence that both somatic and germline cell populations in morphologically feminized FemZZs maintain significant transcriptomic and epigenetic memories of genetic sex.

Transcriptomic and Epigenetic Preservation of Genetic Sex Identity in Estrogen-feminized Male Chicken Embryonic Gonads.

In vitro expansion of human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived PGC model, has proved challenging due to rapid loss of primordial germ cell (PGC)-like identity and limited cell survival/proliferation. Here, we describe long-term culture hPGCLCs (LTC-hPGCLCs), which actively proliferate in a serum-free, feeder-free condition without apparent limit as highly homogeneous diploid cell populations maintaining transcriptomic and epigenomic characteristics of hPGCLCs. Histone proteomics confirmed reduced H3K9me2 and increased H3K27me3 marks in LTC-hPGCLCs compared with induced pluripotent stem cells (iPSCs). LTC-hPGCLCs established from multiple human iPSC clones of both sexes were telomerase positive, senescence-free cells readily passaged with minimal cell death or deviation from the PGC-like identity. LTC-hPGCLCs are capable of differentiating to DAZL-positive M-spermatogonia-like cells in the xenogeneic reconstituted testis (xrTestis) organ culture milieu as well as efficiently producing fully pluripotent embryonic germ cell-like cells in the presence of stem cell factor and fibroblast growth factor 2. Thus, LTC-hPGCLCs provide convenient access to unlimited amounts of high-quality and homogeneous hPGCLCs.

Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers.

Obesogens such as tributyltin (TBT) are xenobiotic compounds that promote obesity, in part by distorting the normal balance of lipid metabolism. The obesogenic effects of TBT can be observed in directly exposed (F1 and F2 generations) and also subsequent generations (F3 and beyond) that were never exposed. To address the effects of TBT exposure on germ cells, we exposed pregnant transgenic OG2 mouse dams (F0), which specifically express EGFP in germline cells, to an environmentally relevant dose of TBT or DMSO throughout gestation through drinking water. When fed with a high-fat diet, F3 male offspring of TBT-exposed F0 dams (TBT-F3) accumulated much more body fat than did DMSO-F3 males. TBT-F3 males also lost more body fluid and lean compositions than did DMSO-F3 males. Expression of genes involved in transcriptional regulation or mesenchymal differentiation was up-regulated in somatic cells of TBT-F1 (but not TBT-F3) E18.5 fetal testes, and promoter-associated CpG islands were hyper-methylated in TBT-F1 somatic cells. Global mRNA expression of protein-coding genes in F1 or F3 fetal testicular cells was unaffected by F0 exposure to TBT; however, expression of a subset of endogenous retroviruses was significantly affected in F1 and F3. We infer that TBT may directly target testicular somatic cells in F1 testes to irreversibly affect epigenetic suppression of endogenous retroviruses in both germline and somatic cells.

Transgenerational Transcriptomic and DNA Methylome Profiling of Mouse Fetal Testicular Germline and Somatic Cells after Exposure of Pregnant Mothers to Tributyltin, a Potent Obesogen.

Burgeoning evidence demonstrates that effects of environmental exposures can be transmitted to subsequent generations through the germline without DNA mutations. This phenomenon remains controversial because underlying mechanisms have not been identified. Therefore, understanding how effects of environmental exposures are transmitted to unexposed generations without DNA mutations is a fundamental unanswered question in biology. Here, we used an established murine model of male-specific transgenerational obesity to show that exposure to the obesogen tributyltin (TBT) elicited heritable changes in chromatin interactions (CIs) in primordial germ cells (PGCs). New CIs were formed within the  gene encoding Insulin Degrading Enzyme in the directly exposed PGCs, then stably maintained in PGCs of the subsequent (unexposed) two generations. Concomitantly,  mRNA expression was decreased in livers of male descendants from the exposed dams. These males were hyperinsulinemic and hyperglycemic, phenocopying -deficient mice that are predisposed to adult-onset, diet-induced obesity. Creation of new CIs in PGCs, suppression of hepatic  mRNA, increased fat mass, hyperinsulinemia and hyperglycemia were male-specific. Our results provide a plausible molecular mechanism underlying transmission of the transgenerational predisposition to obesity caused by gestational exposure to an environmental obesogen. They also provide an entry point for future studies aimed at understanding how environmental exposures alter chromatin structure to influence physiology across multiple generations in mammals.

ArticleTotal : 2	Year
Publication title	2019
Publication title	2025

Article	Year
Relevance of iPSC-derived human PGC-like cells at the surface of embryoid bodies to prechemotaxis migrating PGCs. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29087313	2017
Prenatal Exposure to Bisphenol A Disrupts Naturally Occurring Bimodal DNA Methylation at Proximal Promoter of fggy, an Obesity-Relevant Gene Encoding a Carbohydrate Kinase, in Gonadal White ... Endocrinology\| PubMed ID: 29220483	2018
Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells. Nature methods\| PubMed ID: 30127506	2018
Antiestrogen-resistant subclones of MCF-7 human breast cancer cells are derived from a common monoclonal drug-resistant progenitor. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19706540	2009
Masculine epigenetic sex marks of the CYP19A1/aromatase promoter in genetically male chicken embryonic gonads are resistant to estrogen-induced phenotypic sex conversion. Biology of reproduction\| PubMed ID: 22539680	2012
Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor α protein in MCF-7 cells require the CSK c-Src tyrosine kinase. PloS one\| PubMed ID: 23593342	2013
Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 27486249	2016
DNA methylome of human neonatal umbilical cord: Enrichment of differentially methylated regions compared to umbilical cord blood DNA at transcription factor genes involved in body patterning... PloS one\| PubMed ID: 31063509	2019
Production and Analysis of Human Primordial Germ Cell-Like Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32852762	2021
Transcriptomic and Epigenetic Preservation of Genetic Sex Identity in Estrogen-feminized Male Chicken Embryonic Gonads. Endocrinology\| PubMed ID: 33170207	2021
Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers. Stem cell reports\| PubMed ID: 35148847	2022
Transgenerational Transcriptomic and DNA Methylome Profiling of Mouse Fetal Testicular Germline and Somatic Cells after Exposure of Pregnant Mothers to Tributyltin, a Potent Obesogen. Metabolites\| PubMed ID: 35208169	2022
Heritable changes in chromatin contacts linked to transgenerational obesity. Research square\| PubMed ID: 38077066	2023

Keiko Shioda

Biography

JoVE Journal Publications

Other Publications