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

Juntendo University

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.

Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast toward the amniotic ectoderm, primitive streak/mesoderm, and primordial germ cells were constructed and compared with in vivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells.

Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development.

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and several types of somatic cells. Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), a method of generating granulosa cells has been elusive. Here, we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of hiPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology and may enable the development of therapies for female reproductive health.

Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression.

The generation of germline cells from human induced pluripotent stem cells (hiPSCs) represents a milestone toward in vitro gametogenesis. Methods to recapitulate germline development beyond primordial germ cells in vitro have relied on long-term cell culture, such as 3-dimensional organoid co-culture for ~four months. Using a pipeline with highly parallelized screening, this study identifies combinations of TFs that directly and rapidly convert hiPSCs to induced oogonia-like cells (iOLCs). We demonstrate that co-expression of five TFs - namely, ZNF281, LHX8, SOHLH1, ZGLP1, and ANHX, induces high efficiency DDX4-positive iOLCs in only four days in a feeder-free monolayer culture condition. We also show improved production of human primordial germ cell-like cells (hPGCLCs) from hiPSCs by expression of DLX5, HHEX, and FIGLA. We characterize these TF-based iOLCs and hPGCLCs via gene and protein expression analyses and demonstrate their similarity to in vivo and in vitro-derived oogonia and primordial germ cells. Together, these results identify new regulatory factors that enhance human germ cell specification in vitro, and further establish unique computational and experimental tools for human in vitro oogenesis research.

Article	Year
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
Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development. Cell stem cell\| PubMed ID: 36055194	2022
Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression. eLife\| PubMed ID: 36803359	2023
Rapid human oogonia-like cell specification via transcription factor-directed differentiation. EMBO reports\| PubMed ID: 39849206	2025

Mutsumi Kobayashi

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