Human Germ Cell Research Articles

Specification of human germ cell fate with enhanced progression capability supported by hindgut organoids

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, are specified during weeks 2-3 after fertilization. Few studies on exvivo and invitro cultured human embryos reported plausible hPGCs on embryonic day (E) 12-13 and in an E16-17 gastrulating embryo. Invitro, hPGC-like cells (hPGCLCs) can be specified from the intermediary pluripotent stage or peri-gastrulation precursors. Here, we explore the broad spectrum of hPGCLC precursors and how different precursors impact hPGCLC development. We show that resetting precursors can give rise to hPGCLCs (rhPGCLCs) in response to BMP. Strikingly, rhPGCLCs co-cultured with human hindgut organoids progress at a pace reminiscent of invivo hPGC development, unlike those derived from peri-gastrulation precursors. Moreover, rhPGCLC specification depends on both EOMES and TBXT, not just on EOMES as for peri-gastrulation hPGCLCs. Importantly, our study provides the foundation for developing efficient invitro models of human gametogenesis.

PGCLCs of human 45,XO reveal pathogenetic pathways of neurocognitive and psychosocial disorders

BackgroundNeurocognitive disorders and psychosocial difficulties are common in patients with Turner syndrome and multiple neurodegenerative diseases, yet there is no effective cure. Human primordial germ cells (hPGCs) are pluripotent germline stem cells in early embryo, which pass genetic information from one generation to the next, whereas all somatic cells will die along with the end of life. However, it is not known whether patient hPGCs with Turner syndrome contain information of neurocognitive and psychosocial illness.ResultsIn this report, we used a high-density of culture system of embryoids derived from iPSCs of a patient with Turner syndrome to ask how pathogenetic pathways are associated with onset of neurocognitive and psychosocial disorders. The hPGC-Like Cells (hPGCLCs) were in vitro specified from iPSCs of 45,XO, 46,XX and 46,XY by the high-density induction of embryoids. Amazingly, we found that the specification process of the hPGCLCs in 45,XO, compared to those in 46,XX and 46,XY, enriched several common pathogenetic pathways regulating neurocognitive and psychosocial disorders, that shared among multiple neurodegenerative diseases and Turner syndrome. The downregulated chemical synaptic transmission pathways, including glutamatergic, GABAergic, and nicotine cholinergic synapses, indicated synaptic dysfunctions, while upregulated pathways that were associated with imbalance of mitochondrial respiratory chain complexes and apoptosis, may contribute to neuronal dysfunctions. Notably, downregulation of three types of ubiquitin ligases E1-E2-E3 and lysosome-associated sulfatases and RAB9A, owing to haploinsufficiency and parental preference of the X chromosome expression, indicated that two pathways of cellular degradation, lysosome and ubiquitin–proteasome, were impaired in the specification process of 45,XO hPGCLCs. This would lead to accumulation of undesired proteins and aggregates, which is a typically pathological hallmark in neurodegenerative diseases.ConclusionsOur data suggest that the specification process of the hPGCLCs in 45,XO, compared to those in 46,XX and 46,XY, enriched pathogenetic pathways that are associated with the onset of neurocognitive and psychosocial disorders.

Sorting and Manipulation of Human PGC-LC Using PDPN and Hanging Drop Cultures.

The generation of oocytes from induced pluripotent stem cells (iPSCs) was proven efficient with mouse cells. However, no human iPSCs have yet been reported to generate cells able to complete oogenesis. Additionally, efficient sorting of human Primordial Germ Cell-like Cells (hPGC-LCs) without genomic integration of fluorescent reporter for their downstream manipulation is still lacking. Here, we aimed to develop a model that allows human germ cell differentiation in vitro in order to study the developing human germline. The hPGC-LCs specified from two iPS cell lines were sorted and manipulated using the PDPN surface marker without genetic modification. hPGC-LCs obtained remain arrested at early stages of maturation and no further differentiation nor meiotic onset occurred when these were cultured with human or mouse fetal ovarian somatic cells. However, when cultured independently of somatic ovarian cells, using BMP4 and the hanging drop-transferred EBs system, early hPGC-LCs further differentiate efficiently and express late PGC (DDX4) and meiotic gene markers, although no SYCP3 protein was detected. Altogether, we characterized a tool to sort hPGC-LCs and an efficient in vitro differentiation system to obtain pre-meiotic germ cell-like cells without using a gonadal niche.

Modeling Human Gonad Development in Organoids.

Our learning about human reproductive development is greatly hampered due to the absence of an adequate model. Animal studies cannot truthfully recapitulate human developmental processes, and studies of human fetal tissues are limited by their availability and ethical restrictions. Innovative three-dimensional (3D) organoid technology utilizing human pluripotent stem cells (hPSCs) offered a new approach to study tissue and organ development in vitro. However, a system for modeling human gonad development has not been established, thus, limiting our ability to study causes of infertility. In our study we utilized the 3D hPSC organoid culture in mini-spin bioreactors. Relying on intrinsic self-organizing and differentiation capabilities of stem cells, we explored whether organoids could mimic the development of human embryonic and fetal gonad. We have developed a simple, bioreactor-based organoid system for modeling early human gonad development. Male hPSC-derived organoids follow the embryonic gonad developmental trajectory and differentiate into multipotent progenitors, which further specialize into testicular supporting and interstitial cells. We demonstrated functional activity of the generated cell types by analyzing the expression of cell type-specific markers. Furthermore, the specification of gonadal progenitors in organoid culture was accompanied by the characteristic architectural tissue organization. This organoid system opens the opportunity for detailed studies of human gonad and germ cell development that can advance our understanding of sex development disorders. Implementation of human gonad organoid technology could be extended to modeling causes of infertility and regenerative medicine applications.

The balance between NANOG and SOX17 mediated by TET proteins regulates specification of human primordial germ cell fate

BackgroundHuman primordial germ cells (hPGCs) initiate from the early post-implantation embryo at week 2–3 and undergo epigenetic reprogramming during development. However, the regulatory mechanism of DNA methylation during hPGC specification is still largely unknown due to the difficulties in analyzing early human embryos. Using an in vitro model of hPGC induction, we found a novel function of TET proteins and NANOG in the hPGC specification which was different from that discovered in mice.MethodsUsing the CRISPR–Cas9 system, we generated a set of TET1, TET2 and TET3 knockout H1 human embryonic stem cell (hESC) lines bearing a BLIMP1-2A-mKate2 reporter. We determined the global mRNA transcription and DNA methylation profiles of pluripotent cells and induced hPGC-like cells (hPGCLCs) by RNA-seq and whole-genome bisulfite sequencing (WGBS) to reveal the involved signaling pathways after TET proteins knockout. ChIP-qPCR was performed to verify the binding of TET and NANOG proteins in the SOX17 promoter. Real-time quantitative PCR, western blot and immunofluorescence were performed to measure gene expression at mRNA and protein levels. The efficiency of hPGC induction was evaluated by FACS.ResultsIn humans, TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) impaired the NODAL signaling pathway and impeded hPGC specification in vitro, while the hyperactivated NODAL signaling pathway led to gastrulation failure when Tet proteins were inactivated in mouse. Specifically, TET proteins stimulated SOX17 through the NODAL signaling pathway and directly regulates NANOG expression at the onset of hPGCLCs induction. Notably, NANOG could bind to SOX17 promoter to regulate its expression in hPGCLCs specification. Furthermore, in TKO hESCs, DNMT3B-mediated hypermethylation of the NODAL signaling-related genes and NANOG/SOX17 promoters repressed their activation and inhibited hPGCLC induction. Knockout of DNMT3B in TKO hESCs partially restored NODAL signaling and NANOG/SOX17 expression, and rescued hPGCLC induction.ConclusionOur results show that TETs-mediated oxidation of 5-methylcytosine modulates the NODAL signaling pathway and its downstream genes, NANOG and SOX17, by promoting demethylation in opposition to DNMT3B-mediated methylation, suggesting that the epigenetic balance of DNA methylation and demethylation in key genes plays a fundamental role in early hPGC specification.

DAZL regulates proliferation of human primordial germ cells by direct binding to precursor miRNAs and enhances DICER processing activity.

Understanding the molecular and cellular mechanisms of human primordial germ cells (hPGCs) is essential in studying infertility and germ cell tumorigenesis. Many RNA-binding proteins (RBPs) and non-coding RNAs are specifically expressed and functional during hPGC developments. However, the roles and regulatory mechanisms of these RBPs and non-coding RNAs, such as microRNAs (miRNAs), in hPGCs remain elusive. In this study, we reported a new regulatory function of DAZL, a germ cell-specific RBP, in miRNA biogenesis and cell proliferation. First, DAZL co-localized with miRNA let-7a in human PGCs and up-regulated the levels of >100 mature miRNAs, including eight out of nine let-7 family, miR21, miR22, miR125, miR10 and miR199. Purified DAZL directly bound to the loops of precursor miRNAs with sequence specificity of GUU. The binding of DAZL to the precursor miRNA increased the maturation of miRNA by enhancing the cleavage activity of DICER. Furthermore, cell proliferation assay and cell cycle analysis confirmed that DAZL inhibited the proliferation of in vitro PGCs by promoting the maturation of these miRNAs. Evidently, the mature miRNAs up-regulated by DAZL silenced cell proliferation regulators including TRIM71. Moreover, DAZL inhibited germline tumor cell proliferation and teratoma formation. These results demonstrate that DAZL regulates hPGC proliferation by enhancing miRNA processing.

VASA protein and gene expression analysis of human non-obstructive azoospermia and normal by immunohistochemistry, immunocytochemistry, and bioinformatics analysis

VASA, also known as DDX4, is a member of the DEAD-box proteins and an RNA binding protein with an ATP-dependent RNA helicase. The VASA gene expression, which is required for human germ cell development, may lead to infertility. Immunocytochemistry and immunohistochemistry were used to examine the expression of VASA protein in the human testis sections of azoospermic patients, in-vitro and in-silico models. Some studies of fertile humans showed VASA expression in the basal and adluminal compartments of seminiferous tubules. Our Immunocytochemistry and immunohistochemistry in infertile humans showed expression of VASA in the luminal compartments of the seminiferous tubule. The immunohistochemical analysis of three human cases with different levels of non-obstructive azoospermia revealed a higher expression of VASA-positive cells. For this purpose, Enrichr and Shiny Gene Ontology databases were used for pathway enrichment analysis and gene ontology. STRING and Cytoscape online evaluation were applied to predict proteins' functional and molecular interactions and performed to recognize the master genes, respectively. According to the obtained results, the main molecular functions of the up-regulated and downregulated genes include the meiotic cell cycle, RNA binding, and differentiation. STRING and Cytoscape analyses presented seven genes, i.e., DDX5, TNP2, DDX3Y, TDRD6, SOHL2, DDX31, and SYCP3, as the hub genes involved in infertility with VASA co-function and protein–protein interaction. Our findings suggest that VASA and its interacting hub proteins could help determine the pathophysiology of germ cell abnormalities and infertility.

Localization Patterns of RAB3C Are Associated with Murine and Human Sperm Formation.

Background and Objectives: Septins (SEPTs) are highly conserved GTP-binding proteins and the fourth component of the cytoskeleton. Polymerization of SEPTs contributes to several critical cellular processes such as cytokinesis, cytoskeletal remodeling, and vesicle transportation. In our previous study, we found that SEPT14 mutations resulted in teratozoospermia with >87% sperm morphological defects. SEPT14 interactors were also identified through proteomic assays, and one of the peptides was mapped to RAB3B and RAB3C. Most studies on the RAB3 family have focused on RAB3A, which regulates the exocytosis of neurotransmitters and acrosome reactions. However, the general expression and patterns of the RAB3 family members during human spermatogenesis, and the association between RAB3 and teratozoospermia owing to a SEPT14 mutation, are largely unknown. Materials and Methods: Human sperm and murine male germ cells were collected in this study and immunofluorescence analysis was applied on the collected sperm. Results: In this study, we observed that the RAB3C transcripts were more abundant than those of RAB3A, 3B, and 3D in human testicular tissues. During human spermatogenesis, the RAB3C protein is mainly enriched in elongated spermatids, and RAB3B is undetectable. In mature human spermatozoa, RAB3C is concentrated in the postacrosomal region, neck, and midpiece. The RAB3C signals were delocalized within human spermatozoa harboring the SEPT14 mutation, and the decreased signals were accompanied by a defective head and tail, compared with the healthy controls. To determine whether RAB3C is involved in the morphological formation of the head and tail of the sperm, we separated murine testicular tissue and isolated elongated spermatids for further study. We found that RAB3C is particularly expressed in the manchette structure, which assists sperm head shaping at the spermatid head, and is also localized at the sperm tail. Conclusions: Based on these results, we suggest that the localization of RAB3C proteins in murine and human sperm is associated with SEPT14 mutation-induced morphological defects in sperm.

The President's Hat.

<h3>ABSTRACT</h3> The hominoid-specific endogenous retrovirus LTR5_Hs is transcriptionally activated in human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived cell culture model of PGCs. Here, taking the unique advantage of our novel cell culture method to obtain large amounts of pure hPGCLCs, we performed proteomics profiling of hPGCLCs and detected various viral proteins produced from the LTR5_Hs RNA <i>via</i> ribosomal frameshifting. We also present transmission electron microscopy images of 100-nm diameter virus-like particles (VLPs) assembled at the surface of hPGCLCs. Compared to hPGCLCs, expression of LTR5_Hs RNA is far weaker in human seminomas, the germ cell tumors resembling PGCs. Re-analysis of published single cell RNA-seq data of human embryos revealed strong activation of LTR5_Hs in migrating PGCs but suppressed in PGCs upon they reach the gonadal anlagen. In the microfluidics-supported polarized embryoids mimicking peri-implantation stages of human embryos, LTR5_Hs RNA was detected by RNA in situ hybridization in NANOG⁺/TFAP2C⁺/SOX17⁺ cells resembling freshly emerged PGCs. These results support that human germ cells produce LTR5_Hs proteins and VLPs during their earliest stages of normal development until their settlement in the gonadal anlagen. <h3>SUMMARY STATEMENT</h3> The hominoid-specific endogenous retrovirus LTR5_Hs is activated in a cell culture model resembling early-stage human primordial germ cells, producing not only viral RNA but also retrovirus proteins and virus-like particles.

Cloning and characterization of a novel functional organic anion transporting polypeptide 3A1 isoform highly expressed in the human brain and testis.

Organic anion transporting polypeptide 3A1 (OATP3A1, encoded by the SLCO3A1 gene) is a prostaglandin, oligopeptide, and steroid/thyroid hormone transporter with wide tissue distribution, expressed, e.g., in the human brain and testis. Although the physiological importance of OATP3A1 has not yet been clarified, based on its expression pattern, substrate recognition, and evolutionary conservation, OATP3A1 is a potential pharmacological target. Previously, two isoforms of OATP3A1, termed as V1 and V2, have been characterized. Here, we describe the cloning and functional characterization of a third isoform, OATP3A1_V3. The mRNA of isoform V3 is formed by alternative splicing and results in an OATP3A1 protein with an altered C-terminus compared to isoforms V1 and V2. Based on quantitative PCR, we demonstrate the widespread expression of SLCO3A1_V3 mRNA in human organs, with the highest expression in the brain and testis. By generation of an isoform V3-specific antibody and immunostaining, we show that the encoded protein is expressed in the human choroid plexus, neurons, and both germ and Sertoli cells of the testis. Moreover, we demonstrate that in contrast to isoform V1, OATP3A1_V3 localizes to the apical membrane of polarized MDCKII cells. Using HEK-293 cells engineered to overexpress OATP3A1_V3, we verify the protein’s functionality and identify dehydroepiandrosterone sulfate as a novel OATP3A1 substrate. Based on their distinct expression patterns but overlapping functions, OATP3A1 isoforms may contribute to transcellular (neuro)steroid transport in the central nervous system.

Single‐cell nucleic acid sequencing reinforces to reveal signal‐ dependent pathways contributing to cellular heterogeneity

Single‐cell nucleic acid sequencing reinforces to reveal signal‐ dependent pathways contributing to cellular heterogeneity

Meiosis resumption in human primordial germ cells from induced pluripotent stem cells by in vitro activation and reconstruction of ovarian nests

BackgroundThe differentiation of human induced pluripotent stem cells (iPSCs) into oocytes, which involves the transformation from mitosis to meiosis, has been a hotspot of biological research for many years and represents a desirable experimental model and potential strategy for treating infertility. At present, studies have shown that most cells stagnate in the oogonium stage after differentiation into primordial germ cells (PGCs) from human iPSCs.MethodsiPSCs carrying a SYCP3-mkate2 knock-in reporter were generated by the CRISPR/Cas9 strategy to monitor meiosis status during induced differentiation from iPSCs into oocytes. These induced PGCs/oogonia were activated by small molecules from the Wnt signaling pathway and then cocultured with reconstructed human ovarian nests in vivo for further development.ResultsFirst, human PGCs and oogonia were efficiently induced from iPSCs. Second, induced dormant PGCs resumed meiosis and then differentiated into primary oocytes through the in vitro activation of the Wnt signaling pathway. Finally, a new coculture system involving the reconstruction of ovarian nests in vitro could facilitate the differentiation of oocytes.ConclusionsHuman PGCs/oogonia induced from iPSCs can be activated and used to resume meiosis by molecules of the Wnt signaling pathway. The coculture of activated PGCs and reconstruction of ovarian nests facilitated differentiation into primary oocytes and the generation of haploid human oocytes in vivo. These findings established a new strategy for germline competence in primary oocytes and provided a keystone for human gametogenesis in vitro and in vivo.

Advances in studying human gametogenesis and embryonic development in China†.

Reproductive medicine in China has developed rapidly since 1988 due to support from the government and scientific exploration. However, the success rate of assisted reproduction technology is around 30-40% and many unknown "black boxes" in gametogenesis and embryo development are still present. With the development of single-cell and low-input sequencing technologies, the network of transcriptome and epigenetic regulation (DNA methylation, chromatin accessibility, and histone modifications) during the development of human primordial germ cells, gametes, and embryos has been investigated in depth. Furthermore, preimplantation genetic testing has also rapidly developed. In this review, we summarize and analyze China's outstanding progress in these fields.

O-233 Cell-cell interactions within germ cell nests reveal the mechanism of primordial follicle formation in humans

Abstract Study question How interactions between human fetal female germ cells (hFGCs) and pre-granulosa cells (pre-GCs) affect the hFGCs transition from ovarian cord to primordial follicles (PFs). Summary answer Progressive engulfment of hFGCs by pre-GCs and establishment of cell-cell junctions and cell-cell adhesions between hFGCs and pre-GCs leads to PF formation in humans. What is known already In mice, development of FGCs is synchronized due to formation of cysts. Cysts comprise of sister FGCs that remain connected via intercellular bridges until germ cell nest breakdown is initiated and PFs are formed. In contrast, hFGCs differentiate asynchronously. In second trimester human ovaries, mitotic, RA-responsive, meiotic and oogonia hFGCs are present in designated parts of the ovary that corresponds to their differentiation stage. Meiotic hFGCs enter prophase I that is divided into 4 substages: leptotene, zygotene, pachytene, diplotene/dictyate. hFGCs in diplotene are ready to form a PF with pre-GCs and become dormant. Study design, size, duration We collected in total 17 second trimester human fetal ovaries between 14 and 22 weeks of gestation from elective abortions. We have analyzed cellular structures of hFGC and pre-GCs by transmission electron microscopy and investigated different cell adhesion and cell junction proteins by immunostaining. Participants/materials, setting, methods Materials: human fetal ovaries between 14 and 22 weeks of gestation. Methods: characterization of tissue morphology, immunofluorescence staining, whole mount staining, confocal microscopy, super-resolution microscopy, transmission electron microscopy (TEM), cell counting Main results and the role of chance Meiotic hFGCs formed germ cell cysts consisting of multiple sister hFGCs connected by intercellular bridges. Furthermore, we discovered that a subset of hFGCs formed syncytia containing several nuclei (2–4 nuclei). Syncytium formation was caused by cytokinesis failure when an in intercellular bridge failed to be formed. In addition, in some cases hFGC syncytia were connected to other syncytia or mono-nucleated hFGCs by an intercellular bridges. Those multinucleated syncytia did not display signs of apoptosis or morphological abnormalities. Investigation of physical interactions between hFGCs and pre-GCs revealed that as hFGCs enter prophase I, pre-GCs (FOXL2+, VIM+, CDH1+) form membrane protrusions (MPs) and progressively engulf them. First, CDH1+ and VIM+ domains within a pre-GC are mixed, but as engulfment progresses two distinct domains are formed. MP formation is possible due to mesenchymal-epithelial hybrid state and active Hippo pathway in pre-GCs. Inside the cords, hFGCs highly express CDH2, while CDH2 is lowly expressed in pre-GCs. At diplotene, hFGCs (TP63+) downregulate CDH2 and upregulate CDH1. Expression of CDH1 in both diplotene hFGC and pre-GCs causes increase of cell-cell adhesion and enables PF formation. In addition, progressive formation of tight junctions (TJP1+) and cellular interdigitations between pre-GCs and meiotic hFGCs reinforce PF structure. Limitations, reasons for caution Due to technical limitations and lack of in vitro culture protocols that support meiotic hFGCs development and survival as well as human PF formation in vitro, we cannot experimentally validate roles of cadherins and other cell junction and cell adhesion proteins in PF formation Wider implications of the findings In depth understanding of PF formation process in terms of physical interactions between hFGCs and pre-GCs will be crucial to design in vitro conditions that will support PF formation and our findings will be key to achieve assembling of PFs in artificial human oocytes (gametes) created from pluripotent stem cells. Trial registration number not applicable

Human Testicular Germ Cells, a Reservoir for Zika Virus, Lack Antiviral Response Upon Zika or Poly(I:C) Exposure.

Zika virus (ZIKV) is an emerging teratogenic arbovirus that persists in semen and is sexually transmitted. We previously demonstrated that ZIKV infects the human testis and persists in testicular germ cells (TGCs) for several months after patients’ recovery. To decipher the mechanisms underlying prolonged ZIKV replication in TGCs, we compared the innate immune response of human testis explants and isolated TGCs to ZIKV and to Poly(I:C), a viral RNA analog. Our results demonstrate the weak innate responses of human testis to both ZIKV and Poly(I:C) as compared with other tissues or species. TGCs failed to up-regulate antiviral effectors and type I IFN upon ZIKV or Poly(I:C) stimulation, which might be due to a tight control of PRR signaling, as evidenced by the absence of activation of the downstream effector IRF3 and elevated expression of repressors. Importantly, exogenous IFNβ boosted the innate immunity of TGCs and inhibited ZIKV replication in the testis ex vivo, raising hopes for the prevention of ZIKV infection and persistence in this organ.

Distinct Roles of NANOS1 and NANOS3 in the Cell Cycle and NANOS3-PUM1-FOXM1 Axis to Control G2/M Phase in a Human Primordial Germ Cell Model.

Nanos RNA-binding proteins are critical factors of germline development throughout the animal kingdom and their dysfunction causes infertility. During evolution, mammalian Nanos paralogues adopted divergent roles in germ cell biology. However, the molecular basis behind this divergence, such as their target mRNAs, remains poorly understood. Our RNA-sequencing analysis in a human primordial germ cell model-TCam-2 cell line revealed distinct pools of genes involved in the cell cycle process downregulated upon NANOS1 and NANOS3 overexpression. We show that NANOS1 and NANOS3 proteins influence different stages of the cell cycle. Namely, NANOS1 is involved in the G1/S and NANOS3 in the G2/M phase transition. Many of their cell cycle targets are known infertility and cancer-germ cell genes. Moreover, NANOS3 in complex with RNA-binding protein PUM1 causes 3′UTR-mediated repression of FOXM1 mRNA encoding a transcription factor crucial for G2/M phase transition. Interestingly, while NANOS3 and PUM1 act as post-transcriptional repressors of FOXM1, FOXM1 potentially acts as a transcriptional activator of NANOS3, PUM1, and itself. Finally, by utilizing publicly available RNA-sequencing datasets, we show that the balance between FOXM1-NANOS3 and FOXM1-PUM1 expression levels is disrupted in testis cancer, suggesting a potential role in this disease.

Xenotransplantation of Human Spermatogonia Into Various Mouse Recipient Models.

Spermatogonial stem cells are the foundation of continuous spermatogenesis in adult mammals. Xenograft models have been established to define human SSCs, mostly using infertile and immune-deficient mice as the recipients for human germ cell transplantation. However, it is time-consuming to prepare such recipients using irradiation or chemotherapeutic agents, and this approach may also introduce confounding factors when residual endogenous germ cells recover in transplanted recipients. It remains to be determined whether immune-competent genetically infertile mice can be suitable recipients for xenotransplantation. In this study, we observed similar engraftment efficiencies when using spermatogonia from human biopsied testes across immune-deficient nude mice, immune-competent ICR mice, and genetically infertile Kit w/w-v mice, suggesting minimal immunological rejection from immune-competent mouse recipients upon xenotransplantation of human germ cells. More importantly, we derived EpCAM negative and TNAP positive spermatogonia-like cells (SLCs) from human pluripotent stem cells (PSCs), which highly expressed spermatogonial markers including PLZF, INTERGRINα6, TKTL1, CD90, and DRMT3. We found that upon transplantation, these SLCs proliferated and colonized at the basal membrane of seminiferous tubules in testes of both immune-deficient nude mice and Kit w/w-v mice, though complete spermatogenesis would likely require supporting human signaling factors and microenvironment. Taken together, our study functionally defined the cell identity of PSC-derived SLCs, and supported xenotransplantation using genetically infertile recipients as a convenient model for functionally evaluating spermatogonia derived from different species.

FGFR3 is expressed by human primordial germ cells and is repressed after meiotic initiation to form primordial oocytes.

Human germ cell development is a highly regulated process beginning soon after embryo implantation with the specification of primordial germ cells (PGCs) and ending in adulthood with the differentiation of gametes. Here, we show that fibroblast growth factor receptor 3 (FGFR3) is expressed by human PGCs during the first and second trimester, becoming repressed as PGCs differentiate into primordial oocytes. Using fluorescence-activated cell sorting (FACS) with antibodies that recognize FGFR3 followed by single-cell RNA sequencing, we show that isolating FGFR3-positive cells enriches for human PGCs. Taken together, FGFR3 could be used in future studies as a strategy to identify maturing PGCs in vitro.

Expanding Homogeneous Culture of Human Primordial Germ Cell–Like Cells Maintaining Germline Features Without Serum or Feeder Layers

(Abstracted from Stem Cell Rep 2022;17(3):507–521) Primordial germ cells (PGCs) are embryonic precursors of gametes that are extensively studied in research settings. Human primordial PGC–like cells (hPGCLCs) are a surrogate model for embryonic PGCs that present difficulties in in vitro cell culture and expansion due to rapid loss of PGC-like identity and limited cell survival.

Efficient differentiation of human primordial germ cells through geometric control reveals a key role for Nodal signaling.

Human primordial germ cells (hPGCs) form around the time of implantation and are the precursors of eggs and sperm. Many aspects of hPGC specification remain poorly understood because of the inaccessibility of the early postimplantation human embryo for study. Here, we show that micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 give rise to hPGC-like cells (hPGCLC) and use these as a quantitatively reproducible and simple in vitro model to interrogate this important developmental event. We characterize micropatterned hPSCs up to 96 hr and show that hPGCLC populations are stable and continue to mature. By perturbing signaling during hPGCLC differentiation, we identify a previously unappreciated role for Nodal signaling and find that the relative timing and duration of BMP and Nodal signaling are critical parameters controlling the number of hPGCLCs. We formulate a mathematical model for a network of cross-repressive fates driven by Nodal and BMP signaling, which predicts the measured fate patterns after signaling perturbations. Finally, we show that hPSC colony size dictates the efficiency of hPGCLC specification, which led us to dramatically improve the efficiency of hPGCLC differentiation.