Three induced pluripotent stem cell sublines (hiPSCs) were generated from human dermal human dermal fibroblasts (HDFs) derived from a human skin punch biopsy. The biopsy was donated from a woman with known infertility due to ovarian failure. The hiPSC sublines were created using Sendai virus vectors and were positive for markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA-4. Pluripotency was verified using PluriTest analysis and in vitro differentiation using Taqman Real-Time PCR assays for somatic lineage markers. This participant's monozygotic twin sister also donated a skin-punch biopsy, whose resulting hiPSC lines were published previously as a resource.

Germ cell tumors (GCTs) are a heterogeneous group of tumors occurring in gonadal and extragonadal locations. GCTs are hypothesized to arise from primordial germ cells (PGCs), which fail to differentiate. One recently identified susceptibility loci for human GCT is PR (PRDI-BF1 and RIZ) domain proteins 14 (PRDM14). PRDM14 is expressed in early primate PGCs and is repressed as PGCs differentiate. To examine PRDM14 in human GCTs we profiled human GCT cell lines and patient samples and discovered that PRDM14 is expressed in embryonal carcinoma cell lines, embryonal carcinomas, seminomas, intracranial germinomas and yolk sac tumors, but is not expressed in teratomas. To model constitutive overexpression in human PGCs, we generated PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs) and discovered that elevated expression of PRDM14 does not block early PGC formation. Instead, we show that elevated PRDM14 in PGCLCs causes proliferation and differentiation defects in the germline.

We generated three human induced pluripotent stem cell (hiPSC) sublines from human dermal fibroblasts (HDF) (MZT05) generated from a skin biopsy donated from a previously fertile woman. The skin biopsy was broadly consented for generating hiPSC lines for biomedical research, including unique consent specifically for studying human fertility, infertility and germ cell differentiation. hiPSCs were reprogrammed using Sendai virus vectors and were subsequently positive for markers of self-renewal. Pluripotency was further verified using PluriTest analysis and in vitro differentiation was tested using Taqman Real-Time PCR assays. These sublines serve as controls for hiPSC research projects aimed at understanding the cell and molecular regulation of female fertility.

Six human induced pluripotent stem cell sublines (hiPSCs) were generated from human dermal fibroblasts (HDFs) derived from skin biopsies donated from monozygotic twin women wherein one woman had proven fertility and her sister was infertile due to ovarian failure. Three hiPSC sublines were created from each twin's HDFs. hiPSCs were reprogrammed using Sendai virus vectors and were subsequently positive for markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA-4. Pluripotency was further verified using PluriTest. We show here that the hiPSC lines created from the twins are equivalent in measures of pluripotency and self-renewal, despite their differential diagnosis.

Background

In Central Asia, between 33% and 72% of cumulative HIV infections has been attributed to unsafe injection practices among people who inject drugs (PWID).

Methods

We reviewed the current status and trends of national efforts in Central Asian countries to control HIV among PWID, and also reviewed the key structural and health-systems-related challenges that facilitate drug-use-related HIV risk in Central Asia.

Results

The spectrum and scale of HIV prevention services targeting PWID vary considerably among Central Asian countries. In all countries, the potential impact of these interventions is hindered by several key features: a restrictive legal environment, poor performance of service providers, widespread opposition to harm reduction, deficient human resources and funding mechanisms, poor services integration, insufficient community involvement, and other structural factors.

Conclusions

Scaling up HIV prevention interventions in Central Asia will demand greater attention to the structural, health-care-related and social factors that facilitate HIV risk and impede service utilization among PWID. Multi-level combination prevention interventions should be developed with a focus on the sexual partners and risk networks of PWID, aiming at early detection of HIV, timely enrollment in HIV care, and retention in HIV care.

Naive and primed pluripotent human embryonic stem cells bear transcriptional similarity to pre- and post-implantation epiblast and thus constitute a developmental model for understanding the pluripotent stages in human embryo development. To identify new transcription factors that differentially regulate the unique pluripotent stages, we mapped open chromatin using ATAC-seq and found enrichment of the activator protein-2 (AP2) transcription factor binding motif at naive-specific open chromatin. We determined that the AP2 family member TFAP2C is upregulated during primed to naive reversion and becomes widespread at naive-specific enhancers. TFAP2C functions to maintain pluripotency and repress neuroectodermal differentiation during the transition from primed to naive by facilitating the opening of enhancers proximal to pluripotency factors. Additionally, we identify a previously undiscovered naive-specific POU5F1 (OCT4) enhancer enriched for TFAP2C binding. Taken together, TFAP2C establishes and maintains naive human pluripotency and regulates OCT4 expression by mechanisms that are distinct from mouse.

Three-dimensional (3D) stem cell models of the ovary have the potential to benefit womens reproductive health research. One such model, the reconstituted ovary (rOvary) self-assembles with pluripotent stem cell-derived germ cells creating a 3D ovarian mimic competent to support the differentiation of functional oocytes inside follicles. In this study, we evaluated the cellular composition of the rOvary revealing the capacity to generate multiple follicles surrounded by NR2F2+ stroma cells. However, the rOvary does not develop a surface epithelium, the source of second-wave pre-granulosa cells, or steroidogenic theca. Therefore, the rOvary models represent the self-assembly of activated follicles in a pre-pubertal ovary poised but not yet competent for hormone production.

Development of primordial germ cells (PGCs) is required for reproduction. During PGC development in mammals, major epigenetic remodeling occurs, which is hypothesized to establish an epigenetic landscape for sex-specific germ cell differentiation and gametogenesis. In order to address the role of embryonic ectoderm development (EED) and histone 3 lysine 27 trimethylation (H3K27me3) in this process, we created an EED conditional knockout mouse and show that EED is essential for regulating the timing of sex-specific PGC differentiation in both ovaries and testes, as well as X chromosome dosage decompensation in testes. Integrating chromatin and whole genome bisulfite sequencing of epiblast and PGCs, we identified a poised repressive signature of H3K27me3/DNA methylation that we propose is established in the epiblast where EED and DNMT1 interact. Thus, EED joins DNMT1 in regulating the timing of sex-specific PGC differentiation during the critical window when the gonadal niche cells specialize into an ovary or testis.

Primordial germ cells (PGCs) are the embryonic precursors of sperm and eggs. They transmit genetic and epigenetic information across generations. Given the prominent role of germline defects in diseases such as infertility, detailed understanding of human PGC (hPGC) development has important implications in reproductive medicine and studying human evolution. Yet, hPGC specification remains an elusive process. Here, we report the induction of hPGC-like cells (hPGCLCs) in a bioengineered human pluripotent stem cell (hPSC) culture that mimics peri-implantation human development. In this culture, amniotic ectoderm-like cells (AMLCs), derived from hPSCs, induce hPGCLC specification from hPSCs through paracrine signaling downstream of ISL1. Our data further show functional roles of NODAL, WNT, and BMP signaling in hPGCLC induction. hPGCLCs are successfully derived from eight non-obstructive azoospermia (NOA) participant-derived hPSC lines using this biomimetic platform, demonstrating its promise for screening applications.