SUMMARY Human primordial germ cells (hPGCs) are the first embryonic progenitors in the germ cell lineage, yet the molecular mechanisms required for hPGC formation are not well characterized. To identify regulatory regions in hPGC development, we used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to systematically characterize regions of open chromatin in hPGCs and hPGC-like cells (hPGCLCs) differentiated from human embryonic stem cells (hESCs). We discovered regions of open chromatin unique to hPGCs and hPGCLCs that significantly overlap with TFAP2C-bound enhancers identified in the naive ground state of pluripotency. Using CRISPR/Cas9, we show that deleting the TFAP2C-bound naive enhancer at the OCT4 locus (also called POU5F1) results in impaired OCT4 expression and a negative effect on hPGCLC identity.

The signaling pathways leading to the development of asbestos-associated diseases are poorly understood. Here we used normal and protein kinase C (PKC)-delta knockout (PKCdelta-/-) mice to demonstrate multiple roles of PKC-delta in the development of cell proliferation and inflammation after inhalation of chrysotile asbestos. At 3 days, asbestos-induced peribronchiolar cell proliferation in wild-type mice was attenuated in PKCdelta-/- mice. Cytokine profiles in bronchoalveolar lavage fluids showed increases in interleukin (IL)-1beta, IL-4, IL-6, and IL-13 that were decreased in PKCdelta-/- mice. At 9 days, microarray and quantitative reverse transcriptase-polymerase chain reaction analysis of lung tissues revealed increased mRNA levels of the profibrotic cytokine, IL-4, in asbestos-exposed wild-type mice but not PKCdelta-/- mice. PKCdelta-/- mice also exhibited decreased lung infiltration of polymorphonuclear cells, natural killer cells, and macrophages in bronchoalveolar lavage fluid and lung, as well as increased numbers of B lymphocytes and plasma cells. These changes were accompanied by elevated mRNA levels of immunoglobulin chains. These data show that modulation of PKC-delta has multiple effects on peribronchiolar cell proliferation, proinflammatory and profibrotic cytokine expression, and immune cell profiles in lung. These results also implicate targeted interruption of PKC-delta as a potential therapeutic option in asbestos-induced lung diseases.

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The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis.

To investigate the role of bronchiolar epithelial NF-κB activity in the development of inflammation and fibrogenesis in a murine model of asbestos inhalation, we used transgenic (Tg) mice expressing an IκBα mutant (IκBαsr) resistant to phosphorylation-induced degradation and targeted to bronchial epithelium using the CC10 promoter. Sham and chrysotile asbestos-exposed CC10-IκBαsr Tg+ and Tg− mice were examined for altered epithelial cell proliferation and differentiation, cytokine profiles, lung inflammation, and fibrogenesis at 3, 9, and 40 days. KC, IL-6 and IL-1β were increased (p ≤ 0.05) in bronchoalveolar lavage fluid (BALF) from asbestos-exposed mice, but to a lesser extent (p ≤ 0.05) in Tg+ vs Tg− mice. Asbestos also caused increases in IL-4, MIP-1β, and MCP-1 in BALF that were more elevated (p ≤ 0.05) in Tg+ mice at 9 days. Differential cell counts revealed eosinophils in BALF that increased (p ≤ 0.05) in Tg+ mice at 9 days, a time point corresponding with significantly increased numbers of bronchiolar epithelial cells staining positively for mucus production. At all time points, asbestos caused increased numbers of distal bronchiolar epithelial cells and peribronchiolar cells incorporating the proliferation marker, Ki-67. However, bronchiolar epithelial cell and interstitial cell labeling was diminished at 40 days (p ≤ 0.05) in Tg+ vs Tg− mice. Our findings demonstrate that airway epithelial NF-κB activity plays a role in orchestrating the inflammatory response as well as cell proliferation in response to asbestos.

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.

Human fertility is dependent upon the correct establishment and differentiation of the germline. This is because no other cell type in the body is capable of passing a genome and epigenome from parent to child. Terminally differentiated germline cells in the adult testis and ovary are called gametes. However, the initial specification of germline cells occurs in the embryo around the time of gastrulation. Most of our knowledge regarding the cell and molecular events that govern human germline specification involves extrapolating scientific principles from model organisms, most notably the mouse. However, recent work using next generation sequencing, gene editing and differentiation of germline cells from pluripotent stem cells has revealed that the core molecular mechanisms that regulate human germline development are different from rodents. Here, we will discuss the major molecular pathways required for human germline differentiation and how pluripotent stem cells have revolutionized our ability to study the earliest steps in human embryonic lineage specification to understand human fertility.

During development, human primordial germ cells (hPGCs) transition through a transcriptional and epigenetic state similar to pre-implantation naive ground state epiblast cells. In hPGCs, this state is called naive-like ground state pluripotency. Diagnostic transcription factors that define this state include TFAP2C, KLF4, and TFCP2L1, with TFAP2C necessary for both establishment of the naive-like ground state in hPGC-like cells (hPGCLCs) and establishment of naive ground state human embryonic stem cells (hESCs). Here, we show that KLF4 and TFCP2L1 are not required for hPGC specification or establishment of the naive-like ground state in hPGCLCs. Instead, KLF4 and TFCP2L1 are each required for reversion of primed hESCs to the self-renewing naive ground state. Additionally, TFCP2L1 but not KLF4 function after hPGC specification in the proliferation and survival of hPGCLCs.