Project description:Retention of source cell-type epigenetic memory may mitigate the potential for induced pluripotent stem cells (iPSCs) to fully achieve transitions in cell fate in vitro. While this may not preclude the use of iPSC-derived somatic cell types for therapeutic applications, it becomes a major concern impacting the potential use of iPSC-derived germline cell types for reproductive applications. The transition from a source somatic cell type to iPSCs and then on to germ-cell like cells (GCLCs) recapitulates two major epigenetic reprogramming events that normally occur during development in vivo -embryonic reprogramming in the epiblast and germline reprogramming in primordial germ cells (PGCs). We examined the extent of epigenetic and transcriptomic memory persisting first during the transition from differentiated source cell types to iPSCs, and then during the transition from iPSCs to PGC-like cells (PGCLCs). We derived iPSCs from four differentiated mouse cell types including two somatic and two germ cell types and tested the extent to which each resulting iPSC line resembled a) a validated ES cell reference line, and b) their respective source cell types, on the basis of genome-wide gene expression and DNA methylation patterns. We then induced each iPSC line to form PGCLCs, and assessed epigenomic and transcriptomic memory in each compared to endogenous PGCs/M-prospermatogonia. In each iPSC line, we found residual gene expression and epigenetic programming patterns characteristic of the corresponding source differentiated cell type from which each was derived. However, upon deriving PGCLCs, we found very little evidence of lingering epigenetic or transcriptomic memory of the original source cell type. This result indicates that derivation of iPSCs and then GCLCs from differentiated source cell types in vitro recapitulates the two-phase epigenetic reprogramming that normally occurs in vivo, and that, to a significant extent, germline cell types derived in vitro from pluripotent cells accurately recapitulate epigenetic programming and gene expression patterns corresponding to equivalent endogenous germ cell types, suggesting that they have the potential to form the basis of in vitro gametogenesis as a useful therapeutic strategy for treatment of infertility.

Project description:Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor Tcfap2c / TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of Tcfap2c in primordial germ cell-like cells. We show that loss of Tcfap2c affects many aspects of the genetic network regulating germ cell biology, such as downregulation maturation markers and induction of markers indicative of somatic differentiation, cell cycle, epigenetic remodeling, and pluripotency associated genes. Chromatin-immunoprecipitation analyses demonstrated binding of Tcfap2c to regulatory regions of deregulated genes (Sfrp1, Dmrt1, Nanos3, c-Kit, Cdk6, Cdkn1a, Fgf4, Klf4, Dnmt3b and Dnmt3l) suggesting that these genes are direct transcriptional targets of Tcfap2c in primordial germ cells. Since Tcfap2c deficient primordial germ cell like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the Tcfap2c-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for Tcfap2c develop germ cell cancer with high incidence. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate, that mice with a heterozygous deletion of the Tcfap2c target gene Nanos3 are also prone to develop teratoma. These data highlight Tcfap2c as a critical and dose-sensitive regulator of germ cell fate. 8 samples were analyzed. Ctrl ESC: Control mouse embryonic stem cells (ESCs), 2 biological rep KO ESC: Tcfap2c knock-out mouse embryonic stem cells (ESCs), 2 biological rep Ctrl PGC: Control mouse primordial germ cells (PGCs), 2 biological rep KO PGC: Tcfap2c knock-out mouse primordial germ cells (PGCs), 2 biological rep

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The vertebrate protein Dead end maintains primordial germ cell fate by inhibiting somatic differentiation

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