No abstract

Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications.

CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

These modified fibroblasts are useful as a genetically-defined feeder cell line for reproducible and cost-effective culture of both hESC and iPS cells.