Principles of early human development and germ cell program from conserved model systems

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during week 2-3 of early postimplantation development(1). Using in vitro models of hPGC induction(2-4), recent studies suggest striking mechanistic differences in specification of human and mouse PGCs(5). This may partly be due to the divergence in their pluripotency networks, and early postimplantation development(6-8). Since early human embryos are inaccessible for direct studies, we considered alternatives, including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs (pPGCs) originate from the posterior pre-primitive streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. Together with human and monkey in vitro models simulating peri-gastrulation development, we show conserved principles for epiblast development for competency for PGC fate, followed by initiation of the epigenetic program(9-11), regulated by a balanced SOX17–BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models, provides synthetic insights on early human development

NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers.

Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice, where its precise role is yet unclear. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nature1648

Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines

Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours’ biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription–quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis.

BACKGROUND: Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed. RESULTS: Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated. CONCLUSION: The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells

Carbon-based cores with polyglycerol shells – the importance of core flexibility for encapsulation of hydrophobic guests

Two core–shell nanoparticles with polyglycerol shells and sp3 carbon cores with different flexibilities (soft dendritic polyethylene and hard nanodiamond) were synthesized, their encapsulation capacities were compared, and their ability to transport into tumor cells was investigated. The nanocarrier with a soft core was superior to the hard one

Direct comparison of distinct naive pluripotent states in human embryonic stem cells

Stem cells & developmental biolog