Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression

The transcription factor Oct4 is well defined as a key regulator of embryonic stem (ES) cell pluripotency. In recent years, the role of Oct4 has purportedly extended to the self renewal and maintenance of multipotency in adult stem cell (ASC) populations. This profile has arisen mainly from reports utilising reverse transcription-polymerase chain reaction (RT-PCR) based methodologies and has since come under scrutiny following the discovery that many developmental genes have multiple pseudogenes associated with them. Six known pseudogenes exist for Oct4, all of which exhibit very high sequence homology (three >97%), and for this reason the generation of artefacts may have contributed to false identification of Oct4 in somatic cell populations. While ASC lack a molecular blueprint of transcription factors proposed to be involved with 'stemness' as described for ES cells, it is not unreasonable to assume that similar gene patterns may exist. The focus of this work was to corroborate reports that Oct4 is involved in the regulation of ASC self-renewal and differentiation, using a combination of methodologies to rule out pseudogene interference. Haematopoietic stem cells (HSC) derived from human umbilical cord blood (UCB) and various differentiated cell lines underwent RT-PCR, product sequencing and transfection studies using an Oct4 promoter-driven reporter. In summary, only the positive control expressed Oct4, with all other cell types expressing a variety of Oct4 pseudogenes. Somatic cells were incapable of utilising an exogenous Oct4 promoter construct, leading to the conclusion that Oct4 does not appear involved in the multipotency of human HSC from UCB

Neural Stem Cells Achieve and Maintain Pluripotency without Feeder Cells

Background: Differentiated cells can be reprogrammed into pluripotency by transduction of four defined transcription factors. Induced pluripotent stem cells (iPS cells) are expected to be useful for regenerative medicine as well as basic research. Recently, the report showed that mouse embryonic fibroblasts (MEF) cells are not essential for reprogramming. However, in using fibroblasts as donor cells for reprogramming, individual fibroblasts that had failed to reprogram could function as feeder cells. Methodology/Principal Finding: Here, we show that adult mouse neural stem cells (NSCs), which are not functional feeder cells, can be reprogrammed into iPS cells using defined four factors (Oct4, Sox2, Klf4, and c-Myc) under feeder-free conditions. The iPS cells, generated from NSCs expressing the Oct4-GFP reporter gene, could proliferate for more than two months (passage 20). Generated and maintained without feeder cells, these iPS cells expressed pluripotency markers (Oct4 and Nanog), the promoter regions of Oct4 and Nanog were hypomethylated, could differentiated into to all three germ layers in vitro, and formed a germline chimera. These data indicate that NSCs can achieve and maintain pluripotency under feeder-free conditions. Conclusion/Significance: This study suggested that factors secreted by feeder cells are not essential in the initial/early stages of reprogramming and for pluripotency maintenance. This technology might be useful for a human system, as

Genome editing reveals a role for OCT4 in human embryogenesis.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.DW was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. NK was supported by the University of Oxford Clarendon Fund. AB was supported by a British Heart Foundation PhD Studentship (FS/11/77/39327). LV was supported by core grant funding from the Wellcome Trust and Medical Research Council (PSAG028). J-SK was supported by the Institute for Basic Science (IBS-R021-D1). Work in the KKN and JMAT labs was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust (FC001120 and FC001193)

The FunGenES Database: A Genomics Resource for Mouse Embryonic Stem Cell Differentiation

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the “Functional Genomics in Embryonic Stem Cells” consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in “Expression Waves” and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells

Adipocyte differentiation in human embryonic stem cells transduced with Oct4 shRNA lentivirus

Oct4 is one of the master pluripotency genes that controls differentiation of human embryonic stem cells (hESCs). We generated HES2 and HES3 hESC lines stably transduced with lentivirus carrying Oct4 short hairpin RNA (shRNA) that display 80–90% reduction of Oct4 expression. Analysis of pluripotency marker expression shows that these Oct4 shRNA-transduced hESCs display normal wild-type expression levels of the pluripotency marker CD9 but an absence of GCTM2 expression. These hESC-derived adipocyte precursor cells display a characteristic morphology and can be propagated and cryopreserved as a standard stem cell line. Interestingly, Oct4 shRNA-transduced hESCs display a remarkably high lineage-specific spontaneous differentiation toward adipocytes. After two weeks of spontaneous differentiation under feeder-free conditions, 60–70% of cells display a mature adipocyte morphology as well as the expression of multiple adipocyte-specific mRNAs as assessed by RT-PCR. The upregulation of trophoblast, mesoderm, and endoderm transcripts is, however, also detected in these spontaneously differentiating cultures. These Oct4 shRNA hESCs will be an interesting model system to study human fetal adipogenesis and constitutes a renewable resource for obesity drug screening purposes

Phage display screening reveals an association between germline-specific transcription factor Oct-4 and multiple cellular proteins

Oct-4 is a transcription factor that is specifically expressed in mouse embryonic stem cells and in cell lines derived thereof. In these cells, Oct-4 activates transcription from remote binding sites due to as of yet unknown co-activators. Expression of Oct-4 in differentiated cells is not sufficient to activate transcription from a distance, rather it requires the co-expression of co-activators such as the adenoviral oncoprotein E1A. In this paper, we used phage display to identify Oct-4-interacting proteins. We first analyzed the interaction between Oct-4 and E1A in order to optimize the biochemical conditions that enable Oct-4-specific interactions with other interacting proteins. A panning approach was used to enrich Oct-4 interacting phages from a pool of excess unspecific phages. The biochemical conditions established in our interaction assays were then used to screen a P19 EC cell cDNA expression library in M13 filamentous phage. A number of phage clones displaying portions of unknown and known transcription factors were obtained, from which the HMG-1 transcription factor was identified. HMG-1, and the closely related factor HMG-2, interact with Oct-4 when co-expressed in mammalian cells. In addition, HMG-1 was found to cooperate with Oct-4 in P19 EC cells. These results provide the first evidence of a non-viral factor that enhances Oct-4 distance-dependent transactivation in stem cells