Biology subjects

Withey, S.

Publications and source records attributed to Withey, S..

5 recordsLinked to original sources

bioRxiv2024

Unraveling the Role of MIXL1 Activation in Endoderm Differentiation of Isogenic Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSC) possess the ability to differentiate into a multitude of cell and tissue types but display heterogeneous propensity of differentiation into specific lineage. Characterization of the transcriptome of eleven hiPSC lines showed that activation of MIXL1 at the early stage of stem cell differentiation correlated with higher efficacy in generating definitive endoderm and advancing differentiation and maturation of endoderm derivatives. Enforced expression of MIXL1 in the endoderm-inefficient hiPSCs enhanced the propensity of endoderm differentiation, suggesting that modulation of key drivers of lineage differentiation can re-wire hiPSC to the desired lineage propensity to generate the requisite stem cell products.

developmental biology↗

bioRxiv2022

Thyroid hormone and ALK5 inhibitor improve maturation of human pluripotent stem cell derived hepatocytes

Hepatocytes derived from human pluripotent stem cells (PSCs) hold great promise for modeling human liver disease, in vitro hepatotoxicity testing, and future cellular therapy. However, current protocols generate hepatocyte-like cells (HLCs) that resemble fetal hepatocytes, and thus do not accurately recapitulate the molecular identity and functions of the adult liver. To address this, we compared the transcriptomes of human fetal and adult liver to PSC-derived HLCs during progressive stages of in vitro differentiation. This revealed that during the final stages of in vitro differentiation the hepatic transcription factors HNF4A and CEBPA were sub-optimally expressed. Computational analyses predicted that ALK5i II (TGF-{beta} receptor inhibitor) and thyroid hormone (T3) would be able to rectify this and improve HLC maturation. We next show that application of these molecules during hepatocyte differentiation indeed increases CEBPA and HNF4A mRNA and protein expression, and that these HLCs show enhanced albumin secretion, a 25-fold increase in CYP3A4 activity, and 10 to 100-fold increased expression of mature hepatic markers. We demonstrate that this improved maturation is effective across different cell lines and HLC differentiation protocols, and exemplifies that our approach provides a tractable template for identifying and targeting additional factors that that will fully mature human liver cells from human pluripotent stem cells.

developmental biology↗

bioRxiv2020

Specification and epigenetic resetting of the pig germline exhibit conservation with the human lineage

Investigations on the human germline and programming are challenging due to limited access to embryonic material. However, the pig as a model may provide insight on transcriptional network and epigenetic reprogramming applicable to both species. Here we show that during the pre- and early migratory stages pig primordial germ cells (PGCs) initiate large-scale epigenetic reprogramming, including DNA demethylation involving TET-mediated hydroxylation and potentially base excision repair (BER). There is also macroH2A1 depletion and increased H3K27me3, as well as X chromosome reactivation (XCR) in females. Concomitantly, there is dampening of glycolytic metabolism genes and re-expression of some pluripotency genes like those in preimplantation embryos. We identified evolutionarily young transposable elements and gene coding regions resistant to DNA demethylation in acutely hypomethylated gonadal PGCs, with potential for transgenerational epigenetic inheritance. Detailed insights into the pig germline will likely contribute significantly to advances in human germline biology, including in vitro gametogenesis.

developmental biology↗

bioRxiv2018

Identification of a primitive intestinal transcription factor network shared between oesophageal adenocarcinoma and its pre-cancerous precursor state.

Oesophageal adenocarcinoma (OAC) is one of the most frequent causes of cancer deaths and yet compared to other common cancers, we know relatively little about the molecular composition of this tumour type. To further our understanding of this cancer we have used open chromatin profiling to decipher the transcriptional regulatory networks that are operational in OAC. We have uncovered a transcription factor network that is usually found in primitive intestinal cells during embryonic development, centred on HNF4A and GATA6. These transcription factors work together to control the OAC transcriptome. Importantly, we show that this network is activated in Barretts oesophagus, the putative precursor state to OAC thereby providing novel molecular evidence in support of stepwise malignant transition. Furthermore, we show that HNF4A alone, is sufficient to drive chromatin opening and activation of a Barretts-like chromatin signature when expressed in normal human epithelial cells. Collectively, these data provide a new way to categorise OAC at a genome scale and implicate HNF4A activation as a potential pivotal event in regulating its malignant transition from healthy cells.

cancer biology↗

bioRxiv2018

Lineage segregation, pluripotency and X-chromosome inactivation in the pig pre-gastrulation embryo

High-resolution molecular programs delineating the cellular foundations of mammalian embryogenesis have emerged recently. Similar analysis of human embryos is limited to pre-implantation stages, since early post-implantation embryos are inaccessible. Notwithstanding, we previously suggested conserved principles of pig and human early development. For further insight on pluripotent states and lineage delineation, we analysed pig embryos at single cell resolution. Here we show progressive segregation of inner cell mass and trophectoderm in early blastocysts, and then of epiblast and hypoblast in late blastocysts. We detected distinct pluripotent states, first as a short naive state followed by a protracted primed state. Dosage compensation with respect to the X-chromosome in females is attained via X-inactivation in late epiblasts. Detailed human-pig comparison is a basis towards comprehending early human development and a foundation for further studies of human pluripotent stem cell differentiation in pig interspecies chimeras.

developmental biology↗