Reprogramming to pluripotency using designer TALE transcription factors targeting enhancers

The modular DNA recognition code of the transcription-activator-like effectors (TALEs) from plant pathogenic bacterial genus Xanthomonas provides a powerful genetic tool to create designer transcription factors (dTFs) targeting specific DNA sequences for manipulating gene expression. Previous studies have suggested critical roles of enhancers in gene regulation and reprogramming. Here, we report dTF activator targeting the distal enhancer of the Pou5f1 (Oct4) locus induces epigenetic changes, reactivates its expression, and substitutes exogenous OCT4 in reprogramming mouse embryonic fibroblast cells (MEFs) to induced pluripotent stem cells (iPSCs). Similarly, dTF activator targeting a Nanog enhancer activates Nanog expression and reprograms epiblast stem cells (EpiSCs) to iPSCs. Conversely, dTF repressors targeting the same genetic elements inhibit expression of these loci, and effectively block reprogramming. This study indicates that dTFs targeting specific enhancers can be used to study other biological processes such as transdifferentiation or directed differentiation of stem cells. © 2013 The Authors.Link_to_subscribed_fulltex

Expanded huntingtin CAG repeats disrupt the balance between neural progenitor expansion and differentiation in human cerebral organoids

Huntington disease (HD) manifests in both adult and juvenile forms. Mutant HTT gene carriers are thought to undergo normal brain development followed by a degenerative phase, resulting in progressive clinical manifestations. However, recent studies in children and prodromal individuals at risk for HD have raised the possibility of abnormal neurodevelopment. Although key findings in rodent models support this notion, direct evidence in the context of human physiology remains lacking. Using a panel of isogenic HD human embryonic pluripotent stem cells and cerebral organoids, we investigated the impact of mutant HTT on early neurodevelopment. We find that ventricular zone-like neuroepithelial progenitor layer expansion is blunted in an HTT CAG repeat length-dependent manner due to premature neurogenesis in HD cerebral organoids, driven by cell intrinsic processes. Transcriptional profiling and imaging analysis revealed impaired cell cycle regulatory processes, increased G1 length, and increased asymmetric division of apical progenitors, collectively contributing to premature neuronal differentiation. We demonstrate increased activity of the ATM-p53 pathway, an up-stream regulator of cell cycle processes, and show that treatment with ATM antagonists partially rescues the blunted neuroepithelial progenitor expansion in HD organoids. Our findings suggest that CAG repeat length regulates the balance between neural progenitor expansion and differentiation during early neurodevelopment. Our results further support the view that HD, at least in its early-onset forms, may not be a purely neurodegenerative disorder, and that abnormal neurodevelopment may be a component of HD pathophysiology

Delineating nuclear reprogramming

Nuclear reprogramming is described as a molecular switch, triggered by the conversion of one cell type to another. Several key experiments in the past century have provided insight into the field of nuclear reprogramming. Previously deemed impossible, this research area is now brimming with new findings and developments. In this review, we aim to give a historical perspective on how the notion of nuclear reprogramming was established, describing main experiments that were performed, including (1) somatic cell nuclear transfer, (2) exposure to cell extracts and cell fusion, and (3) transcription factor induced lineage switch. Ultimately, we focus on (4) transcription factor induced pluripotency, as initiated by a landmark discovery in 2006, where the process of converting somatic cells to a pluripotent state was narrowed down to four transcription factors. The conception that somatic cells possess the capacity to revert to an immature status brings about huge clinical implications including personalized therapy, drug screening and disease modeling. Although this technology has potential to revolutioni ze the medical field, it is still impeded by technical and biological obstacles. This review describes the effervescent changes in this field, addresses bottlenecks hindering its advancement and in conclusion, applies the latest findings to overcome these issues. © 2012 Higher Education Press and Springer-Verlag Berlin Heidelberg.Link_to_subscribed_fulltex

Signalling through retinoic acid receptors is required for reprogramming of both mouse embryonic fibroblast cells and epiblast stem cells to induced pluripotent stem cells

© 2014 AlphaMed Press. We previously demonstrated that coexpressing retinoic acid (RA) receptor gamma and liver receptor homolog-1 (LRH1 or NR5A2) with OCT4, MYC, KL F4, and SOX2 (4F) rapidly reprograms mouse embryonic fibroblast cells (MEFs) into induced pluripotent stem cells (iPSCs). Here, we further explore the role of RA in reprogramming and report that the six factors (6F) efficiently and directly reprogram MEFs into integration-free iPSCs in defined medium (N2B27) in the absence of feeder cells. Through genetic and chemical approaches, we find that RA signalling is essential, in a highly dose-sensitive manner, for MEF reprogramming. The removal of exogenous RA from N2B27, the inhibition of endogenous RA synthesis or the expression of a dominant-negative form of RARA severely impedes reprogramming. By contrast, supplementing N2B27 with various retinoids substantially boosts reprogramming. In addition, when coexpressed with LRH1, RA receptors (RARs) can promote reprogramming in the absence of both exogenous and endogenously synthesized RA. Remarkably, the reprogramming of epiblast stem cells into embryonic stem cell-like cells also requires low levels of RA, which can modulate Wnt signalling through physical interactions of RARs with β-catenin. These results highlight the important functions of RA signalling in reprogramming somatic cells and primed stem cells to naïve pluripotency. Stem Cells 2015;33:1390-1404Link_to_subscribed_fulltex

Treatment with the MAO-A inhibitor clorgyline elevates monoamine neurotransmitter levels and improves affective phenotypes in a mouse model of Huntington disease

10.1016/j.expneurol.2016.01.019EXPERIMENTAL NEUROLOGY2784-Oc