Analysis of the transcription factors and their regulatory roles during a step-by-step differentiation of induced pluripotent stem cells into hepatocyte-like cells

Tauran, Yannick; Poulain, Stéphane; Lereau-Bernier, Myriam; Danoy, Mathieu; Shinohara, Masanao; Ségard, Bertrand-David; Kato, Sachi; Kido, Taketomo; Miyajima, Atsushi; Sakai, Yasuyuki; Plessy, Charles; Leclerc, Éric

doi:10.1039/c9mo00122k

Cited by 11 publications

(7 citation statements)

References 52 publications

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“…In addition, we also found upregulation of the translational repressor pum1 , whose expression in planarians seems to be restricted to neoblasts and is related to cell self‐renewal in mammals (Salvetti et al, 2005 ; Spassov & Jurecic, 2003 ). Additional markers of pluripotency from other cell types have also been found upregulated in regenerating P. leidyi individuals, including adaptor‐related protein complex 2 ( ap2a2 ), alpha 2 subunit , CCR4‐NOT transcription complex subunit 6‐like‐B ( CN6LB ), and fam184A (Elmén et al, 2020 ; Kokkaliaris et al, 2012 ; Tauran et al, 2019 ). The presence of genes related to stem cell maintenance and pluripotency, and more specifically those markers exclusive to neoblasts, suggests the presence of stem cells and their relevance during P. leidyi regeneration.…”

Section: Resultsmentioning

confidence: 99%

Distinct patterns of gene expression during regeneration and asexual reproduction in the annelid Pristina leidyi

2022

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Regeneration, the ability to replace lost body parts, is a widespread phenomenon in the animal kingdom often connected to asexual reproduction or fission, since the only difference between the two appears to be the stimulus that triggers them. Both developmental processes have largely been characterized; however, the molecular toolkit and genetic mechanisms underlying these events remain poorly unexplored.Annelids, in particular the oligochaete Pristina leidyi, provide a good model system to investigate these processes as they show diverse ways to regenerate, and can reproduce asexually through fission under laboratory conditions. Here, we used a comparative transcriptomics approach based on RNA-sequencing and differential gene expression analyses to understand the molecular mechanisms involved in anterior regeneration and asexual reproduction. We found 291 genes upregulated during anterior regeneration, including several regeneration-related genes previously reported in other annelids such as frizzled, paics, and vdra. On the other hand, during asexual reproduction, 130 genes were found upregulated, and unexpectedly, many of them were related to germline development during sexual reproduction. We also found important differences between anterior regeneration and asexual reproduction, with the latter showing a gene expression profile more similar to that of control individuals. Nevertheless, we identified 35 genes that were upregulated in both conditions, many of them related to cell pluripotency, stem cells, and cell proliferation. Overall, our results shed light on the molecular mechanisms that control anterior regeneration and asexual reproduction in annelids and reveal similarities with other animals, suggesting that the genetic machinery controlling these processes is conserved across metazoans.

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Section: Resultsmentioning

confidence: 99%

Distinct patterns of gene expression during regeneration and asexual reproduction in the annelid Pristina leidyi

2022

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“…During DE differentiation, human pluripotent stem cells undergo synchronous epithelial-mesenchymal transition (EMT) 22,23,24 . It is known that DE cells show significantly higher migration in 2D experiments as compared to pluripotent cells in a scratch assay 23 , which argues that DE cells exhibit a higher cell mobility than stem cells.…”

Section: Resultsmentioning

confidence: 99%

Label-free imaging of 3D pluripotent stem cell differentiation dynamics on chip

Atwell

Waibel

Boushehri

et al. 2022

Preprint

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The dynamic chemical and architectural microenvironments of 3D stem cell cultures can be controlled by integration into a microfluidic chip. Massive parallelized 3D stem cell cultures for engineering in vitro human cell types require new imaging methods with high time and spatial resolution to fully exploit technological advances in cell culture. Here, we introduce a label-free deep learning method called Bright2Nuc to predict in silico nuclear staining in 3D from bright-field images obtained using traditional confocal microscopy. Bright2Nuc was trained and applied to several hundred 3D human induced pluripotent stem cell cultures differentiating towards definitive endoderm on a microfluidic platform. Combined with existing image analysis tools, Bright2Nuc segmented individual nuclei from bright-field images, quantified their morphological properties, predicted stem cell differentiation state, and tracked the cells over time. Our methods are available in an open-source pipeline that enables researchers to upscale 3D cell phenotyping in stem cell culture.

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“…The observation that more mature HLCs can be generated from PSCs via timed upregulation of HNF6 and PROX1 [15] or enforced adenovirus vector-mediated expression of ATF5, CEBPA and PROX1 [16], suggests that the immature phenotype is at least in part due to the suboptimal expression of key hepatic transcription factors. Mapping gene expression profiles and transcription factor motifs that define the differentiation of stem cells into HLC [17] further supports the idea that key stimuli required to induce, or maintain, appropriate gene expression in mature hepatocytes are lacking from current protocols[18]. Previously random, inefficient screens have been undertaken to identify small molecules that can promote hepatic specification, proliferation and maturation, [19, 20].…”

Section: Introductionmentioning

confidence: 96%

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

Withey

Gerrard

Leeson

et al. 2022

Preprint

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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-β 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.

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Analysis of the transcription factors and their regulatory roles during a step-by-step differentiation of induced pluripotent stem cells into hepatocyte-like cells

Abstract: Human induced pluripotent stem cells have been investigated through a sequential in vitro step-by-step differentiation into hepatocyte-like cells using nanoCAGE, an original method for promoters, transcription factors, and transcriptome analysis.

Cited by 11 publications

References 52 publications

Distinct patterns of gene expression during regeneration and asexual reproduction in the annelid Pristina leidyi

Distinct patterns of gene expression during regeneration and asexual reproduction in the annelid Pristina leidyi

Label-free imaging of 3D pluripotent stem cell differentiation dynamics on chip

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

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