Pancreatic Progenitor Commitment Is Marked by an Increase in Ink4a/Arf Expression

The identification of the molecular mechanisms controlling early cell fate decisions in mammals is of paramount importance as the ability to determine specific lineage differentiation represents a significant opportunity for new therapies. Pancreatic Progenitor Cells (PPCs) constitute a regenerative reserve essential for the maintenance and regeneration of the pancreas. Besides, PPCs represent an excellent model for understanding pathological pancreatic cellular remodeling. Given the lack of valid markers of early endoderm, the identification of new ones is of fundamental importance. Both products of the Ink4a/Arf locus, in addition to being critical cell-cycle regulators, appear to be involved in several disease pathologies. Moreover, the locus’ expression is epigenetically regulated in ES reprogramming processes, thus constituting the ideal candidates to modulate PPCs homeostasis. In this study, starting from mouse embryonic stem cells (mESCs), we analyzed the early stages of pancreatic commitment. By inducing mESCs commitment to the pancreatic lineage, we observed that both products of the Cdkn2a locus, Ink4a and Arf, mark a naïve pancreatic cellular state that resembled PPC-like specification. Treatment with epi-drugs suggests a role for chromatin remodeling in the CDKN2a (Cycline Dependent Kinase Inhibitor 2A) locus regulation in line with previous observations in other cellular systems. Our data considerably improve the comprehension of pancreatic cellular ontogeny, which could be critical for implementing pluripotent stem cells programming and reprogramming toward pancreatic lineage commitment.

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Nicotinamide promotes pancreatic differentiation through the dual inhibition of CK1 and ROCK kinases in human embryonic stem cells

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02426-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yumeng Zhang ◽

Jiaqi Xu ◽

Zhili Ren ◽

Ya Meng ◽

Weiwei Liu ◽

...

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Cell Fate Determination ◽

Rna Seq ◽

Pancreatic Cell ◽

Pancreatic Progenitor ◽

Pancreatic Progenitors ◽

Rock Inhibition

Abstract Background Vitamin B3 (nicotinamide) plays important roles in metabolism as well as in SIRT and PARP pathways. It is also recently reported as a novel kinase inhibitor with multiple targets. Nicotinamide promotes pancreatic cell differentiation from human embryonic stem cells (hESCs). However, its molecular mechanism is still unclear. In order to understand the molecular mechanism involved in pancreatic cell fate determination, we analyzed the downstream pathways of nicotinamide in the derivation of NKX6.1+ pancreatic progenitors from hESCs. Methods We applied downstream modulators of nicotinamide during the induction from posterior foregut to pancreatic progenitors, including niacin, PARP inhibitor, SIRT inhibitor, CK1 inhibitor and ROCK inhibitor. The impact of those treatments was evaluated by quantitative real-time PCR, flow cytometry and immunostaining of pancreatic markers. Furthermore, CK1 isoforms were knocked down to validate CK1 function in the induction of pancreatic progenitors. Finally, RNA-seq was used to demonstrate pancreatic induction on the transcriptomic level. Results First, we demonstrated that nicotinamide promoted pancreatic progenitor differentiation in chemically defined conditions, but it did not act through either niacin-associated metabolism or the inhibition of PARP and SIRT pathways. In contrast, nicotinamide modulated differentiation through CK1 and ROCK inhibition. We demonstrated that CK1 inhibitors promoted the generation of PDX1/NKX6.1 double-positive pancreatic progenitor cells. shRNA knockdown revealed that the inhibition of CK1α and CK1ε promoted pancreatic progenitor differentiation. We then showed that nicotinamide also improved pancreatic progenitor differentiation through ROCK inhibition. Finally, RNA-seq data showed that CK1 and ROCK inhibition led to pancreatic gene expression, similar to nicotinamide treatment. Conclusions In this report, we revealed that nicotinamide promotes generation of pancreatic progenitors from hESCs through CK1 and ROCK inhibition. Furthermore, we discovered the novel role of CK1 in pancreatic cell fate determination.

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Epigenetic regulations follow cell cycle progression during differentiation of human pluripotent stem cells

10.1101/2020.06.26.173211 ◽

2020 ◽

Author(s):

Pedro Madrigal ◽

Siim Pauklin ◽

Kim Jee Goh ◽

Rodrigo Grandy ◽

Anna Osnato ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Fate ◽

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Activator Protein 1 ◽

Cellular Division ◽

Mapk Signalling

AbstractMost mammalian stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the cascade of epigenetic events and molecular mechanisms occurring between successive cell divisions upon differentiation have not yet been described in detail due to technical limitations. Here, we address this question by taking advantage of the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) reporter to develop a culture system allowing the differentiation of human Embryonic Stem Cells (hESCs) synchronised for their cell cycle. Using this approach, we have assessed the epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We first observed that transcription of key markers of differentiation occurs before division suggesting that differentiation is initiated during the progression of cell cycle. Furthermore, ATAC-seq shows a major decrease in chromatin accessibility after pluripotency exit indicating that the first event of differentiation is the inhibition of alternative cell fate. In addition, using digital genomic footprinting we identified novel cell cycle-specific transcription factors with regulatory potential in endoderm specification. Of particular interest, Activator protein 1 (AP-1) controlled p38/MAPK signalling seems to be necessary for blocking endoderm shifting cell fate toward mesoderm lineage. Finally, histone modifications analyses suggest a temporal order between different marks. We can also conclude that enhancers are dynamically and rapidly established / decommissioned between different cell cycle upon differentiation. Overall, these data not only reveal key the successive interplays between epigenetic modifications during differentiation but also provide a valuable resource to investigate novel mechanisms in germ layer specification.

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Regulated Fluctuations in Nanog Expression Mediate Cell Fate Decisions in Embryonic Stem Cells

PLoS Biology ◽

10.1371/journal.pbio.1000149 ◽

2009 ◽

Vol 7 (7) ◽

pp. e1000149 ◽

Cited By ~ 375

Author(s):

Tibor Kalmar ◽

Chea Lim ◽

Penelope Hayward ◽

Silvia Muñoz-Descalzo ◽

Jennifer Nichols ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Embryonic Stem ◽

Cell Fate Decisions ◽

Nanog Expression ◽

Mediate Cell

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17-P023 The role of Sox2 in regulation of self-renewal and early cell fate decisions in mouse embryonic stem cells

Mechanisms of Development ◽

10.1016/j.mod.2009.06.744 ◽

2009 ◽

Vol 126 ◽

pp. S277

Author(s):

Maria Keramari ◽

Christopher Ward ◽

Susan Kimber

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Self Renewal ◽

Cell Fate Decisions

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Genetic control of pluripotency epigenome determines differentiation bias in mouse embryonic stem cells

10.1101/2021.01.15.426861 ◽

2021 ◽

Author(s):

Candice Byers ◽

Catrina Spruce ◽

Haley J. Fortin ◽

Anne Czechanski ◽

Steven C. Munger ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Genetic Regulation ◽

Embryonic Stem ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Embryoid Bodies ◽

Cell Fate Decisions

AbstractGenetically diverse pluripotent stem cells (PSCs) display varied, heritable responses to differentiation cues in the culture environment. By harnessing these disparities through derivation of embryonic stem cells (ESCs) from the BXD mouse genetic reference panel, along with C57BL/6J (B6) and DBA/2J (D2) parental strains, we demonstrate genetically determined biases in lineage commitment and identify major regulators of the pluripotency epigenome. Upon transition to formative pluripotency using epiblast-like cells (EpiLCs), B6 quickly dissolves naïve networks adopting gene expression modules indicative of neuroectoderm lineages; whereas D2 retains aspects of naïve pluripotency with little bias in differentiation. Genetic mapping identifies 6 major trans-acting loci co-regulating chromatin accessibility and gene expression in ESCs and EpiLCs, indicating a common regulatory system impacting cell state transition. These loci distally modulate occupancy of pluripotency factors, including TRIM28, P300, and POU5F1, at hundreds of regulatory elements. One trans-acting locus on Chr 12 primarily impacts chromatin accessibility in ESCs; while in EpiLCs the same locus subsequently influences gene expression, suggesting early chromatin priming. Consequently, the distal gene targets of this locus are enriched for neurogenesis genes and were more highly expressed when cells carried B6 haplotypes at this Chr 12 locus, supporting genetic regulation of biases in cell fate. Spontaneous formation of embryoid bodies validated this with B6 showing a propensity towards neuroectoderm differentiation and D2 towards definitive endoderm, confirming the fundamental importance of genetic variation influencing cell fate decisions.

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miR-142-3p Contributes to Early Cardiac Fate Decision of Embryonic Stem Cells

Stem Cells International ◽

10.1155/2017/1769298 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Zhong-Yan Chen ◽

Fei Chen ◽

Nan Cao ◽

Zhi-Wen Zhou ◽

Huang-Tian Yang

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Marker Gene ◽

Ectopic Expression ◽

Embryonic Stem ◽

Cardiomyocyte Differentiation ◽

Cell Fate Decisions ◽

Mirna Array ◽

Fate Decision

MicroRNAs (miRNAs) play important roles in cell fate decisions. However, the miRNAs and their targets involved in the regulation of cardiac lineage specification are largely unexplored. Here, we report novel functions of miR-142-3p in the regulation of cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). With a miRNA array screen, we identified a number of miRNAs significantly changed during mESC differentiation into the mesodermal and cardiac progenitor cells, and miR-142-3p was one among the markedly downregulated miRNAs. Ectopic expression and inhibition of miR-142-3p did not alter the characteristics of undifferentiated ESCs, whereas ectopic expression of miR-142-3p impaired cardiomyocyte formation. In addition, ectopic expression of miR-142-3p inhibited the expression of a cardiac mesodermal marker gene Mesp1 and downstream cardiac transcription factors Nkx2.5, Tbx5, and Mef2c but not the expression of three germ layer-specific genes. We further demonstrated that miR-142-3p targeted the 3′-untranslated region of Mef2c. These results reveal miR-142-3p as an important regulator of early cardiomyocyte differentiation. Our findings provide new knowledge for further understanding of roles and mechanisms of miRNAs as critical regulators of cardiomyocyte differentiation.

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Bdh2 Deficiency Promotes Endoderm-Biased Early Differentiation of Mouse Embryonic Stem Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.655145 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yuting Fu ◽

Fangyuan Liu ◽

Shuo Cao ◽

Jia Zhang ◽

Huizhi Wang ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Iron Homeostasis ◽

Embryonic Stem ◽

Cell Fate Decisions ◽

Rate Limiting Step ◽

Fate Decision

3-hydroxybutyrate dehydrogenase-2 (Bdh2), a short-chain dehydrogenase, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore, playing a key role in iron homeostasis, energy metabolism and apoptosis. However, the function of Bdh2 in embryonic stem cells (ESCs) remains unknown. To gain insights into the role of Bdh2 on pluripotency and cell fate decisions of mouse ESCs, we generated Bdh2 homozygous knockout lines for both mouse advanced embryonic stem cell (ASC) and ESC using CRISPR/Cas9 genome editing technology. Bdh2 deficiency in both ASCs and ESCs had no effect on expression of core pluripotent transcription factors and alkaline phosphatase activity, suggesting dispensability of Bdh2 for self-renewal and pluripotency of ESCs. Interestingly, cells with Bdh2 deficiency exhibited potency of endoderm differentiation in vitro; with upregulated endoderm associated genes revealed by RNA-seq and RT-qPCR. We further demonstrate that Bdh2 loss inhibited expression of multiple methyltransferases (DNMTs) at both RNA and protein level, suggesting that Bdh2 may be essentially required to maintain DNA methylation in ASCs and ESCs. Overall, this study provides valuable data and resources for understanding how Bdh2 regulate earliest cell fate decision and DNA methylation in ASCs/ESCs.

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O-GlcNAcylation of Sox2 at threonine 258 regulates the self-renewal and early cell fate of embryonic stem cells

Experimental & Molecular Medicine ◽

10.1038/s12276-021-00707-7 ◽

2021 ◽

Author(s):

Dong Keon Kim ◽

Jang-Seok Lee ◽

Eun Young Lee ◽

Hansol Jang ◽

Suji Han ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Rna Sequencing ◽

Cell Fate ◽

Embryonic Stem ◽

Cartilage Tissue ◽

Sequencing Analysis ◽

Post Translational Modification ◽

Self Renewal ◽

Cell Fate Decisions

AbstractSox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions.

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Expression Profiles of MicroRNAs in Stem Cells Differentiation

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021666200219092520 ◽

2020 ◽

Vol 21 (10) ◽

pp. 906-918

Author(s):

Hadi Rajabi ◽

Somayeh Aslani ◽

Alireza Abhari ◽

Davoud Sanajou

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Signaling Pathways ◽

Cell Fate ◽

Adult Stem Cells ◽

Expression Profiles ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Cell Fate Decisions ◽

Effective Diagnosis

Stem cells are undifferentiated cells and have a great potential in multilineage differentiation. These cells are classified into adult stem cells like Mesenchymal Stem Cells (MSCs) and Embryonic Stem Cells (ESCs). Stem cells also have potential therapeutic utility due to their pluripotency, self-renewal, and differentiation ability. These properties make them a suitable choice for regenerative medicine. Stem cells differentiation toward functional cells is governed by different signaling pathways and transcription factors. Recent studies have demonstrated the key role of microRNAs in the pathogenesis of various diseases, cell cycle regulation, apoptosis, aging, cell fate decisions. Several types of stem cells have different and unique miRNA expression profiles. Our review summarizes novel regulatory roles of miRNAs in the process of stem cell differentiation especially adult stem cells into a variety of functional cells through signaling pathways and transcription factors modulation. Understanding the mechanistic roles of miRNAs might be helpful in elaborating clinical therapies using stem cells and developing novel biomarkers for the early and effective diagnosis of pathologic conditions.

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