scholarly journals A dual reporter system identifies an intermediate state and sequential regulators of 2-cell-like-to-pluripotent state transition

2021 ◽  
Author(s):  
Chao Zhang ◽  
Jing Hao ◽  
Ming Shi ◽  
Yu-Xuan Li ◽  
Wang Yao ◽  
...  
Keyword(s):  
Intermediate State ◽  
Molecular Mechanisms ◽  
State Transition ◽  
Embryonic Stem ◽  
Dual Function ◽  
Reporter System ◽  
Cell Stage ◽  
Pluripotent State ◽  
Dual Reporter ◽  
Essential Transcription Factor

Mouse embryonic stem cells (ESCs) cycle in and out of 2-cell-like (2C-like) state in culture. The molecular mechanism governing the exit of 2C-like state remains obscure, partly due to the lack of a reporter system that can genetically mark intermediate states during exiting process. Here, we identify an intermediate state that is marked by the co-expression of MERVL::tdTomato and OCT4-GFP (MERLOT) during 2C-like-to-pluripotent state transition (2CLPT). Transcriptome and epigenome analyses demonstrate that MERLOT cells cluster closely with 8-16 cell stage mouse embryos, suggesting that 2CLPT partly mimics early preimplantation development. Through a CRISPRa screen, we identify an ARRDC3-NEDD4-OCT4 regulatory axis that plays an essential role in controlling 2CLPT. Furthermore, re-evaluating previously reported 2C-like state regulators reveals dual function of Chaf1a in regulating the entry and exit of 2C-like state. Finally, ATAC-Seq footprinting analysis uncovers Klf3 as an essential transcription factor required for efficient 2CLPT. Together, our study identifies a genetically traceable intermediate state during 2CLPT and provides a valuable tool to study molecular mechanisms regulating this process.

scholarly journals A transcriptional roadmap for 2C-like–to–pluripotent state transition

2020 ◽  
Vol 6 (22) ◽  
pp. eaay5181 ◽  
Author(s):  
Xudong Fu ◽  
Mohamed Nadhir Djekidel ◽  
Yi Zhang
Keyword(s):  
Intermediate State ◽  
State Transition ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Small Cell ◽  
Molecular Event ◽  
Cell Stage ◽  
Pluripotent State ◽  
Transcriptional Dynamics ◽  
The One

In mouse embryonic stem cell (ESC), a small cell population displays totipotent features by expressing a set of genes that are transiently active in 2-cell–stage embryos. These 2-cell–like (2C-like) cells spontaneously transit back into the pluripotent state. We previously dissected the transcriptional dynamics of the transition from pluripotency to the totipotent 2C-like state and identified factors that modulate the process. However, how 2C-like cells transit back into the pluripotent state remains largely unknown. In this study, we analyzed the transcriptional dynamics from the 2C-like state to pluripotent ESCs and identified an intermediate state. The intermediate state characterized by two-wave step up-regulation of pluripotent genes is different from the one observed during the 2C-like entry transition. Nonsense-mediated Dux mRNA decay plays an important role in the 2C-like state exit. Thus, our study not only provides a transcriptional roadmap for 2C-like–to–pluripotent state transition but also reveals a key molecular event driving the transition.

scholarly journals A defined glycosylation regulatory network modulates total glycome dynamics during pluripotency state transition

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Federico Pecori ◽  
Ikuko Yokota ◽  
Hisatoshi Hanamatsu ◽  
Taichi Miura ◽  
Chika Ogura ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
State Transition ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Developmentally Regulated ◽  
Polycomb Repressive Complex 2 ◽  
Pluripotent State ◽  
Lineage Decision ◽  
Early Mouse

AbstractEmbryonic stem cells (ESCs) and epiblast-like cells (EpiLCs) recapitulate in vitro the epiblast first cell lineage decision, allowing characterization of the molecular mechanisms underlying pluripotent state transition. Here, we performed a comprehensive and comparative analysis of total glycomes of mouse ESCs and EpiLCs, revealing that overall glycosylation undergoes dramatic changes from early stages of development. Remarkably, we showed for the first time the presence of a developmentally regulated network orchestrating glycosylation changes and identified polycomb repressive complex 2 (PRC2) as a key component involved in this process. Collectively, our findings provide novel insights into the naïve-to-primed pluripotent state transition and advance the understanding of glycosylation complex regulation during early mouse embryonic development.

scholarly journals Long-Range Enhancer Interactions Are Prevalent in Mouse Embryonic Stem Cells and Are Reorganized upon Pluripotent State Transition

Cell Reports ◽  
2018 ◽  
Vol 22 (10) ◽  
pp. 2615-2627 ◽  
Author(s):  
Clara Lopes Novo ◽  
Biola-Maria Javierre ◽  
Jonathan Cairns ◽  
Anne Segonds-Pichon ◽  
Steven W. Wingett ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Long Range ◽  
State Transition ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Pluripotent State

scholarly journals Repression of Nanog Gene Transcription by Tcf3 Limits Embryonic Stem Cell Self-Renewal

2006 ◽  
Vol 26 (20) ◽  
pp. 7479-7491 ◽  
Author(s):  
Laura Pereira ◽  
Fei Yi ◽  
Bradley J. Merrill
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Molecular Mechanisms ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Regulatory Region ◽  
Embryonic Stem ◽  
Dual Function ◽  
Self Renewal ◽  
Nanog Gene

ABSTRACT The dual function of stem cells requires them not only to form new stem cells through self-renewal but also to form lineage-committed cells through differentiation. Embryonic stem cells (ESC), which are derived from the blastocyst inner cell mass, retain properties of self-renewal and the potential for lineage commitment. To balance self-renewal and differentiation, ESC must carefully control the levels of several transcription factors, including Nanog, Sox2, and Oct4. While molecular mechanisms promoting transcription of these genes have been described, mechanisms preventing excessive levels in self-renewing ESC remain unknown. By examining the function of the TCF family of transcription factors in ESC, we have found that Tcf3 is necessary to limit the steady-state levels of Nanog mRNA, protein, and promoter activity in self-renewing ESC. Chromatin immunoprecipitation and promoter reporter assays showed that Tcf3 bound to a promoter regulatory region of the Nanog gene and repressed its transcriptional activity in ESC through a Groucho interaction domain-dependent process. The absence of Tcf3 caused delayed differentiation of ESC in vitro as elevated Nanog levels persisted through 5 days of embryoid body formation. These new data support a model wherein Tcf3-mediated control of Nanog levels allows stem cells to balance the creation of lineage-committed and undifferentiated cells.

scholarly journals Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Wu-Sheng Sun ◽  
Ju-Lan Chun ◽  
Jeong-Tae Do ◽  
Dong-Hwan Kim ◽  
Jin-Seop Ahn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Line ◽  
Germ Line ◽  
Embryonic Stem ◽  
Regulatory Sequences ◽  
Dual Fluorescence ◽  
Distal Enhancer ◽  
Reporter System ◽  
Pluripotent State ◽  
Epiblast Stem Cells

Oct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5′ upstream regulatory sequences (URSs) of the mouse Oct4 gene, which were demonstrated to control Oct4 expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pig Oct4 and identified two similar enhancers that were highly consistent with the mouse DE and PE. A dual-fluorescence reporter was later constructed by combining a DE-free-Oct4-promoter-driven EGFP reporter cassette with a PE-free-Oct4-promoter-driven mCherry reporter cassette. Then, it was tested in a mouse ESC-like cell line (F9) and a mouse EpiSC-like cell line (P19) before it is formally used for pig. As a result, a higher red fluorescence was observed in F9 cells, while green fluorescence was primarily detected in P19 cells. This fluorescence expression pattern in the two cell lines was consistent with that in the early naïve pluripotent state and late primed pluripotent state during differentiation of mouse ESCs. Hence, this reporter system will be a convenient tool for screening out ESC-like naïve pluripotent stem cells from other metastable state cells in a heterogenous population.

scholarly journals Pig embryonic stem cell line with porcine-specific OCT4 upstream region based dual reporter system

2021 ◽  
pp. 102609
Author(s):  
Seung-Hun Kim ◽  
Kwang-Hwan Choi ◽  
Jinsol Jeong ◽  
Mingyun Lee ◽  
Dong-Kyung Lee ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Line ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Upstream Region ◽  
Reporter System ◽  
Stem Cell Line ◽  
Dual Reporter

microRNA regulation of pluripotent state transition

2020 ◽  
Vol 64 (6) ◽  
pp. 947-954 ◽  
Author(s):  
Shao-Hua Wang ◽  
Chao Zhang ◽  
Yangming Wang
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Embryo Development ◽  
State Transition ◽  
Embryonic Stem ◽  
Short Review ◽  
Early Embryo ◽  
Mammalian Development ◽  
Early Embryo Development ◽  
Pluripotent State

Abstract microRNAs (miRNAs) play essential roles in mouse embryonic stem cells (ESCs) and early embryo development. The exact mechanism by which miRNAs regulate cell fate transition during embryo development is still not clear. Recent studies have identified and captured various pluripotent stem cells (PSCs) that share similar characteristics with cells from different stages of pre- and post-implantation embryos. These PSCs provide valuable models to understand miRNA functions in early mammalian development. In this short review, we will summarize recent work towards understanding the function and mechanism of miRNAs in regulating the transition or conversion between different pluripotent states. In addition, we will highlight unresolved questions and key future directions related to miRNAs in pluripotent state transition. Studies in these areas will further our understanding of miRNA functions in early embryo development, and may lead to practical means to control human PSCs for clinical applications in regenerative medicine.

scholarly journals Induced Pluripotent Stem Cells

2018 ◽  
pp. 16-22
Author(s):  
Abdullah El-Sayes
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Scientific Progress ◽  
Ips Cells ◽  
Ips Cell ◽  
Pluripotent State ◽  
Scientific Investigations ◽  
Induced Pluripotent ◽  
Mouse Somatic Cell

The isolation of human embryonic stem cells in 1998 has since fueled the ideology that stem cells may eventually be used for human disease therapies as well as the regeneration of tissues and organs. The transformation of somatic cells to a pluripotent state via somatic nuclear transfer and embryonic stem cell fusion brought the scientific community nearer to understanding the molecular mechanisms that govern cellular pluripotency. In 2006, the first induced pluripotent stem (iPS) cell was reported, where a mouse somatic cell was successfully converted to a pluripotent state via transduction of four essential factors. This cellular breakthrough has allowed for robust scientific investigations of human diseases that were once extremely difficult to study. Scientists and pharmaceuticals now use iPS cells as means for disease investigations, drug development and cell or tissue transplantation. There is little doubt that scientific progress on iPS cells will change many aspects of medicine in the next couple of decades.

scholarly journals Stem cell differentiation is a stochastic process with memory

2017 ◽  
Author(s):  
Patrick S. Stumpf ◽  
Rosanna C. G. Smith ◽  
Michael Lenz ◽  
Andreas Schuppert ◽  
Franz-Josef Müller ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stochastic Process ◽  
Statistical Mechanics ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Pluripotent State

AbstractPluripotent stem cells are able to self-renew indefinitely in culture and differentiate into all somatic cell types in vivo. While much is known about the molecular basis of pluripotency, the molecular mechanisms of lineage commitment are complex and only partially understood. Here, using a combination of single cell profiling and mathematical modeling, we examine the differentiation dynamics of individual mouse embryonic stem cells (ESCs) as they progress from the ground state of pluripotency along the neuronal lineage. In accordance with previous reports we find that cells do not transit directly from the pluripotent state to the neuronal state, but rather first stochastically permeate an intermediate primed pluripotent state, similar to that found in the maturing epiblast in development. However, analysis of rate at which individual cells enter and exit this intermediate metastable state using a hidden Markov model reveals that the observed ESC and epiblast-like ‘macrostates’ conceal a chain of unobserved cellular ‘microstates’, which individual cells transit through stochastically in sequence. These hidden microstates ensure that individual cells spend well-defined periods of time in each functional macrostate and encode a simple form of epigenetic ‘memory’ that allows individual cells to record their position on the differentiation trajectory. To examine the generality of this model we also consider the differentiation of mouse hematopoietic stem cells along the myeloid lineage and observe remarkably similar dynamics, suggesting a general underlying process. Based upon these results we suggest a statistical mechanics view of cellular identities that distinguishes between functionally-distinct macrostates and the many functionally-similar molecular microstates associated with each macrostate. Taken together these results indicate that differentiation is a discrete stochastic process amenable to analysis using the tools of statistical mechanics.

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