Rational design and optimization of synthetic gene switches for controlling cell-fate decisions in pluripotent stem cells

AbstractPluripotent stem cells represent a powerful system to identify the mechanisms governing cell fate decisions during early mammalian development. Covalent attachment of the Small Ubiquitin Like Modifier (SUMO) to proteins has emerged as an important factor in stem cell maintenance. Here we show that SUMO is required to maintain stem cells in their pluripotent state and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells (hiPSCs). Loss of SUMO increases chromatin accessibility and expression of long non-coding RNAs and human endogenous retroviral elements, indicating a role for the SUMO modification of SETDB1 and a large TRIM28 centric network of zinc finger proteins in silencing of these elements. While most protein coding genes are unaffected, the Preferentially Expressed Antigen of Melanoma (PRAME) gene locus becomes more accessible and transcription is dramatically increased after inhibition of SUMO modification. When PRAME is silent, a peak of SUMO over the transcriptional start site overlaps with ChIP-seq peaks for cohesin, RNA pol II, CTCF and ZNF143, with the latter two heavily modified by SUMO. These associations suggest that silencing of the PRAME gene is maintained by the influence of SUMO on higher order chromatin structure. Our data indicate that SUMO modification plays an important role in hiPSCs by repressing genes that disrupt pluripotency networks or drive differentiation.

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Derivation of trophoblast stem cells from naïve human pluripotent stem cells

eLife ◽

10.7554/elife.52504 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 15

Author(s):

Chen Dong ◽

Mariana Beltcheva ◽

Paul Gontarz ◽

Bo Zhang ◽

Pooja Popli ◽

...

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Pluripotent Stem Cells ◽

Chromatin Accessibility ◽

Human Pluripotent Stem Cells ◽

Human Trophoblast ◽

Trophoblast Stem Cells ◽

Cell Fate Decisions ◽

Trophoblast Stem ◽

Normal Human

Naïve human pluripotent stem cells (hPSCs) provide a unique experimental platform of cell fate decisions during pre-implantation development, but their lineage potential remains incompletely characterized. As naïve hPSCs share transcriptional and epigenomic signatures with trophoblast cells, it has been proposed that the naïve state may have enhanced predisposition for differentiation along this extraembryonic lineage. Here we examined the trophoblast potential of isogenic naïve and primed hPSCs. We found that naïve hPSCs can directly give rise to human trophoblast stem cells (hTSCs) and undergo further differentiation into both extravillous and syncytiotrophoblast. In contrast, primed hPSCs do not support hTSC derivation, but give rise to non-self-renewing cytotrophoblasts in response to BMP4. Global transcriptome and chromatin accessibility analyses indicate that hTSCs derived from naïve hPSCs are similar to blastocyst-derived hTSCs and acquire features of post-implantation trophectoderm. The derivation of hTSCs from naïve hPSCs will enable elucidation of early mechanisms that govern normal human trophoblast development and associated pathologies.

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Assessing the bipotency of in vitro-derived neuromesodermal progenitors

F1000Research ◽

10.12688/f1000research.6345.2 ◽

2015 ◽

Vol 4 ◽

pp. 100 ◽

Cited By ~ 8

Author(s):

Anestis Tsakiridis ◽

Valerie Wilson

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Pluripotent Stem Cells ◽

Population Level ◽

Paraxial Mesoderm ◽

Cell Fate Decisions ◽

Epiblast Stem Cells ◽

Axis Elongation ◽

Stimulation Of

Retrospective clonal analysis in the mouse has demonstrated that the posterior spinal cord neurectoderm and paraxial mesoderm share a common bipotent progenitor. These neuromesodermal progenitors (NMPs) are the source of new axial structures during embryonic rostrocaudal axis elongation and are marked by the simultaneous co-expression of the transcription factors T(Brachyury) (T(Bra)) and Sox2. NMP-like cells have recently been derived from pluripotent stem cells in vitro following combined stimulation of Wnt and fibroblast growth factor (FGF) signaling. Under these conditions the majority of cultures consist of T(Bra)/Sox2 co-expressing cells after 48-72 hours of differentiation. Although the capacity of these cells to generate posterior neural and paraxial mesoderm derivatives has been demonstrated at the population level, it is unknown whether a single in vitro-derived NMP can give rise to both neural and mesodermal cells. Here we demonstrate that T(Bra) positive cells obtained from mouse epiblast stem cells (EpiSCs) after culture in NMP-inducing conditions can generate both neural and mesodermal clones. This finding suggests that, similar to their embryonic counterparts, in vitro-derived NMPs are truly bipotent and can thus be exploited as a model for studying the molecular basis of developmental cell fate decisions.

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Signaling Pathways Controlling Pluripotency and Early Cell Fate Decisions of Human Induced Pluripotent Stem Cells

Stem Cells ◽

10.1002/stem.199 ◽

2009 ◽

Vol 27 (11) ◽

pp. 2655-2666 ◽

Cited By ~ 120

Author(s):

Ludovic Vallier ◽

Thomas Touboul ◽

Stephanie Brown ◽

Candy Cho ◽

Bilada Bilican ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Signaling Pathways ◽

Cell Fate ◽

Pluripotent Stem Cells ◽

Cell Fate Decisions ◽

Induced Pluripotent

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Research and therapy with induced pluripotent stem cells (iPSCs): social, legal, and ethical considerations

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1455-y ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

Author(s):

Sharif Moradi ◽

Hamid Mahdizadeh ◽

Tomo Šarić ◽

Johnny Kim ◽

Javad Harati ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Cell Fate ◽

Pluripotent Stem Cells ◽

Cell Types ◽

Healthy Person ◽

New Drugs ◽

Full Potential ◽

Cell Fate Decisions ◽

Induced Pluripotent

AbstractInduced pluripotent stem cells (iPSCs) can self-renew indefinitely in culture and differentiate into all specialized cell types including gametes. iPSCs do not exist naturally and are instead generated (“induced” or “reprogrammed”) in culture from somatic cells through ectopic co-expression of defined pluripotency factors. Since they can be generated from any healthy person or patient, iPSCs are considered as a valuable resource for regenerative medicine to replace diseased or damaged tissues. In addition, reprogramming technology has provided a powerful tool to study mechanisms of cell fate decisions and to model human diseases, thereby substantially potentiating the possibility to (i) discover new drugs in screening formats and (ii) treat life-threatening diseases through cell therapy-based strategies. However, various legal and ethical barriers arise when aiming to exploit the full potential of iPSCs to minimize abuse or unauthorized utilization. In this review, we discuss bioethical, legal, and societal concerns associated with research and therapy using iPSCs. Furthermore, we present key questions and suggestions for stem cell scientists, legal authorities, and social activists investigating and working in this field.

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Modulation of PKM1/2 levels by steric blocking morpholinos alters the metabolic and pluripotent state of murine pluripotent stem cells

10.1101/2021.11.23.469697 ◽

2021 ◽

Author(s):

Joshua G. Dierolf ◽

Hailey L.M. Hunter ◽

Andrew J. Watson ◽

Dean H Betts

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Metabolic Switch ◽

Cell Fate Decisions ◽

Pluripotent State ◽

Primed Cell ◽

Primed Mouse

Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described formative pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 mRNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the formative state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression towards a primed pluripotent state.

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Pluripotency transcription factors at the focus: the phase separation paradigm in stem cells

Biochemical Society Transactions ◽

10.1042/bst20210856 ◽

2021 ◽

Author(s):

Camila Oses ◽

Martin Stortz ◽

Paula Verneri ◽

Alejandra Guberman ◽

Valeria Levi

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Phase Separation ◽

Cell Fate ◽

Pluripotent Stem Cells ◽

New Paradigm ◽

Cell Fate Decisions ◽

Key Players ◽

New Ideas ◽

Nuclear Processes

The transcription factors (TFs) OCT4, SOX2 and NANOG are key players of the gene regulatory network of pluripotent stem cells. Evidence accumulated in recent years shows that even small imbalances in the expression levels or relative concentrations of these TFs affect both, the maintenance of pluripotency and cell fate decisions. In addition, many components of the transcriptional machinery including RNA polymerases, cofactors and TFs such as those required for pluripotency, do not distribute homogeneously in the nucleus but concentrate in multiple foci influencing the delivery of these molecules to their DNA-targets. How cells control strict levels of available pluripotency TFs in this heterogeneous space and the biological role of these foci remain elusive. In recent years, a wealth of evidence led to propose that many of the nuclear compartments are formed through a liquid–liquid phase separation process. This new paradigm early penetrated the stem cells field since many key players of the pluripotency circuitry seem to phase-separate. Overall, the formation of liquid compartments may modulate the kinetics of biochemical reactions and consequently regulate many nuclear processes. Here, we review the state-of-the-art knowledge of compartmentalization in the cell nucleus and the relevance of this process for transcriptional regulation, particularly in pluripotent stem cells. We also highlight the recent advances and new ideas in the field showing how compartmentalization may affect pluripotency preservation and cell fate decisions.

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bHLH Transcription Factor Math6 Antagonizes TGF-β Signalling in Reprogramming, Pluripotency and Early Cell Fate Decisions

Cells ◽

10.3390/cells8060529 ◽

2019 ◽

Vol 8 (6) ◽

pp. 529 ◽

Cited By ~ 1

Author(s):

Satya Srirama Karthik Divvela ◽

Patrick Nell ◽

Markus Napirei ◽

Holm Zaehres ◽

Jiayu Chen ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Cell Fate ◽

Pluripotent Stem Cells ◽

Cellular Reprogramming ◽

Bhlh Transcription Factor ◽

Tgf Beta ◽

Cell Fate Decisions ◽

Helix Loop Helix ◽

Induced Pluripotent

The basic helix-loop-helix (bHLH) transcription factor Math6 (Atonal homolog 8; Atoh8) plays a crucial role in a number of cellular processes during embryonic development, iron metabolism and tumorigenesis. We report here on its involvement in cellular reprogramming from fibroblasts to induced pluripotent stem cells, in the maintenance of pluripotency and in early fate decisions during murine development. Loss of Math6 disrupts mesenchymal-to-epithelial transition during reprogramming and primes pluripotent stem cells towards the mesendodermal fate. Math6 can thus be considered a regulator of reprogramming and pluripotent stem cell fate. Additionally, our results demonstrate the involvement of Math6 in SMAD-dependent TGF beta signalling. We furthermore monitor the presence of the Math6 protein during these developmental processes using a newly generated Math6Flag-tag mouse. Taken together, our results suggest that Math6 counteracts TGF beta signalling and, by this, affects the initiating step of cellular reprogramming, as well as the maintenance of pluripotency and early differentiation.

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