Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells

The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.

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199 DIFFERENTIATION OF HIGHLY ENRICHED OLIGODENDROCYTE PRECURSORS AND MATURE OLIGODENDROCYTES FROM RHESUS MONKEY EMBRYONIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab199 ◽

2006 ◽

Vol 18 (2) ◽

pp. 207

Author(s):

T. Li ◽

Y. Xie ◽

W. Ji

Keyword(s):

Stem Cells ◽

Cell Differentiation ◽

Growth Factor ◽

Rhesus Monkey ◽

Molecular Mechanisms ◽

Es Cells ◽

Embryonic Stem ◽

Oligodendrocyte Differentiation ◽

Culture Systems ◽

Oligodendrocyte Precursors

Generating homologous oligodendrocytes are required for studying the molecular mechanisms of oligodendrogliogenesis and for providing donor cells for transplantation therapies. Previous studies have shown that embryonic stem (ES) cells can be induced to generate neural stem cells with many kinds of culture systems; however, few or no oligodendrocytes were obtained from these culture systems. Here we present a simple method containing five steps for obtaining highly enriched oligodendrocyte precursors (75 � 6.8%) and mature oligodendrocytes (81 � 8.6%) from rhesus monkey embryonic stem (rES) cells. We expanded rES cells on a feeder layer of irradiated MESF (ear skin fibroblasts from a one-week-old rhesus monkey), formed embryoid bodies (EBs), promoted Day 9 (3 days in hanging drop and 6 days in suspension) differentiation into highly enriched (90.2 � 6.1%) neural progenitors (NPs) with hepatocyte growth factor (HGF) and G5 supplement [containing 5 ng/mL (bFGF) and 10 ng/mL epidermal growth factor (EGF)], purified NPs with 0.0625% trypsin in 0.04% EDTA (98% of cells were nestin-positive), amplified those progenitors in HGF and G5 media for two months, and then induced oligodendrocyte precursors differentiation in the absence of G5, but in the presence of 20 ng/mL HGF for 2 days. To obtain terminal oligodendrocytes, neurospheres cultured for 2 months were plated on laminin-coated plates for 3 weeks in the presence of HGF. The results showed that differentiated cells expressed myelin basic protein (MBP) and had typical mature oligodendrocyte morphology. Our studies also revealed that HGF significantly increased the NP proliferation speed (P < 0.05) by both decreasing cell apoptosis rate (P < 0.05) and shortening cell cycle time (P < 0.05) in the presence of G5. Additionally, HGF promoted oligodendrocyte maturation by increasing the length and number of branches and the expression of MBP. To test whether the original HGF had similar functions for oligodendrocyte specification, a series of experiments were evaluated by adding HGF or G5 to differentiation or expansion media at different differentiation stages. The results demonstrated that the ability of HGF responsiveness to initiate oligodendrocyte differentiation was regulated by G5 and by HGF alone without G5-induced rES cell differentiation into neurons. Further studies showed that the crucial time point of G5 action was from EBs to NPs; the early addition of HGF to EBs in the presence of G5 increased oligodendrocyte differentiation rate, but was not necessary, and the treatment during the first 2 days was enough to produce a similar effect; and HGF was required for terminal oligodendrocyte differentiation from NPs. Taken together, these results showed that HGF and G5 cooperatively promote rES cell differentiation into highly enriched oligodendrocyte precursors and mature oligodendrocytes.These observations set the method for obtaining highly enriched oligodendrocytes from ES cells in the nonhuman primate for clinical application and provide a platform to probe the molecular mechanisms that control oligodendrocyte differentiation.

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Abstract P001: HDAC1 Plays an Important Role in the Differentiation of Embryonic Stem Cells and Induced Pluripotent Stem Cells into Cardiovascular Lineages

Circulation Research ◽

10.1161/res.109.suppl_1.ap001 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Eneda Hoxha ◽

Erin Lambers ◽

Veronica Ramirez ◽

Prasanna Krishnamurthy ◽

Suresh Verma ◽

...

Keyword(s):

Stem Cells ◽

Molecular Mechanisms ◽

Es Cells ◽

Critical Role ◽

Embryonic Stem ◽

Ips Cells ◽

Full Potential ◽

Early Differentiation ◽

Increased Risk ◽

Mes Cells

Despite advancements in the treatment of myocardial infarction (MI), the majority of patients are at increased risk for developing heart failure due to the loss of cardiomyocytes and microvasculature. Some of the main obstacles in the realization of the full potential of iPS/ES cells arise from incomplete and poorly understood molecular mechanisms and epigenetic modifications that govern their pluripotency and directed differentiation. Real-time array experiments revealed that HDAC1 is highly expressed in pluripotent cells. Additionally the lack of this molecule is embryonic lethal, suggesting it plays a key role in development. Thus, we hypothesized that HDAC1 plays a critical role in directing cardiovascular differentiation of mES and iPS cells in vitro. HDAC1 was knocked down in mES cells (C57BL/6) and iPS cells using a shRNA vector. Differentiation through embryoid body (EB) was induced in wild type mES cells and iPS cells and in their HDAC1-null counterparts and the ability of these cells to differentiate into three early embryonic lineages and more specifically cardiovascular lineage was monitored. EBs lacking HDAC1 differentiated slower and showed delayed suppression of pluripotent genes such as Oct4 and Sox2. ChiP experiments revealed high histone acetylation levels at the promoter regions of these genes during early differentiation. In addition cells lacking HDAC1 showed reduced expression of early markers for all three germ layers. HDAC1-null EBs also showed delayed and reduced spontaneous beating. Expression of cardiomyocite markers as well as markers of other cardiovascular lineages was repressed in HDAC1 -null cells. However, supplementation with BMP2 during early differentiation recovered the ability in the HDAC1-null cells to differentiate into endodermal and mesodermal lineages, but not ectodermal. We propose that HDAC1 plays a critical role in early development and cardiovascular differentiation of mES and iPS cells by repressing pluripotent genes and allowing for expression of early developmental genes such as SOX17 and BMP2. Further research in the molecular mechanisms involved in this process will greatly aid our understanding of the epigenetic circuitry of pluripotency and differentiation in ES and iPS cells.

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TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing

Bone Marrow Research ◽

10.1155/2014/986571 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Louis Norman ◽

Paul Tarrant ◽

Timothy Chevassut

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Genomic Dna ◽

Es Cells ◽

Embryonic Stem ◽

Gene Promoters ◽

Loss Of Function ◽

Promoter Sequences ◽

Maintenance Methylation

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5′-methylcytosine to 5′-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5′-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5′-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5′-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented.

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Thrombopoietin (TPO) Enhances Heamatopoietic Differentiation of Human Embryonic Stem Cells.

Blood ◽

10.1182/blood.v108.11.4157.4157 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4157-4157

Author(s):

Anand S. Srivastava ◽

Rangrath Mishra ◽

Ewa Carrier

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Es Cells ◽

Embryonic Stem ◽

Cytoplasmic Domain ◽

Embryoid Bodies ◽

Janus Kinase ◽

Human Es Cells

Abstract Recently, it was demonstrated that TPO enhances hematopoietic differentiation of primate ES cells, but its role in differentiating human ES cells is unknown. We sought to investigate the regulatory mechanism of TPO induced signals mediated by the c-mpl cytoplasmic domain during human embryonic stem (hES) cells hematopoietic commitment. We hypothesize that in human embryonic stem cells, binding of TPO to its c-mpl receptor causes three-dimensional alterations which bring the c-mpl cytoplasmic domain and Janus Kinase into close-proximity and thus induces the phosphorylation and dimerization of STAT5 molecule. Dimerized STAT5 molecules detach from the receptors and migrate to the nucleus where they bind GAS site and induce transcription of a set of target, hematopoiesis-related genes. NIH human ES cell lines (WI01) were used in this experiment. In brief, to induce EB formation, cells were incubated in differentiation medium, which consisted of knockout DMEM medium (GIBCO/BRL, Carlsbad, USA), supplemented with 20% non-heat-inactivated fetal bovine serum (FBS, Hyclone, USA), 1% nonessential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Subsequently, DMEM was replaced by IMDM (GIBCO/BRL, USA) with the same supplements and additional two cytokines (100 ng/mL SCF and 100 ng/mL Flt-3 ligand (Flt-3L)) (control group). To investigate the role of TPO and VEGF, cells were additionally treated with 100 ng/mL TPO alone or in combination with 100 ng/mL rhVEGF. All cytokines were from the R&D systems (USA). Significant increase in the numbers of embryoid bodies (EBs) formation in TPO (125/105), TPO/VEGF (150/105 cells) when compared to controls (10/105 planted ES cells) was documented. This corresponded with the increase in CFU-C and the number of CD31/CD34 positive and CD34-positive progenitors. Analysis of gene expression during hematopoietic development demonstrated that TPO/VEGF combination increased mRNA expression of the TPO receptor (TPO-R) and VEGF (VEGF-R) receptors in hematopoietic progenitors obtained from human ES cells. We are in the process of determining the role of JAK/STAT pathway in this process; functional studies involve blocking of TPO/c-mpl using TPO-R-specific antibodies and determining its impact on human ES-derived hematopoiesis.

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Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis

Biochemical Society Transactions ◽

10.1042/bst0331534 ◽

2005 ◽

Vol 33 (6) ◽

pp. 1534-1536 ◽

Cited By ~ 5

Author(s):

S.A. Duncan

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Es Cells ◽

Embryonic Stem ◽

Recipient Mouse ◽

Gata Factors ◽

Embryonic Tissues ◽

Tetraploid Complementation ◽

Traditional Procedure

Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

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Zic3 Is Required for Maintenance of Pluripotency in Embryonic Stem Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-07-0624 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1348-1358 ◽

Cited By ~ 85

Author(s):

Linda Shushan Lim ◽

Yuin-Han Loh ◽

Weiwei Zhang ◽

Yixun Li ◽

Xi Chen ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Embryonic Stem Cells ◽

Regulatory Networks ◽

Molecular Mechanisms ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Lineage Specification ◽

Mouse Es Cells

Embryonic stem (ES) cell pluripotency is dependent upon sustained expression of the key transcriptional regulators Oct4, Nanog, and Sox2. Dissection of the regulatory networks downstream of these transcription factors has provided critical insight into the molecular mechanisms that regulate ES cell pluripotency and early differentiation. Here we describe a role for Zic3, a member of the Gli family of zinc finger transcription factors, in the maintenance of pluripotency in ES cells. We show that Zic3 is expressed in ES cells and that this expression is repressed upon differentiation. The expression of Zic3 in pluripotent ES cells is also directly regulated by Oct4, Sox2, and Nanog. Targeted repression of Zic3 in human and mouse ES cells by RNA interference–induced expression of several markers of the endodermal lineage. Notably, the expression of Nanog, a key pluripotency regulator and repressor of extraembryonic endoderm specification in ES cells, was significantly reduced in Zic3 knockdown cells. This suggests that Zic3 may prevent endodermal marker expression through Nanog-regulated pathways. Thus our results extend the ES cell transcriptional network beyond Oct4, Nanog, and Sox2, and further establish that Zic3 plays an important role in the maintenance of pluripotency by preventing endodermal lineage specification in embryonic stem cells.

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Abstract 134: Rigid Microenvironments Promote Cardiac Differentiation Of Mouse And Human Embryonic Stem Cells

Circulation Research ◽

10.1161/res.113.suppl_1.a134 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Armin Arshi ◽

Yasuhiro Nakashima ◽

Haruko Nakano ◽

Sarayoot Eaimkhong ◽

Denis Evseenko ◽

...

Keyword(s):

Stem Cells ◽

Cardiac Tissue ◽

De Novo ◽

Troponin T ◽

Es Cells ◽

Embryonic Stem ◽

Cell Lineage ◽

Cardiac Differentiation ◽

Expression Ratio

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using an genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of the already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

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Abscission couples cell division to embryonic stem cell fate

10.1101/798165 ◽

2019 ◽

Cited By ~ 3

Author(s):

Agathe Chaigne ◽

Celine Labouesse ◽

Meghan Agnew ◽

Edouard Hannezo ◽

Kevin J Chalut ◽

...

Keyword(s):

Stem Cells ◽

Cell Division ◽

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Strongly Correlated ◽

Cellular Mechanisms ◽

Mouse Es Cells ◽

Cytoplasmic Bridges

Cell fate transitions are key to development and homeostasis. It is thus essential to understand the cellular mechanisms controlling fate transitions. Cell division has been implicated in fate decisions in many stem cells, including neuronal and epithelial progenitors. In other stem cells, such as embryonic stem (ES) cells, the role of division remains unclear. Here we show that exit from naïve pluripotency in mouse ES cells generally occurs after a division. We further show that exit timing is strongly correlated between sister cells, which remain connected by cytoplasmic bridges long after division, and that bridge abscission progressively accelerates as cells exit naïve pluripotency. Finally, interfering with abscission impairs exit from naïve pluripotency. Altogether, our data indicate that a switch in the division machinery leading to faster abscission is crucial for pluripotency exit. Our study identifies abscission as a key step coupling cell division to fate transitions.

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The Key Role of MicroRNAs in Self-Renewal and Differentiation of Embryonic Stem Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21176285 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6285

Author(s):

Giuseppina Divisato ◽

Fabiana Passaro ◽

Tommaso Russo ◽

Silvia Parisi

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Developmental Stages ◽

Embryonic Stem ◽

Self Renewal ◽

Cycle Progression ◽

Epiblast Stem Cells

Naïve pluripotent embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent distinctive developmental stages, mimicking the pre- and the post-implantation events during the embryo development, respectively. The complex molecular mechanisms governing the transition from ESCs into EpiSCs are orchestrated by fluctuating levels of pluripotency transcription factors (Nanog, Oct4, etc.) and wide-ranging remodeling of the epigenetic landscape. Recent studies highlighted the pivotal role of microRNAs (miRNAs) in balancing the switch from self-renewal to differentiation of ESCs. Of note, evidence deriving from miRNA-based reprogramming strategies underscores the role of the non-coding RNAs in the induction and maintenance of the stemness properties. In this review, we revised recent studies concerning the functions mediated by miRNAs in ESCs, with the aim of giving a comprehensive view of the highly dynamic miRNA-mediated tuning, essential to guarantee cell cycle progression, pluripotency maintenance and the proper commitment of ESCs.

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Stem Cells - biological update and cell therapy progress

Medicine and Pharmacy Reports ◽

10.15386/cjmed-483 ◽

2015 ◽

Vol 88 (3) ◽

pp. 265-271 ◽

Cited By ~ 16

Author(s):

Mihai Girlovanu ◽

Sergiu Susman ◽

Olga Soritau ◽

Dan Rus-Ciuca ◽

Carmen Melincovici ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Therapy ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Type I ◽

Differentiation Potential ◽

Induced Pluripotent

In recent years, the advances in stem cell research have suggested that the human body may have a higher plasticity than it was originally expected.Until now, four categories of stem cells were isolated and cultured in vivo: embryonic stem cells, fetal stem cells, adult stem cells and induced pluripotent stem cells (hiPSCs).Although multiple studies were published, several issues concerning the stem cells are still debated, such as: the molecular mechanisms of differentiation, the methods to prevent teratoma formation or the ethical and religious issues regarding especially the embryonic stem cell research.The direct differentiation of stem cells into specialized cells: cardiac myocytes, neural cells, pancreatic islets cells, may represent an option in treating incurable diseases such as: neurodegenerative diseases, type I diabetes, hematologic or cardiac diseases.Nevertheless, stem cell-based therapies, based on stem cell transplantation, remain mainly at the experimental stages and their major limitation is the development of teratoma and cancer after transplantation. The induced pluripotent stem cells (hiPSCs) represent a prime candidate for future cell therapy research because of their significant self-renewal and differentiation potential and the lack of ethical issues.This article presents an overview of the biological advances in the study of stem cells and the current progress made in the field of regenerative medicine.

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