Direct Effect of Chenodeoxycholic Acid on Differentiation of Mouse Embryonic Stem Cells Cultured under Feeder-Free Culture Conditions

Chenodeoxycholic acid (CDCA), a farnesoid X receptor (FXR) ligand, is a member of the nuclear receptor family and is probably involved in regulating the cellular activities of embryonic stem (ES) cells. Recently, although it was reported that the FXR ligand can mediate differentiation, apoptosis, and/or growth arrest in several cell types, it is still not well known how CDCA mediates effects in ES cells. Therefore, we investigated the direct effect of CDCA on mES cells. Feeder-free mES cells were treated in a dose-dependent manner with CDCA (50, 100, and 200 μM) for 72 h, and then a 100 μM CDCA treatment was performed for an additional 72 h. We analyzed the morphology, cell growth, cell characteristics, immunocytochemistry, and RT-PCR. In CDCA-treated cells, we observed the disappearance of pluripotent stem cell markers including alkaline phosphatase, Oct4, and Nanog and a time- and dose-dependent increase in expression of nestin, PAX6, andα-smooth muscle actin, but notα-fetoprotein. The 100 μM CDCA-treated cells in their second passage continued this differentiation pattern similar to those in the controls. In conclusion, these results suggest that CDCA can guide mES cells by an FXR-independent pathway to differentiate into ectoderm and/or mesoderm, but not endoderm.

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Different Kinetics and Function of Vascular Endothelial Growth Factor Recepotor-1 and −2 during Hemangioblast Development from Primate Embryonic Stem Cells.

Blood ◽

10.1182/blood.v108.11.3920.3920 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3920-3920

Author(s):

Katsutsugu Umeda ◽

Toshio Heike ◽

Gen Shinoda ◽

Akira Niwa ◽

Masato Arai ◽

...

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Cell Development ◽

Dependent Manner ◽

Vascular Endothelial ◽

Blocking Antibody ◽

Cd34 Cells ◽

Low Levels ◽

Endothelial Growth Factor ◽

Dose Dependent

Abstract The close developmental association between hematopoietic and endothelial cells suggests that both lineage cells share a common precursor, the hemangioblast. The vascular endothelial growth factor (VEGF)-A system has been proven to have roles in the embryonic development of hemangioblast in mouse by the use of embryos or embryonic stem (ES) cells deprived of the genes encoding its ligand or receptors (VEGFR-1 and VEGFR-2). On the other hand, there have been only a few reports on the hemangioblast development during primate (human and monkey) embryogenesis. We have previously demonstrated that the hemangioblast is highly enriched in the VEGFR-2high CD34+ cell fraction, differentiated from cynomolgus monkey ES cells by 6-day coculture with OP9 stromal cells. In the current study, we examined whether the VEGF-A system was involved in the development of hemangioblast induced from primate ES cells by using the coculture system. VEGFR-1 and −2 were expressed by undifferentiated monkey ES cells at low levels. The VEGFR-2low cells gradually decreased while VEGFR-2high cells could be detected on day 6. On the other hand, VEGFR-1 was constantly expressed at low levels on day 6 and thereafter. Exogenous VEGF-A165, the action of which is mediated VEGFR-1 and VEGFR-2, increased the proportion of VEGFR-2high CD34+ cells in a dose-dependent manner. Addition of VEGF-E (VEGFR-2-specific agonist) and VEGFR-1 blocking antibody resulted in the increase of VEGFR-2high CD34+ cells in a dose-dependent manner, while VEGFR-2 blocking antibody suppressed their development. Thus, VEGF-A/VEGFR-2 interaction promoted the development of hemangioblast, while VEGF-A/VEGFR-1 interaction acted as a negative regulator. We then examined when VEGF-related signals work on VEGFR-2high cell development by changing factors on day 4. The proportion of VEGFR-2high CD34+ cells with the last 2-day VEGF-A165 and -E treatment were almost equivalent to that with continuous exposure to the VEGF. On the contrary, initial 4-day VEGF-A165 and -E treatment did not any stimulatory effects on VEGFR-2high CD34+ cell development. Our previous results show that during differentiation, Brachyury (early mesodermal marker) is first expressed on day 4, followed by the up-regulation of genes, such as SCL, LMO2, and MYB, representing hematopoietic and/or endothelial potentials on day 6. Collectively, VEGFR-2-mediated signals might contribute to the commitment and/or expansion of the hemangioblast rather than their development. This coculture system provides an opportunity to better understand the regulative mechanisms on early hematopoietic and endothelial cell development, which remains unresolved by experiments using human embryos.

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Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells

Development ◽

10.1242/dev.121.9.2853 ◽

1995 ◽

Vol 121 (9) ◽

pp. 2853-2859 ◽

Cited By ~ 1

Author(s):

A. Weng ◽

T. Magnuson ◽

U. Storb

Keyword(s):

De Novo ◽

Es Cells ◽

Embryonic Stem ◽

Dependent Manner ◽

Mouse Development ◽

Partial Methylation ◽

Distal Chromosome ◽

Before And After ◽

Endogenous Genes ◽

De Novo Methylation

A murine transgene, HRD, is methylated only when carried in certain inbred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain-specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryos revealed that strain-specific methylation is initiated prior to embryonic day (E) 6.5. Strain-specific transgene methylation is all-or-none in pattern and occurs exclusively in the primitive ectoderm lineage. A strain-independent pattern of partial methylation occurs in the primitive endoderm and trophectoderm lineages. To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and examined for transgene methylation before and after differentiation. Though the transgene had already acquired some methylation in undifferentiated ES cells, differentiation induced further, de novo methylation in a strain-dependent manner. Analysis of methylation in ES cultures suggests that the transgene and endogenous genes (such as immunoglobulin genes) are synchronously methylated during early development. These results are interpreted in the context of a model in which Ssm1-like modifier genes produce alterations in chromatin structure during and/or shortly after implantation, thereby marking target loci for de novo methylation with the rest of the genome during gastrulation.

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SUMO conjugation susceptibility of Akt/protein kinase B affects the expression of the pluripotency transcription factor Nanog in embryonic stem cells

PLoS ONE ◽

10.1371/journal.pone.0254447 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0254447

Author(s):

Marcos Francia ◽

Martin Stortz ◽

Camila Vazquez Echegaray ◽

Camila Oses ◽

Paula Verneri ◽

...

Keyword(s):

Transcription Factor ◽

Es Cells ◽

Embryonic Stem ◽

Dependent Manner ◽

Cellular Functions ◽

Cellular Processes ◽

Sumo Conjugation ◽

Heterologous System ◽

Canonical Pathways ◽

The Impact

Akt/PKB is a kinase involved in the regulation of a wide variety of cell processes. Its activity is modulated by diverse post-translational modifications (PTMs). Particularly, conjugation of the small ubiquitin-related modifier (SUMO) to this kinase impacts on multiple cellular functions, such as proliferation and splicing. In embryonic stem (ES) cells, this kinase is key for pluripotency maintenance. Among other functions, Akt is known to promote the expression of Nanog, a central pluripotency transcription factor (TF). However, the relevance of this specific PTM of Akt has not been previously analyzed in this context. In this work, we study the effect of Akt1 variants with differential SUMOylation susceptibility on the expression of Nanog. Our results demonstrate that both, the Akt1 capability of being modified by SUMO conjugation and a functional SUMO conjugase activity are required to induce Nanog gene expression. Likewise, we found that the common oncogenic E17K Akt1 mutant affected Nanog expression in ES cells also in a SUMOylatability dependent manner. Interestingly, this outcome takes places in ES cells but not in a non-pluripotent heterologous system, suggesting the presence of a crucial factor for this induction in ES cells. Remarkably, the two major candidate factors to mediate this induction, GSK3-β and Tbx3, are non-essential players of this effect, suggesting a complex mechanism probably involving non-canonical pathways. Furthermore, we found that Akt1 subcellular distribution does not depend on its SUMOylatability, indicating that Akt localization has no influence on the effect on Nanog, and that besides the membrane localization of E17K Akt mutant, SUMOylation is also required for its hyperactivity. Our results highlight the impact of SUMO conjugation in the function of a kinase relevant for a plethora of cellular processes, including the control of a key pluripotency TF.

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NPM1 is a Novel Therapeutic Target and Prognostic Biomarker for Ewing Sarcoma

Frontiers in Genetics ◽

10.3389/fgene.2021.771253 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yangfan Zhou ◽

Yuan Fang ◽

Junjie Zhou ◽

Yulian Liu ◽

Shusheng Wu ◽

...

Keyword(s):

Ewing Sarcoma ◽

Es Cells ◽

Differentially Expressed Gene ◽

Bioinformatic Analysis ◽

Dependent Manner ◽

Hub Gene ◽

Immune Infiltration ◽

Proliferation And Apoptosis ◽

Immune Related Genes ◽

Dose Dependent

Ewing sarcoma (ES) is a cancer that may originate from stem mesenchymal or neural crest cells and is highly prevalent in children and adolescents. In recent years, targeted therapies against immune-related genes have shown good efficacy in a variety of cancers. However, effective targets for immunotherapy in ES are yet to be developed. In our study, we first identified the immune-associated differential hub gene NPM1 by bioinformatics methods as a differentially expressed gene, and then validated it using real time-PCR and western blotting, and found that this gene is not only closely related to the immune infiltration in ES, but also can affect the proliferation and apoptosis of ES cells, and is closely related to the survival of patients. The results of our bioinformatic analysis showed that NPM1 can be a hub gene in ES and an immunotherapeutic target to reactivate immune infiltration in patients with ES. In addition, treatment with NPM1 promoted apoptosis and inhibited the proliferation of ES cells. The NPM1 inhibitor NSC348884 can induce apoptosis of ES cells in a dose-dependent manner and is expected to be a potential therapeutic agent for ES.

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Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro

Blood ◽

10.1182/blood.v95.7.2275 ◽

2000 ◽

Vol 95 (7) ◽

pp. 2275-2283 ◽

Cited By ~ 65

Author(s):

Naoki Nakayama ◽

Jae Lee ◽

Laura Chiu

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Bone Morphogenetic Protein ◽

Es Cells ◽

Embryonic Stem ◽

Dependent Manner ◽

Vascular Endothelial ◽

Morphogenetic Protein ◽

Endothelial Growth Factor

Abstract The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.

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Piezo1 forms a slowly-inactivating mechanosensory channel in mouse embryonic stem cells

eLife ◽

10.7554/elife.33149 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 24

Author(s):

Josefina Inés del Mármol ◽

Kouki K Touhara ◽

Gist Croft ◽

Roderick MacKinnon

Keyword(s):

Motor Neurons ◽

Es Cells ◽

Embryonic Stem ◽

Inactivation Kinetics ◽

Fast Inactivation ◽

Cell Stage ◽

Slow Kinetics ◽

Aminoacid Sequence ◽

Reduced Rate ◽

Mes Cells

Piezo1 is a mechanosensitive (MS) ion channel with characteristic fast-inactivation kinetics. We found a slowly-inactivating MS current in mouse embryonic stem (mES) cells and characterized it throughout their differentiation into motor-neurons to investigate its components. MS currents were large and slowly-inactivating in the stem-cell stage, and became smaller and faster-inactivating throughout the differentiation. We found that Piezo1 is expressed in mES cells, and its knockout abolishes MS currents, indicating that the slowly-inactivating current in mES cells is carried by Piezo1. To further investigate its slow inactivation in these cells, we cloned Piezo1 cDNA from mES cells and found that it displays fast-inactivation kinetics in heterologous expression, indicating that sources of modulation other than the aminoacid sequence determine its slow kinetics in mES cells. Finally, we report that Piezo1 knockout ES cells showed a reduced rate of proliferation but no significant differences in other markers of pluripotency and differentiation.

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Stem cell markers in gastrointestinal cancer

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.21095 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 21095-21095

Author(s):

M. Valladares-Ayerbes ◽

S. Díaz Prado ◽

V. Medina ◽

P. Iglesias ◽

B. Rodríguez ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Colon Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Gastrointestinal Cancer ◽

Es Cells ◽

Embryonic Stem ◽

Stem Cell Markers ◽

Continuous Growth

21095 Background: Cancer cells with stem cells (CSC) properties have been identified in different tumors. It is suggested that CSC are responsible for the continuous growth of tumors, metastasis and drug-resistance. Markers for stem cells have been described. Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. ABCG2 transporter (MDR1) gene expression has been described as surrogate for the side-population phenotype. PTTG1 has also been recently identified as a component of the molecular signature of human (hu) ES cell-lines. Methods: Using Digital Northern we have demonstrated a significant tag counts for PTTG1 and reticulocalbin 2 (RCN2) in 11 huES cell-lines of the CGAP. The objective of our work has been to assess gene expression of these SC markers in a panel of new gastrointestinal cancer (GC) cells lines (CL) developed in our laboratory. Quantitative assessment was obtained by real-time PCR relative to normal bone marrow (BM), colonic mucosa and established cell-lines. GCCLs have been developed from ascitic fluid obtained of pancreatic carcinoma (MBQ-OJC1) and colon cancer (JJPF-OJC4, LCM-OJC5 and JAC-OJC6). GCCLs had been fully characterized by cytomorphology, epithelial and tumor markers (keratins, EGFR, EpCAM, p53), karyotype and tumor spheroids cultures. Results: Expression for ABCG2, Nanog, Oct4, PTTG1 and RCN2 were clearly detected in all the GCCL. Relative levels for each mRNA shown wide variety. For example, ABCG2 mRNA was highly expressed (2–26 fold) in colon cancer CL relative to BM. RCN2 was overexpressed (more than 2 x 102 fold) in 3 GCCL. Conclusions: Our results show that expressions of different “stemness” genes are maintained in cultured cancer cells. These data suggest that CSC are present in metastatic sites and can be maintained in continuous culture. We hypothesized that PTTG1 and RCN2 could be tested as a new cancer stem cells markers. No significant financial relationships to disclose.

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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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Effects of Synthetic Neural Adhesion Molecule Mimetic Peptides and Related Proteins on the Cardiomyogenic Differentiation of Mouse Embryonic Stem Cells

Cellular Physiology and Biochemistry ◽

10.1159/000374044 ◽

2015 ◽

Vol 35 (6) ◽

pp. 2437-2450 ◽

Cited By ~ 2

Author(s):

Ruodan Xu ◽

Sureshkumar Perumal Srinivasan ◽

Poornima Sureshkumar ◽

Erastus Nembu Nembo ◽

Christoph Schäfer ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Synthetic Peptides ◽

Es Cells ◽

Embryonic Stem ◽

Dependent Manner ◽

Es Cell ◽

Cardiomyogenic Differentiation ◽

Cellular Environment ◽

Structural And Functional Properties

Background/Aims: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. Methods: In the present study, using a transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain (α-MHC) promoter (pαMHC-EGFP), we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGLL, hNgf_C2, EnkaminE, Plannexin and C3) on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. Results: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF) peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. Conclusion: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to cardiac pathologies where BDNF levels are impaired.

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Redirecting differentiation of hematopoietic progenitors by a transcription factor, GATA-2

Blood ◽

10.1182/blood-2005-06-2527 ◽

2006 ◽

Vol 107 (5) ◽

pp. 1857-1863 ◽

Cited By ~ 54

Author(s):

Kenji Kitajima ◽

Makoto Tanaka ◽

Jie Zheng ◽

Hilo Yen ◽

Ayuko Sato ◽

...

Keyword(s):

Transcription Factor ◽

Es Cells ◽

Critical Role ◽

Embryonic Stem ◽

Erythroid Differentiation ◽

Dependent Manner ◽

Hematopoietic Differentiation ◽

Macrophage Differentiation ◽

Conditional Gene Expression

GATA-2 is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells. We analyzed the function of GATA-2 by a combined method of tetracycline-dependent conditional gene expression and in vitro hematopoietic differentiation from mouse embryonic stem (ES) cells using OP9 stroma cells (OP9 system). In the presence of macrophage colony-stimulating factor (M-CSF), the OP9 system induced macrophage differentiation. GATA-2 expression in this system inhibited macrophage differentiation and redirected the fate of hematopoietic differentiation to other hematopoietic lineages. GATA-2 expression commencing at day 5 or day 6 induced megakaryocytic or erythroid differentiation, respectively. Expression levels of PU.1, a hematopoietic transcription factor that interferes with GATA-2, appeared to play a critical role in differentiation to megakaryocytic or erythroid lineages. Transcription of PU.1 was affected by histone acetylation induced by binding of GATA-2 to the PU.1 promoter region. This study demonstrates that the function of GATA-2 is modified in a context-dependent manner by expression of PU.1, which in turn is regulated by GATA-2.

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