Reorganization of Metabolism during Cardiomyogenesis Implies Time-Specific Signaling Pathway Regulation

Understanding the cell differentiation process involves the characterization of signaling and regulatory pathways. The coordinated action involved in multilevel regulation determines the commitment of stem cells and their differentiation into a specific cell lineage. Cellular metabolism plays a relevant role in modulating the expression of genes, which act as sensors of the extra-and intracellular environment. In this work, we analyzed mRNAs associated with polysomes by focusing on the expression profile of metabolism-related genes during the cardiac differentiation of human embryonic stem cells (hESCs). We compared different time points during cardiac differentiation (pluripotency, embryoid body aggregation, cardiac mesoderm, cardiac progenitor and cardiomyocyte) and showed the immature cell profile of energy metabolism. Highly regulated canonical pathways are thoroughly discussed, such as those involved in metabolic signaling and lipid homeostasis. We reveal the critical relevance of retinoic X receptor (RXR) heterodimers in upstream retinoic acid metabolism and their relationship with thyroid hormone signaling. Additionally, we highlight the importance of lipid homeostasis and extracellular matrix component biosynthesis during cardiomyogenesis, providing new insights into how hESCs reorganize their metabolism during in vitro cardiac differentiation.

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Activation of the amino acid response modulates lineage specification during differentiation of murine embryonic stem cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00136.2013 ◽

2013 ◽

Vol 305 (3) ◽

pp. E325-E335 ◽

Cited By ~ 6

Author(s):

Jixiu Shan ◽

Takashi Hamazaki ◽

Tiffany A. Tang ◽

Naohiro Terada ◽

Michael S. Kilberg

Keyword(s):

Stem Cells ◽

Amino Acid ◽

Embryonic Stem Cells ◽

Embryoid Body ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Specific Cell ◽

Lineage Specification ◽

Amino Acid Response

In somatic cells, a collection of signaling pathways activated by amino acid limitation have been identified and referred to as the amino acid response (AAR). Despite the importance of possible detrimental effects of nutrient limitation during in vitro culture, the AAR has not been investigated in embryonic stem cells (ESC). AAR activation caused the expected increase in transcription factors that mediate specific AAR pathways, as well as the induction of asparagine synthetase, a terminal AAR target gene. Neither AAR activation nor stable knockdown of activating transcription factor (Atf) 4, a transcriptional mediator of the AAR, adversely affected ESC self-renewal or pluripotency. Low-level induction of the AAR over a 12-day period of embryoid body differentiation did alter lineage specification such that the primitive endodermal, visceral endodermal, and endodermal lineages were favored, whereas mesodermal and certain ectodermal lineages were suppressed. Knockdown of Atf4 further enhanced the AAR-induced increase in endodermal formation, suggesting that this phenomenon is mediated by an Atf4-independent mechanism. Collectively, the results indicate that, during differentiation of mouse embryoid bodies in culture, the availability of nutrients, such as amino acids, can influence the formation of specific cell lineages.

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Activin A Modulates CRIPTO-1/HNF4α+ Cells to Guide Cardiac Differentiation from Human Embryonic Stem Cells

Stem Cells International ◽

10.1155/2017/4651238 ◽

2017 ◽

Vol 2017 ◽

pp. 1-17 ◽

Cited By ~ 6

Author(s):

Robin Duelen ◽

Guillaume Gilbert ◽

Abdulsamie Patel ◽

Nathalie de Schaetzen ◽

Liesbeth De Waele ◽

...

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Heart Development ◽

Embryoid Body ◽

Embryonic Stem ◽

Activin A ◽

Embryoid Bodies ◽

Cardiac Differentiation ◽

Potential Applications

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-, atrial-, and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives, which in turn promoted cardiomyocyte differentiation. Moreover, a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation, improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation, measuring action potentials, and intracellular Ca2+ dynamics. These findings are relevant for improving our understanding on human heart development, and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes, similar to those observed in adult cardiac myocytes.

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AW551984: a novel regulator of cardiomyogenesis in pluripotent embryonic cells

Biochemical Journal ◽

10.1042/bj20110520 ◽

2011 ◽

Vol 437 (2) ◽

pp. 345-355 ◽

Cited By ~ 3

Author(s):

Satoshi Yasuda ◽

Tetsuya Hasegawa ◽

Tetsuji Hosono ◽

Mitsutoshi Satoh ◽

Kei Watanabe ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Embryoid Body ◽

Es Cells ◽

Embryonic Stem ◽

Cardiac Differentiation ◽

Marker Genes ◽

Embryonic Cells ◽

Transcript Levels

An understanding of the mechanism that regulates the cardiac differentiation of pluripotent stem cells is necessary for the effective generation and expansion of cardiomyocytes as cell therapy products. In the present study, we have identified genes that modulate the cardiac differentiation of pluripotent embryonic cells. We isolated P19CL6 cell sublines that possess distinct properties in cardiomyogenesis and extracted 24 CMR (cardiomyogenesis-related candidate) genes correlated with cardiomyogenesis using a transcriptome analysis. Knockdown of the CMR genes by RNAi (RNA interference) revealed that 18 genes influence spontaneous contraction or transcript levels of cardiac marker genes in EC (embryonal carcinoma) cells. We also performed knockdown of the CMR genes in mouse ES (embryonic stem) cells and induced in vitro cardiac differentiation. Three CMR genes, AW551984, 2810405K02Rik (RIKEN cDNA 2810405K02 gene) and Cd302 (CD302 antigen), modulated the cardiac differentiation of both EC cells and ES cells. Depletion of AW551984 attenuated the expression of the early cardiac transcription factor Nkx2.5 (NK2 transcription factor related locus 5) without affecting transcript levels of pluripotency and early mesoderm marker genes during ES cell differentiation. Activation of Wnt/β-catenin signalling enhanced the expression of both AW551984 and Nkx2.5 in ES cells during embryoid body formation. Our findings indicate that AW551984 is a novel regulator of cardiomyogenesis from pluripotent embryonic cells, which links Wnt/β-catenin signalling to Nkx2.5 expression.

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Human Pluripotent Stem Cell Culture: Current Status, Challenges, and Advancement

Stem Cells International ◽

10.1155/2018/7396905 ◽

2018 ◽

Vol 2018 ◽

pp. 1-17 ◽

Cited By ~ 30

Author(s):

Sushrut Dakhore ◽

Bhavana Nayer ◽

Kouichi Hasegawa

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Pluripotent Stem Cells ◽

Large Scale ◽

Embryonic Stem ◽

Current Status ◽

Specific Cell ◽

Therapeutic Applications ◽

Proliferative Ability

Over the past two decades, human embryonic stem cells (hESCs) have gained attention due to their pluripotent and proliferative ability which enables production of almost all cell types in the human body in vitro and makes them an excellent tool to study human embryogenesis and disease, as well as for drug discovery and cell transplantation therapies. Discovery of human-induced pluripotent stem cells (hiPSCs) further expanded therapeutic applications of human pluripotent stem cells (PSCs). hPSCs provide a stable and unlimited original cell source for producing suitable cells and tissues for downstream applications. Therefore, engineering the environment in which these cells are grown, for stable and quality-controlled hPSC maintenance and production, is one of the key factors governing the success of these applications. hPSCs are maintained in a particular niche using specific cell culture components. Ideally, the culture should be free of xenobiotic components to render hPSCs suitable for therapeutic applications. Substantial efforts have been put to identify effective components, and develop culture conditions and protocols, for their large-scale expansion without compromising on quality. In this review, we discuss different media, their components and functions, including specific requirements to maintain the pluripotent and proliferative ability of hPSCs. Understanding the role of culture components would enable the development of appropriate conditions to promote large-scale, quality-controlled expansion of hPSCs thereby increasing their potential applications.

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The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells

Biomaterials ◽

10.1016/j.biomaterials.2009.11.033 ◽

2010 ◽

Vol 31 (7) ◽

pp. 1885-1893 ◽

Cited By ~ 139

Author(s):

Jeffrey C. Mohr ◽

Jianhua Zhang ◽

Samira M. Azarin ◽

Andrew G. Soerens ◽

Juan J. de Pablo ◽

...

Keyword(s):

Stem Cells ◽

Body Size ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryoid Body ◽

Embryonic Stem ◽

Cardiac Differentiation

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Expression of 4-Integrin Defines the Earliest Precursor of Hematopoietic Cell Lineage Diverged From Endothelial Cells

Blood ◽

10.1182/blood.v93.4.1168 ◽

1999 ◽

Vol 93 (4) ◽

pp. 1168-1177 ◽

Cited By ~ 65

Author(s):

Minetaro Ogawa ◽

Masami Kizumoto ◽

Satomi Nishikawa ◽

Tetsuhiro Fujimoto ◽

Hiroaki Kodama ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Cell Population ◽

Embryonic Stem ◽

Cell Lineage ◽

Hematopoietic Cell ◽

Hematopoietic Stem ◽

Hematopoietic Cell Lineage ◽

E Cadherin

Abstract Embryonic stem cells can differentiate in vitro into hematopoietic cells through two intermediate stages; the first being FLK1+ E-cadherin− proximal lateral mesoderm and the second being CD45− VE-cadherin+endothelial cells. To further dissect the CD45−VE-cadherin+ cells, we have examined distribution of 4-integrin on this cell population, because 4-integrin is the molecule expressed on hematopoietic stem cells. During culture of FLK1+ E-cadherin− cells, CD45− VE-cadherin+4-integrin− cells differentiate first, followed by 4-integrin+ cells appearing in both CD45− VE-cadherin+ and CD45−VE-cadherin− cell populations. In the CD45−VE-cadherin+ cell population, 4-integrin+ subset but not 4-integrin− subset had the potential to differentiate to hematopoietic lineage cells, whereas endothelial cell progenitors were present in both subsets. The CD45−VE-cadherin− 4-integrin+ cells also showed hematopoietic potential. Reverse transcription-polymerase chain reaction analyses showed that differential expression of the Gata2 and Myb genes correlated with the potential of the 4-integrin+ cells to give rise to hematopoietic cell differentiation. Hematopoietic CD45−VE-cadherin+ 4-integrin+ cells were also present in the yolk sac and embryonic body proper of 9.5 day postcoitum mouse embryos. Our results suggest that the expression of 4-integrin is a marker of the earliest precursor of hematopoietic cell lineage that was diverged from endothelial progenitors.

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A Novel Population of Oct-4+ SSEA-1+ CXCR4+ CD34+ CD133+ Lin− CD45− Very Small Embryonic-Like (VSEL) Stem Cells Identified in Human Cord Blood.

Blood ◽

10.1182/blood.v108.11.3195.3195 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3195-3195

Author(s):

Magda Kucia ◽

Maciej Halasa ◽

Marcin Wysoczynski ◽

Magda Baskiewicz-Masiuk ◽

Ewa Zuba-Surma ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cord Blood ◽

Mononuclear Cells ◽

Embryonic Stem ◽

Specific Cell ◽

Human Cord Blood ◽

Organ Tissue ◽

Hypotonic Lysis

Abstract Mononuclear cells (MNC) isolated from bone marrow (BM) or cord blood (CB) contributes to organ/tissue regeneration, however, the identity of the specific cell type(s) involved remains unknown. Recently we identified in murine BM a homogenous population of rare (~0.01% of BM MNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1, highly express Rif-1 telomerase protein and display several features typical for primary embryonic stem cells such as a small size (~2–4 um in diameter), a large nuclei surrounded by a narrow rim of cytoplasm, and open-type chromatin (euchromatin) that is typical for embryonic stem cells (Leukemia2006;20,857–869). These cells were named very small embryonic-like (VSELs) stem cells. We will present a new two step isolation procedure to purify a similar population of cells from human CB, which is based on isolation of CB mononuclear cells (CB MNC) by hypotonic lysis and multiparameter FACS sorting. Accordingly, we perform hypotonic lysis of CB to remove erythrocytes and to enrich for CB MNC combined with multiparameter sorting for CXCR4+AC133+CD34+lin−CD45− CB MNC. CB-derived VSELs (CB-VSELs) isolated this way similarly as those isolated from adult murine BM are very small (3–5 um), possess large nuclei containing unorganized euchromatin, express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4. In vitro cultures CB-VSELs are able to grow neurospheres that gave rise to neuronal lineages (beta-III tubulin+, nestin+, O4+, MBP+, GFAP+) and cardiomyocytes (beta-myosin heavy chain+, alpha-sarcomeric actin. Based on this we conclude that CB contains VSELs and that the majority of these CB VSELs are lost during routine procedures employed currently for banking of CB MNC. Thus based on our observations, new more efficient methods of CB banking are needed that will enrich/preserve these cells in CB units during preparation before storage. Furthermore, we conclude that CB tissue/organ regenerating potential may be much higher than initially postulated if the proper fraction of CB MNC is employed and we are currently testing this hypothesis in animal models.

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Expression of 4-Integrin Defines the Earliest Precursor of Hematopoietic Cell Lineage Diverged From Endothelial Cells

Blood ◽

10.1182/blood.v93.4.1168.404k12_1168_1177 ◽

1999 ◽

Vol 93 (4) ◽

pp. 1168-1177 ◽

Cited By ~ 4

Author(s):

Minetaro Ogawa ◽

Masami Kizumoto ◽

Satomi Nishikawa ◽

Tetsuhiro Fujimoto ◽

Hiroaki Kodama ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Cell Population ◽

Embryonic Stem ◽

Cell Lineage ◽

Hematopoietic Cell ◽

Hematopoietic Stem ◽

Hematopoietic Cell Lineage ◽

E Cadherin

Embryonic stem cells can differentiate in vitro into hematopoietic cells through two intermediate stages; the first being FLK1+ E-cadherin− proximal lateral mesoderm and the second being CD45− VE-cadherin+endothelial cells. To further dissect the CD45−VE-cadherin+ cells, we have examined distribution of 4-integrin on this cell population, because 4-integrin is the molecule expressed on hematopoietic stem cells. During culture of FLK1+ E-cadherin− cells, CD45− VE-cadherin+4-integrin− cells differentiate first, followed by 4-integrin+ cells appearing in both CD45− VE-cadherin+ and CD45−VE-cadherin− cell populations. In the CD45−VE-cadherin+ cell population, 4-integrin+ subset but not 4-integrin− subset had the potential to differentiate to hematopoietic lineage cells, whereas endothelial cell progenitors were present in both subsets. The CD45−VE-cadherin− 4-integrin+ cells also showed hematopoietic potential. Reverse transcription-polymerase chain reaction analyses showed that differential expression of the Gata2 and Myb genes correlated with the potential of the 4-integrin+ cells to give rise to hematopoietic cell differentiation. Hematopoietic CD45−VE-cadherin+ 4-integrin+ cells were also present in the yolk sac and embryonic body proper of 9.5 day postcoitum mouse embryos. Our results suggest that the expression of 4-integrin is a marker of the earliest precursor of hematopoietic cell lineage that was diverged from endothelial progenitors.

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Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo

10.1101/158931 ◽

2017 ◽

Cited By ~ 3

Author(s):

Luca Tosti ◽

James Ashmore ◽

Boon Siang Nicholas Tan ◽

Benedetta Carbone ◽

Tapan K Mistri ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Binding Sites ◽

Mammalian Cells ◽

Regulatory Networks ◽

Embryonic Stem ◽

Specific Cell ◽

Dna Interactions

AbstractThe identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein-DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps or chemical protein-DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical impediments. Here we describe an optimised DamID method coupled with next generation sequencing (DamID-seq) in mouse cells, and demonstrate the identification of the binding sites of two TFs, OCT4 and SOX2, in as few as 1,000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. Oct4 DamID-seq in the gastrulating mouse embryo at 7.5 days post coitum (dpc) successfully identified multiple Oct4 binding sites proximal to genes involved in embryo development, neural tube formation, mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigations of TF-DNA interactions and GRNs in specific cell types with limited availability in mammals including in vivo samples.

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Human Fetal Liver Derived Stem Cells Can Be Support the Maintenance of Human Embryonic Stem Cells.

Blood ◽

10.1182/blood.v104.11.4264.4264 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4264-4264

Author(s):

Jin-Young Baek ◽

Yun-Hee Rhee ◽

Kwang-Yul Cha ◽

Hyung-Min Chung

Keyword(s):

Stem Cells ◽

Mononuclear Cells ◽

Embryoid Body ◽

Fetal Liver ◽

Es Cells ◽

Embryonic Stem ◽

Human Fetal Liver ◽

Human Es Cells ◽

Cells Cultured

Abstract Prolonged propagation of human embryonic stem (ES) cells is currently achieved by co-culture with primary or immortalized mouse embryonic fibroblast (MEF) cells. In order to replace the heterologous with homologous co-culture systems, an attempt was made using mononuclear cells derived from human fetal liver. Human fetal liver-derived mesenchymal-like stem cells (FL-MLSC) can be maintained for the prolonged period of time. They showed the characteristics of mesenchymal stem cells in various aspects. They retained a normal diploid karyotype and growth characteristics over the successive culture. Human ES cells cultured on human FL-MLSC cells up to 8 passages displayed the unique morphology and molecular markers characteristic for undifferentiated human ES cells as cultured on MEF cells. Alkaline phosphatase activity was detected in human ES cells co-cultured on human FL-MLSC. Immunocytochemical analyses showed that expressions of stage-specific embryonic antigen-3, -4 and Oct-4 were not altered on human ES cells cultured on human FLDSC. Reverse-transcriptase PCR analyses showed that similar expressions of Oct-4 and Nanog genes, markers for undifferentiated ES cells, were also observed in human ES cells cultured on both human FL-MLSC and MEF cells. Furthermore, human ES cells cultured on human FL-MLSC retained unique differentiation potentials in culture when allowed to form embryoid body. Results of this study suggest that human FL-MLSC can support the maintenance of human ES cell in vitro.

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