293 MASS PRODUCTION OF Nkx2.5-POSITIVE CARDIAC PROGENITOR CELLS DERIVED FROM MOUSE EMBRYONIC STEM CELLS IN SLOW-TURNING LATERAL VESSEL FOR CELL TRANSPLANTATION AND DRUG TESTING

2011 ◽  
Vol 23 (1) ◽  
pp. 244
Author(s):  
S. Rungarunlert ◽  
N. Klincumhom ◽  
C. Nemes ◽  
M. Techakumphu ◽  
M. K. Pirity ◽  
...  
Keyword(s):  
Suspension Culture ◽  
Progenitor Cells ◽  
Mass Production ◽  
Large Scale ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cardiac Progenitor Cells ◽  
Scale Production ◽  
Culture Methods ◽  
Mouse Es Cells

Regenerative cell therapy against cardiovascular disease would require mass production and purification of specific cell types before transplantation. To enable large-scale production of embryonic stem (ES)-derived pure cardiomyocytes, we developed an animal model for a single-step scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled slow-turning lateral vessel (STLV, Synthecon, Inc., Houston, TX, USA) bioreactor following inoculation with a single cell suspension of mouse ES cells. To enhance the yield of cardiac progenitor cells, mouse ES cells (HM1; 129Sv/Ola, Magin et al. 1992 Nucl. Acids Res. 20, 3795–3796) were targeted with the cardiac-specific mouse Nkx2.5 promoter driven enhanced fluorescent green protein (EGFP). Among 15 targeted colonies, which were characterised based on morphology, the ability to form EB, EGFP expression, and in vitro differentiation ability toward cardiomyocytes, 3 lines were further evaluated for the efficiency of cardiomyocyte production. The 3 lines were cultured in STLV bioreactor and compared with classical hanging drop (HD) and static suspension culture methods. Embryonic bodies at day 3 to 8 were collected and analysed by using fluorescence-activated cell sorting for markers of pluripotency (e.g. Oct-4, SSEA1, Nanog) and cardiac (e.g. Nkx2.5, Troponin T) lineage commitments. Data was analysed by one-way ANOVA and t-tests. The results showed that both level and kinetics of Nkx2.5 expression was dependent on culture conditions. The STLV and static suspension culture methods produced higher rates of Nkx2.5-positive cells on day 5 than that of HD (70 and 54 v. 30%, respectively). The STLV method produced a highly uniform population of efficiently differentiating EB in large quantities and resulted in the highest, 108 yield of cardiomyocytes in a single 110-mL STLV on day 4. In conclusion, the STLV method provides a technological platform for controlled large-scale generation of ES-cell-derived cardiomyocytes for clinical and industrial applications. In vivo transplantation tests of cardiomyocytes produced via STLV are currently underway. This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205 and NKTH/KPI (NKFP_07_1-ES2HEART-HU OM-00202-2007), CHE-TRF senior scholarship, No. RTA 5080010 (M.T.), and the Thailand Commission on Higher Education [CHE-PhD-SW-2005-100 (S.R.), CHE-PhD-SW-RG-2007 (N.K.)].

scholarly journals Human Fetal Liver: AnIn VitroModel of Erythropoiesis

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Guillaume Pourcher ◽  
Christelle Mazurier ◽  
Yé Yong King ◽  
Marie-Catherine Giarratana ◽  
Ladan Kobari ◽  
...  
Keyword(s):  
Stem Cells ◽  
Large Scale ◽  
Adult Stem Cells ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Scale Production ◽  
Cloning Efficiency ◽  
Hematopoietic Stem

We previously described the large-scale production of RBCs from hematopoietic stem cells (HSCs) of diverse sources. Our present efforts are focused to produce RBCs thanks to an unlimited source of stem cells. Human embryonic stem (ES) cells or induced pluripotent stem cell (iPS) are the natural candidates. Even if the proof of RBCs production from these sources has been done, their amplification ability is to date not sufficient for a transfusion application. In this work, our protocol of RBC production was applied to HSC isolated from fetal liver (FL) as an intermediate source between embryonic and adult stem cells. We studied the erythroid potential of FL-derived CD34+cells. In thisin vitromodel, maturation that is enucleation reaches a lower level compared to adult sources as observed for embryonic or iP, but, interestingly, they (i) displayed a dramaticin vitroexpansion (100-fold more when compared to CB CD34+) and (ii) 100% cloning efficiency in hematopoietic progenitor assays after 3 days of erythroid induction, as compared to 10–15% cloning efficiency for adult CD34+cells. This work supports the idea that FL remains a model of study and is not a candidate forex vivoRBCS production for blood transfusion as a direct source of stem cells but could be helpful to understand and enhance proliferation abilities for primitive cells such as ES cells or iPS.

scholarly journals Ten-Eleven Translocation 1 (Tet1) Is Regulated by O-Linked N-Acetylglucosamine Transferase (Ogt) for Target Gene Repression in Mouse Embryonic Stem Cells

2013 ◽  
Vol 288 (29) ◽  
pp. 20776-20784 ◽  
Author(s):  
Feng-Tao Shi ◽  
Hyeung Kim ◽  
Weisi Lu ◽  
Quanyuan He ◽  
Dan Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Target Genes ◽  
Es Cells ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Mouse Es Cells ◽  
Chromatin Regulators ◽  
Glcnac Transferase

As a member of the Tet (Ten-eleven translocation) family proteins that can convert 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC), Tet1 has been implicated in regulating global DNA demethylation and gene expression. Tet1 is highly expressed in embryonic stem (ES) cells and appears primarily to repress developmental genes for maintaining pluripotency. To understand how Tet1 may regulate gene expression, we conducted large scale immunoprecipitation followed by mass spectrometry of endogenous Tet1 in mouse ES cells. We found that Tet1 could interact with multiple chromatin regulators, including Sin3A and NuRD complexes. In addition, we showed that Tet1 could also interact with the O-GlcNAc transferase (Ogt) and be O-GlcNAcylated. Depletion of Ogt led to reduced Tet1 and 5hmC levels on Tet1-target genes, whereas ectopic expression of wild-type but not enzymatically inactive Ogt increased Tet1 levels. Mutation of the putative O-GlcNAcylation site on Tet1 led to decreased O-GlcNAcylation and level of the Tet1 protein. Our results suggest that O-GlcNAcylation can positively regulate Tet1 protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt.

Roles of vascular endothelial growth factor receptor 3 signaling in differentiation of mouse embryonic stem cell–derived vascular progenitor cells into endothelial cells

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2372-2379 ◽  
Author(s):  
Hiroyuki Suzuki ◽  
Tetsuro Watabe ◽  
Mitsuyasu Kato ◽  
Keiji Miyazawa ◽  
Kohei Miyazono
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Endothelial Differentiation ◽  
Mouse Es Cells ◽  
Vascular Progenitor Cells ◽  
Vegfr2 Signaling ◽  
Endothelial Growth Factor

AbstractVascular endothelial growth factor receptor 2 (VEGFR2/Flk-1)–positive cells derived from embryonic stem (ES) cells serve as vascular progenitors, which differentiate into endothelial cells (ECs) in the presence of VEGF-A. VEGFR3/Flt-4 (fms-like tyrosine kinase 4) signaling is known to be important for the development of lymphatic endothelial cells (LECs). To elucidate the roles of VEGFR3 signaling in the differentiation of vascular progenitor cells into ECs, we introduced various types of VEGFR3 cDNAs into mouse ES cells. VEGF-C, a ligand for VEGFR2 and VEGFR3, stimulated the endothelial differentiation of the VEGFR2+ cells transfected with the VEGFR3 cDNA but not those transfected with kinasenegative mutants of VEGFR3. The VEGFR3-transfected ECs exhibited high expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), one of the markers of LECs, and showed efficient binding of hyaluronan. VEGF-C(C152S), which is able to activate VEGFR3 but not VEGFR2, failed to induce the endothelial differentiation of mock- and VEGFR3-transfected VEGFR2+ cells, suggesting the essential role of VEGFR2 signaling for endothelial differentiation. Furthermore, kinase-negative mutants of VEGFR3 prevented the VEGF-C–mediated endothelial differentiation of the vascular progenitor cells. Thus, VEGFR2 signaling is required for the endothelial differentiation of mouse ES cells induced by VEGF-C, and VEGFR3 signaling may confer lymphatic endothelial-like phenotypes to ECs.

scholarly journals Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

2019 ◽  
Author(s):  
Aseda Tena ◽  
Yuxiang Zhang ◽  
Nia Kyritsis ◽  
Anne Devorak ◽  
Jeffrey Zurita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Progenitors ◽  
Neural Genes ◽  
Genome Wide ◽  
Primary Mouse

ABSTRACTMild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

Human embryonic stem cells

2000 ◽  
Vol 113 (1) ◽  
pp. 5-10 ◽  
Author(s):  
M.F. Pera ◽  
B. Reubinoff ◽  
A. Trounson
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Embryo ◽  
Carcinoma Cells ◽  
Mouse Es Cells ◽  
Undifferentiated State ◽  
Human Es Cells

Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Candidate ES and EG cell lines from the human blastocyst and embryonic gonad can differentiate into multiple types of somatic cell. The phenotype of the blastocyst-derived cell lines is very similar to that of monkey ES cells and pluripotent human embryonal carcinoma cells, but differs from that of mouse ES cells or the human germ-cell-derived stem cells. Although our understanding of the control of growth and differentiation of human ES cells is quite limited, it is clear that the development of these cell lines will have a widespread impact on biomedical research.

2. Embryonic stem cells

2021 ◽  
pp. 21-37
Author(s):  
Jonathan Slack
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Practical Importance ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Human Embryos ◽  
Mouse Es Cells ◽  
Stage Embryo

‘Embryonic stem cells’ focuses on embryonic stem (ES) cells, which are grown in tissue culture from the inner cell mass of a mammalian blastocyst-stage embryo. Human ES cells offer a potential route to making the kinds of cells needed for cell therapy. ES cells were originally prepared from mouse embryos. Although somewhat different, cells grown from inner cell masses of human embryos share many properties with mouse ES cells, such as being able to grow without limit and to generate differentiated cell types. Mouse ES cells have so far been of greater practical importance than those of humans because they have enabled a substantial research industry based on the creation of genetically modified mice.

scholarly journals DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands

2020 ◽  
Vol 6 (35) ◽  
pp. eabb5820 ◽  
Author(s):  
Zhiming Li ◽  
Xu Hua ◽  
Albert Serra-Cardona ◽  
Xiaowei Xu ◽  
Songlin Gan ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Es Cells ◽  
Embryonic Stem ◽  
Endogenous Retroviruses ◽  
Histone Binding ◽  
Dna Polymerase Α ◽  
Mouse Es Cells ◽  
Dna Strands ◽  
Histone Deposition

How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication–linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.

scholarly journals Expansion of Human Pluripotent Stem Cell-derived Early Cardiovascular Progenitor Cells by a Cocktail of Signaling Factors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sadaf Vahdat ◽  
Sara Pahlavan ◽  
Elena Mahmoudi ◽  
Maryam Barekat ◽  
Hassan Ansari ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Large Scale ◽  
Culture Medium ◽  
Gene Expression Pattern ◽  
Extended Period ◽  
Scale Production ◽  
Chemical Screening ◽  
Wide Range ◽  
Cell Sources

Abstract Cardiovascular progenitor cells (CPCs) derived from human pluripotent stem cells (hPSCs) are proposed to be invaluable cell sources for experimental and clinical studies. This wide range of applications necessitates large-scale production of CPCs in an in vitro culture system, which enables both expansion and maintenance of these cells. In this study, we aimed to develop a defined and efficient culture medium that uses signaling factors for large-scale expansion of early CPCs, called cardiogenic mesodermal cells (CMCs), which were derived from hPSCs. Chemical screening resulted in a medium that contained a reproducible combination of three factors (A83-01, bFGF, and CHIR99021) that generated 1014 CMCs after 10 passages without the propensity for tumorigenicity. Expanded CMCs retained their gene expression pattern, chromosomal stability, and differentiation tendency through several passages and showed both the safety and possible cardio-protective potentials when transplanted into the infarcted rat myocardium. These CMCs were efficiently cryopreserved for an extended period of time. This culture medium could be used for both adherent and suspension culture conditions, for which the latter is required for large-scale CMC production. Taken together, hPSC-derived CMCs exhibited self-renewal capacity in our simple, reproducible, and defined medium. These cells might ultimately be potential, promising cell sources for cardiovascular studies.

scholarly journals Recql5 and Blm RecQ DNA Helicases Have Nonredundant Roles in Suppressing Crossovers

2005 ◽  
Vol 25 (9) ◽  
pp. 3431-3442 ◽  
Author(s):  
Yiduo Hu ◽  
Xincheng Lu ◽  
Ellen Barnes ◽  
Min Yan ◽  
Hua Lou ◽  
...  
Keyword(s):  
Cancer Susceptibility ◽  
Es Cells ◽  
Embryonic Stem ◽  
Bloom Syndrome ◽  
Embryonic Fibroblasts ◽  
Dna Helicases ◽  
Mouse Es Cells ◽  
Elevated Rate ◽  
Wild Type Control ◽  
Mitotic Cells

ABSTRACT In eukaryotes, crossovers in mitotic cells can have deleterious consequences and therefore must be suppressed. Mutations in BLM give rise to Bloom syndrome, a disease that is characterized by an elevated rate of crossovers and increased cancer susceptibility. However, simple eukaryotes such as Saccharomyces cerevisiae have multiple pathways for suppressing crossovers, suggesting that mammals also have multiple pathways for controlling crossovers in their mitotic cells. We show here that in mouse embryonic stem (ES) cells, mutations in either the Bloom syndrome homologue (Blm) or the Recql5 genes result in a significant increase in the frequency of sister chromatid exchange (SCE), whereas deleting both Blm and Recql5 lead to an even higher frequency of SCE. These data indicate that Blm and Recql5 have nonredundant roles in suppressing crossovers in mouse ES cells. Furthermore, we show that mouse embryonic fibroblasts derived from Recql5 knockout mice also exhibit a significantly increased frequency of SCE compared with the corresponding wild-type control. Thus, this study identifies a previously unknown Recql5-dependent, Blm-independent pathway for suppressing crossovers during mitosis in mice.

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