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

2013 ◽  
Vol 305 (3) ◽  
pp. E325-E335 ◽  
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.

scholarly journals Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway

2020 ◽  
Vol 21 (23) ◽  
pp. 8945
Author(s):  
Mercedes Fernández ◽  
Micaela Pannella ◽  
Vito Antonio Baldassarro ◽  
Alessandra Flagelli ◽  
Giuseppe Alastra ◽  
...  
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Embryonic Stem Cells ◽  
Thyroid Hormone ◽  
Nuclear Receptors ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Postnatal Life ◽  
Gene And Protein Expression

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TRα and TRβ), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.

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 ◽  
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.

Induction of Cardiomyocyte Differentiation From Mouse Embryonic Stem Cells in a Confined Microfluidic Environment

2009 ◽  
Author(s):  
Chen-rei Wan ◽  
Seok Chung ◽  
Ryo Sudo ◽  
Roger D. Kamm
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mechanical Stretch ◽  
Embryoid Bodies ◽  
Negative Regulator ◽  
Differentiation Process ◽  
Cardiomyocyte Differentiation

Embryonic stem cell derived cardiomyocytes are deemed an attractive treatment option for myocardial infarction. Their clinical efficacy, however, has not been unequivocally demonstrated. There is a need for better understanding and characterization of the cardiogenesis process. A microfluidic platform in vitro is used to dissect and better understand the differentiation process. Through this study, we find that while embryoid bodies (EBs) flatten out in a well plate system, differentiated EBs self-assemble into complex 3D structures. The beating regions of EBs are also different. Most beating areas are observed in a ring pattern on 2D well plates around the center, self-assembled beating large 3D aggregates are found in microfluidic devices. Furthermore, inspired by the natural mechanical environment of the heart, we applied uniaxial cyclic mechanical stretch to EBs. Results suggest that prolonged mechanical stimulation acts as a negative regulator of cardiogenesis. From this study, we conclude that the culture environments can influence differentiation of embryonic stem cells into cardiomycytes, and that the use of microfluidic systems can provide new insights into the differentiation process.

Hematopoietic Differentiation of Human Embryonic Stem Cells in Vitro : Comparison of Three Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4787-4787
Author(s):  
Marion Brenot ◽  
Annelise Bennaceur-Griscelli ◽  
Marc Peschanski ◽  
Maria Teresa Mitjavila-Garcia
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Human Embryonic Stem Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem

Abstract Human embryonic stem cells (hES) isolated from the inner cell mass of a blastocyst have the ability to self renew indefinitely while maintaining their pluripotency to differentiate into multiple cell lineages. Therefore, hES represent an important source of cells for perspective cell therapies and serve as an essential tool for fundamental research, specifically for understanding pathophysiological mechanisms of human diseases for the development of novel pharmacological drugs. The generation of hematopoietic stem cells from hES may serve as an alternative source of cells for hematopoietic reconstitution following bone marrow transplantation and an interesting approach to understand early stages of hematopoietic development which are difficult to study in human embryos. Using two different methods, we have differentiated three hES cell lines (SA01, H1 and H9) into hematopoietic cells by generating embryoid bodies and co-culturing on the murine Op9 cell line. In both experimental approaches, we obtain cells expressing CD34 and when cultured in hematopoietic conditions, SA01 and H1 cell lines differentiate into various hematopoietic lineages as demonstrated by BFU-E, CFU-GM and CFU-GEMM colony formation, whereas H9 have almost exclusively granulo-macrophage differentiation. Cells composing these hematopoietic colonies express CD45, CD11b, CD31, CD41 and CD235 and staining with May Grundwald-Giemsa demonstrate neutrophil and erythrocyte morphology. These results demonstrate the capacity of hES to differentiate into mature hematopoietic cells in vitro. Nevertheless, there exist some quantitative and qualitative differences about hematopoietic differentiation between the hES cell lines used. However, we still have to evaluate their capacity to reconstitute hematopoiesis in vivo in an immune deficient mouse model. We will also be interested in developing in vitro methods to expand these hematopoietic precursor cells derived from hES which may be used as a viable source for future cell therapy.

scholarly journals Cultivation of Human Embryonic Stem Cells Without the Embryoid Body Step Enhances Osteogenesis In Vitro

Stem Cells ◽  
2006 ◽  
Vol 24 (4) ◽  
pp. 835-843 ◽  
Author(s):  
Jeffrey M. Karp ◽  
Lino S. Ferreira ◽  
Ali Khademhosseini ◽  
Albert H. Kwon ◽  
Judy Yeh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Osteogenesis In Vitro

scholarly journals Differentiation of embryoid-body cells derived from embryonic stem cells into hepatocytes in alginate microbeads in vitro

2007 ◽  
Vol 28 (12) ◽  
pp. 1924-1930 ◽  
Author(s):  
Sheng FANG ◽  
Yu-dong QIU ◽  
Liang MAO ◽  
Xiao-lei SHI ◽  
De-cai YU ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryoid Body ◽  
Embryonic Stem

scholarly journals Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells

2008 ◽  
Vol 205 (8) ◽  
pp. 1917-1927 ◽  
Author(s):  
Hidekazu Nishikii ◽  
Koji Eto ◽  
Noriko Tamura ◽  
Koichi Hattori ◽  
Beate Heissig ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Blood Platelets ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Normal Platelet ◽  
Metalloproteinase Inhibitors ◽  
Flow Cytometric Sorting

Embryonic stem cells (ESCs) could potentially compensate for the lack of blood platelets available for use in transfusions. Here, we describe a new method for generating mouse ESC-derived platelets (ESPs) that can contribute to hemostasis in vivo. Flow cytometric sorting of cells from embryoid bodies on day 6 demonstrated that c-Kit+ integrin αIIb (αIIb)+ cells, but not CD31+ cells or vascular endothelial cadherin+ cells, are capable of megakaryopoiesis and the release of platelet-like structures by day 12. αIIbβ3-expressing ESPs exhibited ectodomain shedding of glycoprotein (GP)Ibα, GPV, and GPVI, but not αIIbβ3 or GPIbβ. ESPs showed impaired αIIbβ3 activation and integrin-mediated actin reorganization, critical events for normal platelet function. However, the administration of metalloproteinase inhibitors GM6001 or TAPI-1 during differentiation increased the expression of GPIbα, improving both thrombogenesis in vitro and posttransfusion recovery in vivo. Thus, the regulation of metalloproteinases in culture could be useful for obtaining high-quality, efficacious ESPs as an alternative platelet source for transfusions.

scholarly journals Synthesis and Organization of Hyaluronan and Versican by Embryonic Stem Cells Undergoing Embryoid Body Differentiation

2009 ◽  
Vol 58 (4) ◽  
pp. 345-358 ◽  
Author(s):  
Shreya Shukla ◽  
Rekha Nair ◽  
Marsha W. Rolle ◽  
Kathleen R. Braun ◽  
Christina K. Chan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Epithelial Mesenchymal Transition ◽  
Embryoid Body ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Proteolytic Fragment ◽  
Mesenchymal Transition ◽  
Hyaluronan Synthases

Embryonic stem cells (ESCs) provide a convenient model to probe the molecular and cellular dynamics of developmental cell morphogenesis. ESC differentiation in vitro via embryoid bodies (EBs) recapitulates many aspects of early stages of development, including the epithelial–mesenchymal transition (EMT) of pluripotent cells into more differentiated progeny. Hyaluronan and versican are important extracellular mediators of EMT processes, yet the temporal expression and spatial distribution of these extracellular matrix (ECM) molecules during EB differentiation remains undefined. Thus, the objective of this study was to evaluate the synthesis and organization of hyaluronan and versican by using murine ESCs during EB differentiation. Hyaluronan and versican (V0 and V1 isoforms), visualized by immunohistochemistry and evaluated biochemically, accumulated within EBs during the course of differentiation. Interestingly, increasing amounts of a 70-kDa proteolytic fragment of versican were also detected over time, along with ADAMTS-1 and −5 protein expression. ESCs expressed each of the hyaluronan synthases (HAS) −1, −2, and −3 and versican splice variants (V0, V1, V2, and V3) throughout EB differentiation, but HAS-2, V0, and V1 were expressed at significantly increased levels at each time point examined. Hyaluronan and versican exhibited overlapping expression patterns within EBs in regions of low cell density, and versican expression was excluded from clusters of epithelial (cytokeratin-positive) cells but was enriched within the vicinity of mesenchymal (N-cadherin-positive) cells. These results indicate that hyaluronan and versican synthesized by ESCs within EB microenviron-ments are associated with EMT processes and furthermore suggest that endogenously produced ECM molecules play a role in ESC differentiation. This manuscript contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.

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