scholarly journals The Lysine Methylase SMYD3 Modulates Mesendodermal Commitment during Development

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1233
Author(s):  
Raffaella Fittipaldi ◽  
Pamela Floris ◽  
Valentina Proserpio ◽  
Franco Cotelli ◽  
Monica Beltrame ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Model Systems ◽  
Embryonic Stem Cell Differentiation ◽  
In Vitro Differentiation ◽  
Cancer Models ◽  
Lineage Markers

SMYD3 (SET and MYND domain containing protein 3) is a methylase over-expressed in cancer cells and involved in oncogenesis. While several studies uncovered key functions for SMYD3 in cancer models, the SMYD3 role in physiological conditions has not been fully elucidated yet. Here, we dissect the role of SMYD3 at early stages of development, employing mouse embryonic stem cells (ESCs) and zebrafish as model systems. We report that SMYD3 depletion promotes the induction of the mesodermal pattern during in vitro differentiation of ESCs and is linked to an upregulation of cardiovascular lineage markers at later stages. In vivo, smyd3 knockdown in zebrafish favors the upregulation of mesendodermal markers during zebrafish gastrulation. Overall, our study reveals that SMYD3 modulates levels of mesendodermal markers, both in development and in embryonic stem cell differentiation.

scholarly journals Extracellular Matrix and Integrins in Embryonic Stem Cell Differentiation

2015 ◽  
Vol 8s2 ◽  
pp. BCI.S30377 ◽  
Author(s):  
Han Wang ◽  
Xie Luo ◽  
Jake Leighton
Keyword(s):  
Extracellular Matrix ◽  
Pancreatic Beta Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
In Vitro Differentiation ◽  
Integrin Receptors

Embryonic stem cells (ESCs) are pluripotent cells with great therapeutic potentials. The in vitro differentiation of ESC was designed by recapitulating embryogenesis. Significant progress has been made to improve the in vitro differentiation protocols by toning soluble maintenance factors. However, more robust methods for lineage-specific differentiation and maturation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) cues should be considered besides growth factor cues. ECM proteins bind to cells and act as ligands of integrin receptors on cell surfaces. Here, we summarize the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM–integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuroectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. In the future, ECM combinations for the optimal ESC differentiation environment will require substantial study.

Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Anichavezhi Devendran ◽  
Rasheed Bailey ◽  
Sumanta Kar ◽  
Francesca Stillitano ◽  
Irene Turnbull ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Mononuclear Cells ◽  
Embryonic Stem ◽  
Model Systems ◽  
Patient Specific ◽  
Induced Pluripotent

Background: Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca ++ ) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca ++ concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca ++ signalling. Methodology and Results: Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation in-vivo, we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific). Conclusion: Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the in-vitro observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for in-vivo gene editing and testing of biochemical signalling pathways.

Bio-mimicking Shear Stress Environments for Enhancing Mesenchymal Stem Cell Differentiation

2020 ◽  
Vol 15 (5) ◽  
pp. 414-427
Author(s):  
Seep Arora ◽  
Akshaya Srinivasan ◽  
Chak Ming Leung ◽  
Yi-Chin Toh
Keyword(s):  
Stem Cells ◽  
Shear Stress ◽  
Stem Cell ◽  
Stem Cell Differentiation ◽  
In Vitro Differentiation ◽  
Msc Differentiation ◽  
Biophysical Cues

Mesenchymal stem cells (MSCs) are multipotent stromal cells, with the ability to differentiate into mesodermal (e.g., adipocyte, chondrocyte, hematopoietic, myocyte, osteoblast), ectodermal (e.g., epithelial, neural) and endodermal (e.g., hepatocyte, islet cell) lineages based on the type of induction cues provided. As compared to embryonic stem cells, MSCs hold a multitude of advantages from a clinical translation perspective, including ease of isolation, low immunogenicity and limited ethical concerns. Therefore, MSCs are a promising stem cell source for different regenerative medicine applications. The in vitro differentiation of MSCs into different lineages relies on effective mimicking of the in vivo milieu, including both biochemical and mechanical stimuli. As compared to other biophysical cues, such as substrate stiffness and topography, the role of fluid shear stress (SS) in regulating MSC differentiation has been investigated to a lesser extent although the role of interstitial fluid and vascular flow in regulating the normal physiology of bone, muscle and cardiovascular tissues is well-known. This review aims to summarise the current state-of-the-art regarding the role of SS in the differentiation of MSCs into osteogenic, cardiovascular, chondrogenic, adipogenic and neurogenic lineages. We will also highlight and discuss the potential of employing SS to augment the differentiation of MSCs to other lineages, where SS is known to play a role physiologically but has not yet been successfully harnessed for in vitro differentiation, including liver, kidney and corneal tissue lineage cells. The incorporation of SS, in combination with biochemical and biophysical cues during MSC differentiation, may provide a promising avenue to improve the functionality of the differentiated cells by more closely mimicking the in vivo milieu.

scholarly journals Defining the earliest step of cardiovascular progenitor specification during embryonic stem cell differentiation

2011 ◽  
Vol 192 (5) ◽  
pp. 751-765 ◽  
Author(s):  
Antoine Bondue ◽  
Simon Tännler ◽  
Giuseppe Chiapparo ◽  
Samira Chabab ◽  
Mirana Ramialison ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Fluorescent Protein ◽  
Transcriptional Profiling ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
Heart Fields

During embryonic development and embryonic stem cell (ESC) differentiation, the different cell lineages of the mature heart arise from two types of multipotent cardiovascular progenitors (MCPs), the first and second heart fields. A key question is whether these two MCP populations arise from differentiation of a common progenitor. In this paper, we engineered Mesp1–green fluorescent protein (GFP) ESCs to isolate early MCPs during ESC differentiation. Mesp1-GFP cells are strongly enriched for MCPs, presenting the ability to differentiate into multiple cardiovascular lineages from both heart fields in vitro and in vivo. Transcriptional profiling of Mesp1-GFP cells uncovered cell surface markers expressed by MCPs allowing their prospective isolation. Mesp1 is required for MCP specification and the expression of key cardiovascular transcription factors. Isl1 is expressed in a subset of early Mesp1-expressing cells independently of Mesp1 and acts together with Mesp1 to promote cardiovascular differentiation. Our study identifies the early MCPs residing at the top of the cellular hierarchy of cardiovascular lineages during ESC differentiation.

scholarly journals Convergence of Notch and β-catenin signaling induces arterial fate in vascular progenitors

2010 ◽  
Vol 189 (2) ◽  
pp. 325-338 ◽  
Author(s):  
Kohei Yamamizu ◽  
Taichi Matsunaga ◽  
Hideki Uosaki ◽  
Hiroyuki Fukushima ◽  
Shiori Katayama ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Gene Promoter ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
Target Gene Promoter ◽  
Vascular Progenitor Cells ◽  
Molecular Machinery

Molecular mechanisms controlling arterial–venous specification have not been fully elucidated. Previously, we established an embryonic stem cell differentiation system and demonstrated that activation of cAMP signaling together with VEGF induces arterial endothelial cells (ECs) from Flk1+ vascular progenitor cells. Here, we show novel arterial specification machinery regulated by Notch and β-catenin signaling. Notch and GSK3β-mediated β-catenin signaling were activated downstream of cAMP through phosphatidylinositol-3 kinase. Forced activation of Notch and β-catenin with VEGF completely reconstituted cAMP-elicited arterial EC induction, and synergistically enhanced target gene promoter activity in vitro and arterial gene expression during in vivo angiogenesis. A protein complex with RBP-J, the intracellular domain of Notch, and β-catenin was formed on RBP-J binding sites of arterial genes in arterial, but not venous ECs. This molecular machinery for arterial specification leads to an integrated and more comprehensive understanding of vascular signaling.

scholarly journals Diverse but unique astrocytic phenotypes during embryonic stem cell differentiation, culturing and aging

2021 ◽  
Author(s):  
Kiara Freitag ◽  
Pascale Eede ◽  
Andranik Ivanov ◽  
Shirin Schneeberger ◽  
Tatiana Borodina ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Calcium Signalling ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
The Central Nervous System ◽  
Transcriptomic Level

Astrocytes are resident glia cells of the central nervous system (CNS) that play complex and heterogeneous roles in brain development, homeostasis and disease. Since their vast involvement in health and disease is becoming increasingly recognized, suitable and reliable tools for studying these cells in vivo and in vitro are of utmost importance. One of the key challenges hereby is to adequately mimic their context-dependent in vivo phenotypes and functions in vitro. To better understand the spectrum of astrocytic variations in defined settings we performed a side-by-side-comparison of embryonic stem cell (ESC)-derived astrocytes as well as primary neonatal and adult astrocytes, revealing major differences on a functional and transcriptomic level, specifically on proliferation, migration, calcium signalling and cilium activity. Our results highlight the need to carefully consider the choice of astrocyte origin and phenotype with respect to age, isolation and culture protocols based on the respective biological question.

The murine H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1105-1114 ◽  
Author(s):  
F. Poirier ◽  
C.T. Chan ◽  
P.M. Timmons ◽  
E.J. Robertson ◽  
M.J. Evans ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Embryonic Stem Cell Differentiation ◽  
Cdna Clones ◽  
Stage Of Development ◽  
Before And After

The differentiation in vitro of murine embryonic stem cells to embryoid bodies mimics events that occur in vivo shortly before and after embryonic implantation. We have used this system, together with differential cDNA cloning, to identify genes the expression of which is regulated during early embryogenesis. Here we describe the isolation of several such cDNA clones, one of which corresponds to the gene H19. This gene is activated in extraembryonic cell types at the time of implantation, suggesting that it may play a role at this stage of development, and is subsequently expressed in all of the cells of the mid-gestation embryo with the striking exception of most of those of the developing central and peripheral nervous systems. After birth, expression of this gene ceases or is dramatically reduced in all tissues.

scholarly journals The dynamic emergence of GATA1 complexes identified in in vitro embryonic stem cell differentiation and in vivo mouse fetal liver

Haematologica ◽  
2019 ◽  
Vol 105 (7) ◽  
pp. 1802-1812
Author(s):  
Xiao Yu ◽  
Andrea Martella ◽  
Petros Kolovos ◽  
Mary Stevens ◽  
Ralph Stadhouders ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Embryonic Stem Cell ◽  
Fetal Liver ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation
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