Regulation of the differentiation and behaviour of extra-embryonic endodermal cells by basement membranes

Both the extracellular matrix and parathyroid hormone-related peptide (PTHrP) have been implicated in the differentiation and migration of extra-embryonic endodermal cells in the pre-implantation mammalian blastocyst. In order to define the individual roles and interactions between these factors in endodermal differentiation, we have used embryoid bodies derived from Lamc1(-/-) embryonic stem cells that lack basement membranes. The results show that in the absence of basement membranes, increased numbers of both visceral and parietal endodermal cells differentiate, but they fail to form organised epithelia. Furthermore, although parietal endodermal cells only migrate away from control embryoid bodies in the presence of PTHrP, they readily migrate from Lamc1(-/-) embryoid bodies in the absence of PTHrP, and this migration is unaffected by PTHrP. Thus, the basement membrane between epiblast and extra-embryonic endoderm is required for the proper organisation of visceral and parietal endodermal cells and also restricts their differentiation to maintain the population of primitive endodermal stem cells. Moreover, this basement membrane inhibits migration of parietal endodermal cells, the role of PTHrP being to stimulate delamination of parietal endodermal cells from the basement membrane rather than promoting migration per se.

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Absence of Basement Membranes after Targeting the LAMC1 Gene Results in Embryonic Lethality Due to Failure of Endoderm Differentiation

The Journal of Cell Biology ◽

10.1083/jcb.144.1.151 ◽

1999 ◽

Vol 144 (1) ◽

pp. 151-160 ◽

Cited By ~ 322

Author(s):

Neil Smyth ◽

H. Seda Vatansever ◽

Patricia Murray ◽

Michael Meyer ◽

Christian Frie ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Basement Membrane ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Basement Membranes ◽

Embryoid Bodies ◽

Endoderm Differentiation

The LAMC1 gene coding for the laminin γ1 subunit was targeted by homologous recombination in mouse embryonic stem cells. Mice heterozygous for the mutation had a normal phenotype and were fertile, whereas homozygous mutant embryos did not survive beyond day 5.5 post coitum. These embryos lacked basement membranes and although the blastocysts had expanded, primitive endoderm cells remained in the inner cell mass, and the parietal yolk sac did not develop. Cultured embryonic stem cells appeared normal after targeting both LAMC1 genes, but the embryoid bodies derived from them also lacked basement membranes, having disorganized extracellular deposits of the basement membrane proteins collagen IV and perlecan, and the cells failed to differentiate into stable myotubes. Secretion of the linking protein nidogen and a truncated laminin α1 subunit did occur, but these were not deposited in the extracellular matrix. These results show that the laminin γ1 subunit is necessary for laminin assembly and that laminin is in turn essential for the organization of other basement membrane components in vivo and in vitro. Surprisingly, basement membranes are not necessary for the formation of the first epithelium to develop during embryogenesis, but first become required for extra embryonic endoderm differentiation.

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Regulation of Programmed Cell Death by Basement Membranes in Embryonic Development

The Journal of Cell Biology ◽

10.1083/jcb.150.5.1215 ◽

2000 ◽

Vol 150 (5) ◽

pp. 1215-1221 ◽

Cited By ~ 87

Author(s):

Patricia Murray ◽

David Edgar

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Embryonic Stem ◽

Basement Membranes ◽

Embryoid Bodies ◽

Wild Type ◽

Mammalian Embryo ◽

Epithelial Development ◽

Endodermal Cells

The formation of the proamniotic cavity in the mammalian embryo is the earliest of many instances throughout development in which programmed cell death and the formation of epithelia play fundamental roles (Coucouvanis, E., and G.R. Martin. 1995. Cell. 83:279–287). To determine the role of the basement membrane (BM) in cavitation, we use embryoid bodies derived from mouse embryonic stem cells in which the LAMC1 genes have been inactivated to prevent BM deposition (Smyth, N., H.S. Vatansever, P. Murray, M. Meyer, C. Frie, M. Paulsson, and D. Edgar. 1999. J. Cell Biol. 144:151–610). We demonstrate here that LAMC1−/− embryoid bodies are unable to cavitate, and do not form an epiblast epithelium in the absence of a BM, although both embryonic ectodermal cells and extraembryonic endodermal cells do differentiate, as evidenced by the expression of cell-specific markers. Acceleration or rescue of BM deposition by exogenous laminin in wild-type or LAMC1−/− embryoid bodies, respectively, results in cavitation that is temporally and spatially associated with restoration of epiblast epithelial development. We conclude that the BM not only directly regulates development of epiblast epithelial cells, but also indirectly regulates the programmed cell death necessary for cavity formation.

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Role of estrogen receptor beta in neural differentiation of mouse embryonic stem cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714094114 ◽

2017 ◽

Vol 114 (48) ◽

pp. E10428-E10437 ◽

Cited By ~ 10

Author(s):

Mukesh K. Varshney ◽

José Inzunza ◽

Diana Lupu ◽

Vaidheeswaran Ganapathy ◽

Per Antonson ◽

...

Keyword(s):

Stem Cells ◽

Estrogen Receptor ◽

Embryonic Stem Cells ◽

Neurodegenerative Diseases ◽

Estrogen Receptor Beta ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Embryoid Bodies ◽

And Migration ◽

Receptor Beta

The ability to propagate mature cells and tissue from pluripotent stem cells offers enormous promise for treating many diseases, including neurodegenerative diseases. Before such cells can be used successfully in neurodegenerative diseases without causing unwanted cell growth and migration, genes regulating growth and migration of neural stem cells need to be well characterized. Estrogen receptor beta (ERβ) is essential for migration of neurons and glial cells in the developing mouse brain. To examine whether ERβ influences differentiation of mouse embryonic stem cells (mESC) into neural lineages, we compared control and ERβ knockout (BERKO) mESCs at defined stages of neural development and examined the effects of an ERβ-selective ligand (LY3201) with a combination of global and targeted gene-expression profiling and the expression of key pluripotency markers. We found that ERβ was induced in embryoid bodies (EBs) and neural precursor cells (NPCs) during development. Proliferation was higher in BERKO NPCs and was inhibited by LY3201. Neurogenesis was reduced in BERKO ES cells, and oligodendrogliogenesis was enhanced. BERKO EBs expressed higher levels of key ectodermal and neural progenitor markers and lower levels of markers for mesoderm and endoderm lineages. ERβ-regulated factors are involved in cell adhesion, axon guidance, and signaling of Notch and GABA receptor pathways, as well as factors important for the differentiation of neuronal precursors into dopaminergic neurons (Engrailed 1) and for the oligodendrocyte fate acquisition (Olig2). Our data suggest that ERβ is an important component for differentiation into midbrain neurons as well as for preventing precocious oligodendrogliogenesis.

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Neural Differentiation Is Inhibited through HIF1α/β-Catenin Signaling in Embryoid Bodies

Stem Cells International ◽

10.1155/2017/8715798 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Josef Večeřa ◽

Jana Kudová ◽

Jan Kučera ◽

Lukáš Kubala ◽

Jiří Pacherník

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Neural Differentiation ◽

Embryonic Stem ◽

Direct Interaction ◽

Embryoid Bodies ◽

Nuclear Fraction ◽

Wild Type ◽

Spontaneous Differentiation

Extensive research in the field of stem cells and developmental biology has revealed evidence of the role of hypoxia as an important factor regulating self-renewal and differentiation. However, comprehensive information about the exact hypoxia-mediated regulatory mechanism of stem cell fate during early embryonic development is still missing. Using a model of embryoid bodies (EBs) derived from murine embryonic stem cells (ESC), we here tried to encrypt the role of hypoxia-inducible factor 1α (HIF1α) in neural fate during spontaneous differentiation. EBs derived from ESC with the ablated gene for HIF1α had abnormally increased neuronal characteristics during differentiation. An increased neural phenotype in Hif1α−/− EBs was accompanied by the disruption of β-catenin signaling together with the increased cytoplasmic degradation of β-catenin. The knock-in of Hif1α, as well as β-catenin ectopic overexpression in Hif1α−/− EBs, induced a reduction in neural markers to the levels observed in wild-type EBs. Interestingly, direct interaction between HIF1α and β-catenin was demonstrated by immunoprecipitation analysis of the nuclear fraction of wild-type EBs. Together, these results emphasize the regulatory role of HIF1α in β-catenin stabilization during spontaneous differentiation, which seems to be a crucial mechanism for the natural inhibition of premature neural differentiation.

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Thrombopoietin (TPO) Enhances Heamatopoietic Differentiation of Human Embryonic Stem Cells.

Blood ◽

10.1182/blood.v108.11.4157.4157 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4157-4157

Author(s):

Anand S. Srivastava ◽

Rangrath Mishra ◽

Ewa Carrier

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Es Cells ◽

Embryonic Stem ◽

Cytoplasmic Domain ◽

Embryoid Bodies ◽

Janus Kinase ◽

Human Es Cells

Abstract Recently, it was demonstrated that TPO enhances hematopoietic differentiation of primate ES cells, but its role in differentiating human ES cells is unknown. We sought to investigate the regulatory mechanism of TPO induced signals mediated by the c-mpl cytoplasmic domain during human embryonic stem (hES) cells hematopoietic commitment. We hypothesize that in human embryonic stem cells, binding of TPO to its c-mpl receptor causes three-dimensional alterations which bring the c-mpl cytoplasmic domain and Janus Kinase into close-proximity and thus induces the phosphorylation and dimerization of STAT5 molecule. Dimerized STAT5 molecules detach from the receptors and migrate to the nucleus where they bind GAS site and induce transcription of a set of target, hematopoiesis-related genes. NIH human ES cell lines (WI01) were used in this experiment. In brief, to induce EB formation, cells were incubated in differentiation medium, which consisted of knockout DMEM medium (GIBCO/BRL, Carlsbad, USA), supplemented with 20% non-heat-inactivated fetal bovine serum (FBS, Hyclone, USA), 1% nonessential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Subsequently, DMEM was replaced by IMDM (GIBCO/BRL, USA) with the same supplements and additional two cytokines (100 ng/mL SCF and 100 ng/mL Flt-3 ligand (Flt-3L)) (control group). To investigate the role of TPO and VEGF, cells were additionally treated with 100 ng/mL TPO alone or in combination with 100 ng/mL rhVEGF. All cytokines were from the R&D systems (USA). Significant increase in the numbers of embryoid bodies (EBs) formation in TPO (125/105), TPO/VEGF (150/105 cells) when compared to controls (10/105 planted ES cells) was documented. This corresponded with the increase in CFU-C and the number of CD31/CD34 positive and CD34-positive progenitors. Analysis of gene expression during hematopoietic development demonstrated that TPO/VEGF combination increased mRNA expression of the TPO receptor (TPO-R) and VEGF (VEGF-R) receptors in hematopoietic progenitors obtained from human ES cells. We are in the process of determining the role of JAK/STAT pathway in this process; functional studies involve blocking of TPO/c-mpl using TPO-R-specific antibodies and determining its impact on human ES-derived hematopoiesis.

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Insufficient Folding of Type IV Collagen and Formation of Abnormal Basement Membrane-like Structure in Embryoid Bodies Derived from Hsp47-Null Embryonic Stem Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e04-01-0050 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4467-4475 ◽

Cited By ~ 37

Author(s):

Yasuhiro Matsuoka ◽

Hiroshi Kubota ◽

Eijiro Adachi ◽

Naoko Nagai ◽

Toshihiro Marutani ◽

...

Keyword(s):

Basement Membrane ◽

Type Iv Collagen ◽

Type I Collagen ◽

Embryonic Stem ◽

Basement Membranes ◽

Embryoid Bodies ◽

Null Mouse ◽

Type I ◽

Type Iv ◽

Collagen Secretion

Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47-/- embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47-/- embryoid bodies were often disorganized, a result that suggested abnormal function of the basement membrane under the visceral endoderm. Rate of type IV collagen secretion by Hsp47-/- cells was fourfold lower than that of Hsp47+/+ cells. Furthermore, type IV collagen secreted from Hsp47-/- cells was much more sensitive to protease digestion than was type IV collagen secreted from Hsp47+/+ cells, which suggested insufficient or incorrect triple helix formation in type IV collagen in the absence of Hsp47. These results indicate for the first time that Hsp47 is required for the molecular maturation of type IV collagen and suggest that misfolded type IV collagen causes abnormal morphology of embryoid bodies.

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The Absence of Nidogen 1 Does Not Affect Murine Basement Membrane Formation

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.7007-7012.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 7007-7012 ◽

Cited By ~ 100

Author(s):

Monzur Murshed ◽

Neil Smyth ◽

Nicolai Miosge ◽

Jörg Karolat ◽

Thomas Krieg ◽

...

Keyword(s):

Basement Membrane ◽

Embryonic Stem ◽

Collagen Iv ◽

Basement Membranes ◽

Null Mutation ◽

Membrane Structures ◽

Membrane Formation ◽

Nidogen 1

ABSTRACT Nidogen 1 is a highly conserved protein in mammals,Drosophila melanogaster, Caenorhabditis elegans, and ascidians and is found in all basement membranes. It has been proposed that nidogen 1 connects the laminin and collagen IV networks, so stabilizing the basement membrane, and integrates other proteins, including perlecan, into the basement membrane. To define the role of nidogen 1 in basement membranes in vivo, we produced a null mutation of the NID-1 gene in embryonic stem cells and used these to derive mouse lines. Homozygous animals produce neither nidogen 1 mRNA nor protein. Surprisingly, they show no overt abnormalities and are fertile, their basement membrane structures appearing normal. Nidogen 2 staining is increased in certain basement membranes, where it is normally only found in scant amounts. This occurs by either redistribution from other extracellular matrices or unmasking of nidogen 2 epitopes, as its production does not appear to be upregulated. The results show that nidogen 1 is not required for basement membrane formation or maintenance.

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Abstract 395: Transient Inactivation of Retinoblastoma Induces De-Novo Cardiomyogenesis From Human Myocardial Progenitors but Not Pre-Existing Cardiomyocyte Replication

Circulation Research ◽

10.1161/res.117.suppl_1.395 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Konstantinos E Hatzistergos ◽

Julien Sage ◽

Jamie F Conklin ◽

Michael Bellio ◽

Krystalenia Valasaki ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

De Novo ◽

Embryonic Stem ◽

Fold Increase ◽

Embryoid Bodies ◽

Cardiac Cell ◽

Cardiac Progenitors ◽

Proliferation And Differentiation

Introduction: Activation of cardiac cell cycle re-entry is considered the primary therapeutic strategy for cardiomyocyte (CM) regeneration. However, the role of cardiac cell-cycle control in cardiomyogenesis remains elusive. Here, we combined RNA interference and stem cell modeling to investigate the role of Retinoblastoma (RB) in human cardiomyogenesis. Hypothesis: RB regulates proliferation and differentiation of cardiac progenitors (CPCs) but not CM replication. Methods: H9 human embryonic stem cells (hESCs) stably expressing tetracycline (tet)-inducible shRNAs against RB (hESCshRB) or hemagglutinin-tagged RB (hESCHA-RB) were tet-induced at selected time-points during or after CM differentiation. Results: Analysis of ser-608 illustrated stage-specific differences in the degree of RB inactivation during normal hESCs-cardiogenesis. Transient shRB knockdown in hESCshRB-derived embryoid bodies (EBs) during the CPC-stage (EB-days 5-8), significantly upregulated GATA4, ISL1, CTNNI, and cKit transcription (p<0.05), while increasing the yield of beating EBs by 2.4-fold (n=6/group, p<0.0001 vs. vehicle). Gene-expression arrays of 22 RB-related genes, illustrated that shRB-knockdown upregulated CCND1, CCND2, CCND3, and CDK4, CDK6 (p<0.05), followed by a 3.6-fold increase in E2F3 (p<0.05) expression. Moreover, expression of p107 and p130, p27, p57, ARF and CDKN3 were also significantly increased (p<0.05), whereas TP53 and MDM2 remained unchanged. Ectopic HA-RB in CPCs did not significantly affect cardiogenesis (n=18). Conversely, shRB knockdown in EB-day 60-derived CMs (n=15) did not stimulate cell cycle re-entry, as assessed by analysis of EdU incorporation and Aurora-B kinase (AurB). Remarkably, co-culture of hESCHA-RB-derived CMs with adult cardiac (CSCs) and/or mesenchymal (MSCs) stem cells (n=15/group), increased cell-cycle re-entry ~2.8-fold, assessed by ser-10 Histone H3 (p=0.0002) and AurB (p<0.0001). Conclusions: These findings suggest that RB regulates proliferation and differentiation of human CPCs in a cell-autonomous manner, via a CCND-CDK4/6-E2F3 mechanism. Conversely, CM replication may be enhanced via cell-cell interactions with MSCs and/or CSCs, but not cell-autonomously via RB inactivation.

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THE ROLE OF SUBSTRATE STIFFNESS IN MAINTAINING PLURIPOTENCY OF EMBRYONIC STEM CELLS IN VITRO CULTURE

Fiziolohichnyĭ zhurnal ◽

10.15407/fz67.03.027 ◽

2021 ◽

Vol 67 (3) ◽

pp. 27-34

Author(s):

D.I. Bilko ◽

◽

Y.B. Chaikovsky ◽

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Embryonic Stem Cells ◽

In Vitro Culture ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Polyacrylamide Gel ◽

Substrate Stiffness

Murine embryonic stem cells (ESCm) cultured in vitro in the presence of LIF (leukemia inhibitory factor) maintain pluripotency. However, when LIF is removed from the media, an active differentiation into various specialized somatic cells is observed. The aim of the study was to determine the role of substrate stiffness in maintaining of pluripotency of embryonic stem cells in vitro culture. To this aim, we used the method of culturing pluripotent stem cells in vitro, the method of “hanging drop”, the determination of the Young’s modulus for polyacrylamide gel of different hardness, the immunocytochemical alkaline phosphatase (AP) streptavidin-biotin method, microscopy. By culturing ESCm on a soft, medium and hard density polyacrylamide gel as a substrate (0.8, 4.0, 8.0 кPа), we found that on a soft gel ESCm differentiation does not occur even in the absence of LIF. ESCm cultured on a soft substrate continue to show signs of pluripotency, namely, create round compact colonies with high alkaline phosphatase activity and form embryoid bodies (EB), the efficiency of which (87.5 ± 3.2 per 100 cells seeded) did not decrease even after LIF withdrawal. In the absence of LIF, ESCs cultured on a hard base showed a low level of EB formation (23.5 ± 2.24). The results of our observations demonstrate that the process of EB formation may be influenced not only by a composition of nutrient medium, but also by complex interaction between the physical forces of the matrix and the mechanical properties of 3D cell aggregates. The model is considered as a tool to study early events in embryogenesis in the search of conditions for effective culture of progenitor cells and differentiated cells for transplantation.

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