Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Laminin isoforms in human embryonic stem cells: synthesis, receptor usage and growth support.

ABSTRACT: To reveal the functional intrinsic niche of human embryonic stem cells (hESC) we examined the production of basement membrane (BM) proteins and the presence of their receptors in feeder-free cell culture conditions. In addition, we investigated binding of hESCs to purified human BM proteins and identified the receptors mediating these contacts. Also, we tested whether purified human laminin (Lm) isoforms have a role in hESC self-renewal and growth in short-term cultures. The results show that hESCs synthesize Lm alpha(1) and Lm alpha(5) chains together with Lm beta(1) and gamma(1) chains suggesting the production of Lms-111 and -511 into the culture medium and deposits on cells. hESCs contain functionally important integrin (Int) subunits, Int beta(1), alpha(3), alpha(6), alpha(5), beta(5) and alpha(V), as well as the Lm alpha(5) receptor, Lutheran (Lu) glycoprotein and its truncated form, basal cell adhesion molecule (B-CAM). In cell adhesion experiments, Int beta(1) was crucial for adhesion to most of the purified human BM proteins. Lu/B-CAM mediated adhesion to Lm-511 together with Int alpha(3)beta(1), and was essential for the adhesion of hESCs to embryonic feeder cells. Adhesion to Lm-411 was mediated by Int alpha(6)beta(1). Lm-511 supported hESC growth in defined medium equally well as Matrigel. These results provide consequential information of the biological role of BM in hESCs, warranting further investigation of BM biology of human pluripotent stem cells.

SUBMITTER: Vuoristo S

PROVIDER: S-EPMC6529980 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Microenvironmental regulation of telomerase isoforms in human embryonic stem cells.

Project description:Recent evidence points to extra-telomeric, noncanonical roles for telomerase in regulating stem cell function. In this study, human embryonic stem cells (hESCs) were cultured in 20% or 2% O2 microenvironments for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% O2-cultured hESCs despite no significant difference in telomerase activity compared with their high-O2-cultured counterparts. Pharmacological inhibition of telomerase activity using a synthetic tea catechin resulted in spontaneous hESC differentiation, while telomerase inhibition with a phosphorothioate oligonucleotide telomere mimic did not. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed variations in transcript levels of full-length and alternate splice variants of TERT in hESCs cultured under varying O2 atmospheres. Steric-blocking of ?? and ?? hTERT splicing using morpholino oligonucleotides altered the hTERT splicing pattern and rapidly induced spontaneous hESC differentiation that appeared biased toward endomesodermal and neuroectodermal cell fates, respectively. Together, these results suggest that post-transcriptional regulation of TERT under varying O2 microenvironments may help regulate hESC survival, self-renewal, and differentiation capabilities through expression of extra-telomeric telomerase isoforms.

| S-EPMC4142780 | biostudies-literature

Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

Project description:In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2), and transforming growth factor ? (TGF-?), thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

| S-EPMC3012692 | biostudies-literature

Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

Project description:Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain-145 kDa-accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

| S-EPMC4574950 | biostudies-literature

Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells.

Project description:Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to provide an infinite source of tissues for regenerative medicine. Although defined xeno-free media have been developed, culture conditions for reliable propagation of hESCs still require considerable improvement. Here we show that recombinant E8 fragments of laminin isoforms (LM-E8s), which are the minimum fragments conferring integrin-binding activity, promote greater adhesion of hESCs and hiPSCs than do Matrigel and intact laminin isoforms. Furthermore, LM-E8s sustain long-term self-renewal of hESCs and hiPSCs in defined xeno-free media with dissociated cell passaging. We successfully maintained three hESC and two hiPSC lines on LM-E8s in three defined media for 10 passages. hESCs maintained high level expression of pluripotency markers, had a normal karyotype after 30 passages and could differentiate into all three germ layers. This culture system allows robust proliferation of hESCs and hiPSCs for therapeutic applications.

| S-EPMC3535336 | biostudies-literature

Recombinant laminin α5 LG1-3 domains support the stemness of human mesenchymal stem cells.

Project description:The extracellular matrix components laminin and elastin serve key roles in stem cell therapy. Elastin-like polypeptides (ELPs), derived from a soluble form of elastin, affect the proliferation and differentiation of various types of cells. In the present study, a novel protein was designed containing globular domains 1-3 of laminin α5 (Lα5LG1-3) fused to ELPs (Lα5LG1-3/ELP). Lα5LG1-3/ELP was expressed in Escherichia coli and displayed a molecular size of ~70 kDa on 12% SDS-polyacrylamide gels. The cellular activities, such as cellular adhesion (adhesion assay) and proliferation (MTT cytotoxicity assay), of human mesenchymal stem cells (hMSCs) treated with 1 µg/ml of Lα5LG1-3/ELP were enhanced compared with those of untreated cells. Additionally, the number of undifferentiated hMSCs and their degree of stemness were assessed based on the gene expression levels of the stem cell markers cluster differentiation 90 (CD90), endoglin (CD105) and CD73. The expression levels of these markers were upregulated by 2.42-, 2.29- and 1.92-fold, respectively, in the hMSCs treated with Lα5LG1-3/ELP compared with the levels in untreated controls. Thus, Lα5LG1-3/ELP may be used to enhance the viability of hMSCs and preserve their undifferentiated state, whereby the clinical applications of hMSCs may be improved.

| S-EPMC7792491 | biostudies-literature

Combinatorial fibronectin and laminin signaling promote highly efficient cardiac differentiation of human embryonic stem cells.

Project description:Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies-primarily based on the optimization of growth factors-have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4(+) and ITGB5(+) cells.

| S-EPMC4120929 | biostudies-literature

Codon usage optimization in pluripotent embryonic stem cells.

Project description:BACKGROUND:The uneven use of synonymous codons in the transcriptome regulates the efficiency and fidelity of protein translation rates. Yet, the importance of this codon bias in regulating cell state-specific expression programmes is currently debated. Here, we ask whether different codon usage controls gene expression programmes in self-renewing and differentiating embryonic stem cells. RESULTS:Using ribosome and transcriptome profiling, we identify distinct codon signatures during human embryonic stem cell differentiation. We find that cell state-specific codon bias is determined by the guanine-cytosine (GC) content of differentially expressed genes. By measuring the codon frequencies at the ribosome active sites interacting with transfer RNAs (tRNA), we further discover that self-renewing cells optimize translation of codons that depend on the inosine tRNA modification in the anticodon wobble position. Accordingly, inosine levels are highest in human pluripotent embryonic stem cells. This effect is conserved in mice and is independent of the differentiation stimulus. CONCLUSIONS:We show that GC content influences cell state-specific mRNA levels, and we reveal how translational mechanisms based on tRNA modifications change codon usage in embryonic stem cells.

| S-EPMC6555954 | biostudies-literature

Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling.

Project description:Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1beta (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.

| S-EPMC2190616 | biostudies-literature

Recombinant Extracellular Matrix Protein Fragments Support Human Embryonic Stem Cell Chondrogenesis.

Project description:We previously developed a 14-day culture protocol under potentially GMP, chemically defined conditions, to generate chondroprogenitors from human embryonic stem cells (hESCs). In vivo work has confirmed the cartilage repair capacity of these cells in a nude rat osteochondral defect model. Aiming to enhance hESC-chondrogenesis, we screened a range of extracellular matrix (ECM) molecules for their ability to support differentiation of hESCs toward chondrocytes. We identified two novel ECM protein fragments that supported hESC-chondrogenesis: Fibronectin III (fibronectin 7-14 protein fragments, including the RGD domain, syndecan-binding domain, and heparin-binding domain) and fibrillin-1 (FBN1) fragment PF8 (encoded by exons 30-38, residues 1238-1605, which contains the RGD motif but not heparin-binding site). These two protein fragments support hESC-chondrogenesis compared with the substrates routinely used previously (a mixture of fibronectin and gelatin) in our directed chondrogenic protocol. We have identified recombinant fibronectin fragment (FN III) and FBNI fragment (PF8) as alternative coating substrates to promote expression of genes known to regulate chondrocytes and code for chondrocyte ECM components. These recombinant protein fragments are likely to have better batch to batch stability than full-length molecules, especially where extracted from tissue/serum.

| S-EPMC5984563 | biostudies-literature

Laminin enhances the growth of human neural stem cells in defined culture media.

Project description:Human neural stem cells (hNSC) have the potential to provide novel cell-based therapies for neurodegenerative conditions such as multiple sclerosis and Parkinson's disease. In order to realise this goal, protocols need to be developed that allow for large quantities of hNSC to be cultured efficiently. As such, it is important to identify factors which enhance the growth of hNSC. In vivo, stem cells reside in distinct microenvironments or niches that are responsible for the maintenance of stem cell populations. A common feature of niches is the presence of the extracellular matrix molecule, laminin. Therefore, this study investigated the effect of exogenous laminin on hNSC growth.To measure hNSC growth, we established culture conditions using B27-supplemented medium that enable neurospheres to grow from human neural cells plated at clonal densities. Limiting dilution assays confirmed that neurospheres were derived from single cells at these densities. Laminin was found to increase hNSC numbers as measured by this neurosphere formation. The effect of laminin was to augment the proliferation/survival of the hNSC, rather than promoting the undifferentiated state. In agreement, apoptosis was reduced in dissociated neurospheres by laminin in an integrin beta1-dependent manner.The addition of laminin to the culture medium enhances the growth of hNSC, and may therefore aid their large-scale production.

| S-EPMC2496909 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data