Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Small-Molecule Induction Promotes Corneal Endothelial Cell Differentiation From Human iPS Cells.

ABSTRACT: Purpose: Corneal endothelial cells (CECs) serve as a barrier and foothold for the corneal stroma to maintain the function and transparency of the cornea. Loss of CECs during aging or disease states leads to blindness, and cell replacement therapy using either donated or artificially differentiated CECs remains the only curative approach. Methods: Human induced pluripotent stem cells (hiPSCs) that were cultured in chemically defined medium were induced with dual-SMAD inhibition to differentiate into neural crest cells (NCCs). A small-molecule library was screened to differentiate the NCCs into corneal endothelial-like cells. The characteristics of these cells were identified with real-time PCR and immunofluorescence. Western blotting was applied to detect the signaling pathways and key factors regulated by the small molecules. Results: We developed an effective protocol to differentiate hiPSCs into CECs with defined small molecules. The hiPSC-CECs were characterized by ZO-1, AQP1, Vimentin and Na⁺/K⁺-ATPase. Based on our small-molecule screen, we identified a small-molecule combination, A769662 and AT13148, that enabled the most efficient production of CECs. The combination of A769662 and AT13148 upregulated the PKA/AKT signaling pathway, FOXO1 and PITX2 to promote the conversion of NCCs to CECs. Conclusion: We established an efficient small molecule-based method to differentiate hiPSCs into corneal endothelial-like cells, which might facilitate drug discovery and the development of cell-based therapies for corneal diseases.

SUBMITTER: Chen J

PROVIDER: S-EPMC8714889 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Small-molecule induction promotes corneal epithelial cell differentiation from human induced pluripotent stem cells.

Project description:Human induced pluripotent stem cells (hiPSCs) offer unique opportunities for developing novel cell-based therapies and disease modeling. In this study, we developed a directed differentiation method for hiPSCs toward corneal epithelial progenitor cells capable of terminal differentiation toward mature corneal epithelial-like cells. In order to improve the efficiency and reproducibility of our method, we replicated signaling cues active during ocular surface ectoderm development with the help of two small-molecule inhibitors in combination with basic fibroblast growth factor (bFGF) in serum-free and feeder-free conditions. First, small-molecule induction downregulated the expression of pluripotency markers while upregulating several transcription factors essential for normal eye development. Second, protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63 positive after 5 weeks of differentiation. Third, corneal epithelial-like cells were obtained upon further maturation.

| S-EPMC3923224 | biostudies-literature

High-efficient serum-free differentiation of endothelial cells from human iPS cells.

Project description:IntroductionEndothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs' physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications.MethodHuman induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h.ResultThis combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4).ConclusionThe differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs.

| S-EPMC9188069 | biostudies-literature

Extracellular Vesicles Derived From Human Corneal Endothelial Cells Inhibit Proliferation of Human Corneal Endothelial Cells.

Project description:Corneal endothelial cells (CEnCs) are a monolayer of hexagonal cells that are responsible for maintaining the function and transparency of the cornea. Damage or dysfunction of CEnCs could lead to blindness. Human CEnCs (HCEnCs) have shown limited proliferative capacity in vivo hence, their maintenance is crucial. Extracellular vesicles (EVs) are responsible for inter- and intra-cellular communication, proliferation, cell-differentiation, migration, and many other complex biological processes. Therefore, we investigated the effect of EVs (derived from human corneal endothelial cell line-HCEC-12) on corneal endothelial cells. HCEC-12 cells were starved with serum-depleted media for 72 h. The media was ultracentrifuged at 100,000xg to isolate the EVs. EV counting, characterization, internalization and localization were performed using NanoSight, flow cytometry, Dil labeling and confocal microscopy respectively. HCEC-12 and HCEnCs were cultured with media supplemented with EVs. Extracted EVs showed a homogeneous mixture of exosomes and microvesicles. Cells with EVs decreased the proliferation rate; increased apoptosis and cell size; showed poor wound healing response in vitro and on ex vivo human, porcine, and rabbit CECs. Thirteen miRNAs were found in the EV sample using next generation sequencing. We observed that increased cellular uptake of EVs by CECs limit the proliferative capacity of HCEnCs. These preliminary data may help in understanding the pathology of corneal endothelial dysfunction and provide further insights in the development of future therapeutic treatment options.

| S-EPMC8854366 | biostudies-literature

Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling.

Project description:Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here, we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34(+)CD31(+) endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable, and endothelial progenitor differentiation was β-catenin dependent. Furthermore, by clonal analysis, we showed that CD34(+)CD31(+)CD117(+)TIE-2(+) endothelial progenitors were multipotent, capable of differentiating into calponin-expressing smooth muscle cells and CD31(+)CD144(+)vWF(+)I-CAM1(+) endothelial cells. These endothelial cells were capable of 20 population doublings, formed tube-like structures, imported acetylated low-density lipoprotein, and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/β-catenin signaling as a primary regulator for generating vascular cells from hPSCs.

| S-EPMC4235141 | biostudies-literature

Human iPS cells engender corneal epithelial stem cells with holoclone-forming capabilities.

Project description:Human induced pluripotent stem cells (hiPSCs) can generate a multiplicity of organoids, yet no compelling evidence currently exists as to whether or not these contain tissue-specific, holoclone-forming stem cells. Here, we show that a subpopulation of cells in a hiPSC-derived corneal epithelial cell sheet is positive for ABCB5 (ATP-binding cassette, sub-family B, member 5), a functional marker of adult corneal epithelial stem cells. These cells possess remarkable holoclone-forming capabilities, which can be suppressed by an antibody-mediated ABCB5 blockade. The cell sheets are generated from ABCB5+ hiPSCs that first emerge in 2D eye-like organoids around six weeks of differentiation and display corneal epithelial immunostaining characteristics and gene expression patterns, including sustained expression of ABCB5. The findings highlight the translational potential of ABCB5-enriched, hiPSC-derived corneal epithelial cell sheets to recover vision in stem cell-deficient human eyes and represent the first report of holoclone-forming stem cells being directly identified in an hiPSC-derived organoid.

| S-EPMC8233200 | biostudies-literature

Temporal Dynamics of Gene Expression During Endothelial Cell Differentiation From Human iPS Cells: A Comparison Study of Signalling Factors and Small Molecules.

Project description:Endothelial cell (EC) therapy may promote vascular growth or reendothelization in a variety of disease conditions. However, the production of a cell therapy preparation containing differentiated, dividing cells presenting typical EC phenotype, functional properties and chemokine profile is challenging. We focused on comparative analysis of seven small molecule-mediated differentiation protocols of ECs from human induced pluripotent stem cells. Differentiated cells showed a typical surface antigen pattern of ECs as characterized with flow cytometry analysis, functional properties, such as tube formation and ability to uptake acetylated LDL. Gene expression analysis by RNA sequencing revealed an efficient silencing of pluripotency genes and upregulation of genes related to cellular adhesion during differentiation. In addition, distinct patterns of transcription factor expression were identified during cellular reprogramming providing targets for more effective differentiation protocols in the future. Altogether, our results suggest that the most optimal EC differentiation protocol includes early inhibition of Rho-associated coiled-coil kinase and activation of cyclic AMP signaling, and inhibition of transforming growth factor beta signaling after mesodermal stage. These findings provide the first systematic characterization of the most potent signalling factors and small molecules used to generate ECs from human induced pluripotent stem cells and, consequently, this work improves the existing EC differentiation protocols and opens up new avenues for controlling cell fate for regenerative EC therapy.

| S-EPMC5861200 | biostudies-literature

Directed Differentiation of Human Pluripotent Stem Cells towards Corneal Endothelial-Like Cells under Defined Conditions.

Project description:The most crucial function of corneal endothelial cells (CEnCs) is to maintain optical transparency by transporting excess fluid out of stroma. Unfortunately, CEnCs are not able to proliferate in vivo in the case of trauma or dystrophy. Visually impaired patients with corneal endothelial deficiencies that are waiting for transplantation due to massive global shortage of cadaveric corneal transplants are in a great need of help. In this study, our goal was to develop a defined, clinically applicable protocol for direct differentiation of CEnCs from human pluripotent stem cells (hPSCs). To produce feeder-free hPSC-CEnCs, we used small molecule induction with transforming growth factor (TGF) beta receptor inhibitor SB431542, GSK-3-specific inhibitor CHIR99021 and retinoic acid to guide differentiation through the neural crest and periocular mesenchyme (POM). Cells were characterized by the morphology and expression of human (h)CEnC markers with immunocytochemistry and RT-qPCR. After one week of induction, we observed the upregulation of POM markers paired-like homeodomain transcription factor 2 (PITX2) and Forkhead box C1 (FOXC1) and polygonal-shaped cells expressing CEnC-associated markers Zona Occludens-1 (ZO-1), sodium-potassium (Na+/K+)-ATPase, CD166, sodium bicarbonate cotransporter 1 (SLC4A4), aquaporin 1 (AQP1) and N-cadherin (NCAD). Furthermore, we showed that retinoic acid induced a dome formation in the cell culture, with a possible indication of fluid transport by the differentiated cells. Thus, we successfully generated CEnC-like cells from hPSCs with a defined, simple and fast differentiation method.

| S-EPMC7915025 | biostudies-literature

Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells.

Project description:The differentiation of pluripotent stem cells to hepatocytes is well established, yet current methods suffer from several drawbacks. These include a lack of definition and reproducibility, which in part stems from continued reliance on recombinant growth factors. This has remained a stumbling block for the translation of the technology into industry and the clinic for reasons associated with cost and quality. We have devised a growth-factor-free protocol that relies on small molecules to differentiate human pluripotent stem cells toward a hepatic phenotype. The procedure can efficiently direct both human embryonic stem cells and induced pluripotent stem cells to hepatocyte-like cells. The final population of cells demonstrates marker expression at the transcriptional and protein levels, as well as key hepatic functions such as serum protein production, glycogen storage, and cytochrome P450 activity.

| S-EPMC4437467 | biostudies-literature

A small molecule inhibitor of SRC family kinases promotes simple epithelial differentiation of human pluripotent stem cells.

Project description:Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs, the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of β-catenin during retinoic acid (RA)--induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated β-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8), which are expressed in simple epithelial cells, while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4- simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications.

| S-EPMC3603942 | biostudies-literature

Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

Project description:AimTo generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies.Materials and methodsFeeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.ResultshESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.ConclusionhESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

| S-EPMC4686926 | 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