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

ABSTRACT: AIM:To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. MATERIALS AND METHODS:Feeder-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. RESULTS:hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPase?1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIII?1 and VIII?2 (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. CONCLUSION:hESC-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.

SUBMITTER: McCabe KL

PROVIDER: S-EPMC4686926 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

McCabe Kathryn L KL Kunzevitzky Noelia J NJ Chiswell Brian P BP Xia Xin X Goldberg Jeffrey L JL Lanza Robert R

PloS one 20151221 12

<h4>Aim</h4>To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies.<h4>Materials and methods</h4>Feeder-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.<h4>Results</h4>hESC-CECs were nearly identical morphologically to primary human corneal endotheli ...[more]

PMID: 26689688

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Project description:BackgroundCorneal transplantation is the most effective clinical treatment for irreversible corneal endothelial decompensation. However, while visual rehabilitation can be achieved by corneal transplantation, transplant rejection, poor postoperative visual acuity, and lack of suitable donor tissue are currently the greatest obstacles to corneal transplantation. As a result, endothelial cell-based therapy has emerged as an alternative to corneal transplantation treatment. Human induced pluripotent stem cells (hiPSCs) were induced to differentiate into human corneal endothelial cell (hCEC)-like cells in our study, which aimed to provide an experimental basis for studying the clinical translation and application of induced PSC (iPSC) to corneal endothelial cell (CEC) differentiation.MethodsA two-step method to convert hiPSCs into hCEC-like cells was applied in the study. First, the transforming growth factor beta (TGF-β) and Wnt signaling pathways were regulated by adding SB4315542 and CHIR99021, and hiPSCs were induced to differentiate into neural crest cells (NCCs) by a chemically defined and serum-free in vitro induction method. Subsequently, NCCs were induced to differentiate into hCEC-like cells by adding B27, platelet-derived growth factor (PDGF)-BB, and XAV939 (Wnt pathway inhibitor) to CEC culture medium.ResultsIPSCs were directionally differentiated into NCCs. During the process of differentiation, iPSCs gradually lost the monoclonal morphology of PSCs. Moreover, β-catenin and SRY-related HMG-box gene 10 (SOX10) proteins were immunohistochemically expressed on the 7th day of differentiation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) results demonstrated that the cells expressed SOX9, SOX10, nerve growth factor receptor (NGFR), human natural killer-1 (HNK-1), and β-catenin, indicating that they were successfully directionally differentiated from iPSCs to NCCs. After 5-7 days of differentiation, the cells demonstrated a hexagonal morphology of monolayer tight junctions. Immunofluorescence results demonstrated that the cells expressed CEC indicator zonula occludens-1 (ZO-1) protein. QRT-PCR results demonstrated that the cells expressed collagen type IV alpha 1 (COL4A1), COL8A2, COL8A1, and ZO-1, which indicated that they were successfully directionally differentiated from NCC to CEC.ConclusionsOur study successfully provided a simple and efficient method with clear chemical composition and serum-free media to directionally differentiate hiPSCs into hCEC-like cells, thereby advancing the results of previous studies on directional transformation into epithelial cells.

Dataset Information

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

Publications

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

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