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Treatment with retinoic acid and lens epithelial cell-conditioned medium in vitro directed the differentiation of pluripotent stem cells towards corneal endothelial cell-like cells.

ABSTRACT: Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have extensive self-renewal capacity and the potential to differentiate into all tissue-specific cell lineages, including corneal endothelial cells (CECs). They are a promising prospect for the future of regenerative medicine. The method of derivation of CECs from ESCs and iPSCs, however, remains to be elucidated. In this study, mouse ESCs and iPSCs were induced to differentiate into CECs using CEC embryonic development events as a guide. All-trans retinoic acid (RA) treatment during the embryoid body (EB) differentiation step was used to promote neural crest (NC) cell differentiation as first step and was followed by a second induction in CEC- or lens epithelial cell (LEC)-conditioned medium (CM) to ultimately generate CEC-like cells. During the corresponding differentiation stages, NC developmental markers and CEC differentiation markers were detected at the protein level using immunocytochemistry (ICC) and at the mRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). During the first stage, the data indicated that 4 days of treatment with 1 ?M RA starting on day 4 of EB formation favored NC cell differentiation and that plating on gelatin-coated plates led to cell migration out of the EBs. The second-stage differentiation results showed that the CM, particularly the LEC-CM, enhanced the yield of polygonal cells with CEC-specific marker expression shown by ICC and RT-qPCR. This study demonstrates that mouse ESCs and iPSCs were induced and expressed CEC differentiation markers when subjected to a two-step inducement process, suggesting that they are a promising resource for corneal endothelium failure replacement therapy in the future.

SUBMITTER: Chen P

PROVIDER: S-EPMC4280952 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Treatment with retinoic acid and lens epithelial cell-conditioned medium <i>in vitro</i> directed the differentiation of pluripotent stem cells towards corneal endothelial cell-like cells.

Chen Ping P Chen Jun-Zhao JZ Shao Chun-Yi CY Li Chuan-Yin CY Zhang Yi-Dan YD Lu Wen-Juan WJ Fu Yao Y Gu Ping P Fan Xianqun X

Experimental and therapeutic medicine 20141203 2

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have extensive self-renewal capacity and the potential to differentiate into all tissue-specific cell lineages, including corneal endothelial cells (CECs). They are a promising prospect for the future of regenerative medicine. The method of derivation of CECs from ESCs and iPSCs, however, remains to be elucidated. In this study, mouse ESCs and iPSCs were induced to differentiate into CECs using CEC embryonic development event ...[more]

PMID: 25574197

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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.

Project description:Corneal endothelial dysfunction causes severe impairment of vision. The only solution is corneal transplantation. However, this treatment is hampered by a worldwide shortage of donor corneas. New therapies may replace the conventional donor corneal transplantation alongside the developments in regenerative medicine and tissue engineering, but sufficient functional corneal endothelial cells (CECs) are essential. The aim of this study was to promote the expansion and function of human corneal endothelial cells (HCECs) in vitro and in vivo.The phenotypes of human orbital adipose-derived stem cells (OASCs) were detected by flow cytometry and immunofluorescence. HCECs were isolated and cultured using a conditioned medium obtained from OASCs (OASC-CM) in vitro. Related cell markers of HCECs were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence. The cell counting kit-8 (CCK-8) assay and the wound healing assay were performed to evaluate the proliferation ability of the cells. The cultured HCECs were then transplanted into rabbit and monkey corneal endothelial dysfunction models by cell injection.CD29, CD105, CD49e, CD166, and vimentin were highly expressed in cultured human OASCs. The CEC-relative markers zonula occludens-1 (ZO-1), Na+/K+ ATPase, N-cadherin, Col8a2, and SLC4A4 were expressed in HCECs cultured by OASC-CM. The HCECs were able to maintain polygonal cell morphology and good proliferative capacity. In animal experiments, corneal transparency was achieved after the injection of HCECs, which demonstrated the good repair capacity of the cells.The proliferation abilities of the cells were significantly enhanced, and related functional markers were strongly positive, while HCEC morphology was maintained using OASC-CM. HCECs obtained some stem cell-like properties. This preclinical study confirmed the therapeutic ability of the HCECs in vivo. Our findings demonstrated that cultured HCECs with OASC-CM might be a promising source for research and clinical treatment.

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Treatment with retinoic acid and lens epithelial cell-conditioned medium in vitro directed the differentiation of pluripotent stem cells towards corneal endothelial cell-like cells.

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Treatment with retinoic acid and lens epithelial cell-conditioned medium <i>in vitro</i> directed the differentiation of pluripotent stem cells towards corneal endothelial cell-like cells.

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