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Characterization of the iPSC-derived conditioned medium that promotes the growth of bovine corneal endothelial cells.

ABSTRACT: Corneal endothelial cells (CECs) maintain corneal transparency and visual acuity. However, the limited proliferative capability of these cells in vitro has prompted researchers to find efficient culturing techniques for them. The aim of our study was to evaluate the use of conditioned medium (CM) obtained from induced pluripotent stem cells (iPSCs) as a source for the effective proliferation of bovine CECs (B-CECs). In our study, the proliferative ability of B-CECs was moderately enhanced when the cells were grown in 25% iPSC conditioned medium (iPSC-CM). Additionally, hexagonal cell morphology was maintained until passage 4, as opposed to the irregular and enlarged shape observed in control corneal endothelial medium (CEM). B-CECs in both the 25% iPSC-CM and CEM groups expressed and Na⁺-K⁺-ATPase. The gene expression levels of NIFK, Na⁺-K⁺-ATPase, Col4A and Col8A and the percentage of cells entering S and G2 phases were higher in the iPSC-CM group. The number of apoptotic cells also decreased in the iPSC-CM group. In comparison to the control cultures, iPSC-CM facilitated cell migration, and these cells showed better barrier functions after several passages. The mechanism of cell proliferation mediated by iPSC-CM was also investigated, and phosphorylation of Akt was observed in B-CECs after exposure to iPSC-CM and showed sustained phosphorylation induced for up to 180 min in iPSC-CM. Our findings indicate that iPSC-CM may employ PI3-kinase signaling in regulating cell cycle progression, which can lead to enhanced cellular proliferation. Effective component analysis of the CM showed that in the iPSC-CM group, the expression of activin-A was significantly increased. If activin-A is added as a supplement, it could help to maintain the morphology of the cells, similar to that of CM. Hence, we conclude that activin-A is one of the effective components of CM in promoting cell proliferation and maintaining cell morphology.

SUBMITTER: Liu Q

PROVIDER: S-EPMC6474332 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Characterization of the iPSC-derived conditioned medium that promotes the growth of bovine corneal endothelial cells.

Liu Qing Q Guo Yonglong Y Liu Shiwei S Wang Peiyuan P Xue Yunxia Y Cui Zekai Z Chen Jiansu J

PeerJ 20190416

Corneal endothelial cells (CECs) maintain corneal transparency and visual acuity. However, the limited proliferative capability of these cells in vitro has prompted researchers to find efficient culturing techniques for them. The aim of our study was to evaluate the use of conditioned medium (CM) obtained from induced pluripotent stem cells (iPSCs) as a source for the effective proliferation of bovine CECs (B-CECs). In our study, the proliferative ability of B-CECs was moderately enhanced when t ...[more]

PMID: 31024764

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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.

Project description:Purpose:Corneal endothelial cells (CECs) are critical in maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived CECs. Methods:We isolated PBMCs and programmed the mononuclear cells to generate iPSCs, which were differentiated to CECs through the neural crest cells (NCCs). The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent and corneal endothelium (CE)-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSC-derived CECs and human corneal endothelium (hCE) was examined by mass spectrometry-based proteome sequencing. Results:The PBMC-originated, iPSC-derived CECs were tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CE-associated markers expressed at significantly higher levels in iPSC-derived CECs at days 13, 20, and 30 compared with their respective levels in iPSCs. It is of importance that only residual expression levels of pluripotency markers were detected in iPSC-derived CECs. Cryopreservation of iPSC-derived CECs did not affect the tight adherence of CECs and their hexagonal-like shape while expressing high levels of CE-associated markers. Mass spectrometry-based proteome sequencing identified 10,575 proteins in the iPSC-derived CEC proteome. In parallel, we completed proteome profiling of the hCE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSC-derived CECs, suggesting that 90.82% of the hCE proteome overlaps with the iPSC-derived CEC proteome. Conclusions:We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the hCE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSC-derived CECs.

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Characterization of the iPSC-derived conditioned medium that promotes the growth of bovine corneal endothelial cells.

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Characterization of the iPSC-derived conditioned medium that promotes the growth of bovine corneal endothelial cells.

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