Regulation of functional corneal endothelial cells isolated from sphere colonies by Rho-associated protein kinase inhibitor

Bi,Yan-Long; Zhou,Qi; Du,Fei; Wu,Ming-Feng; Xu,Guo-Tong; Sui,Gui-Qin

doi:10.3892/etm.2012.816

February 2013 Volume 5 Issue 2

Full Size Image

Journals

International Journal of Molecular Medicine

International Journal of Molecular Medicine is an international journal devoted to molecular mechanisms of human disease.

International Journal of Oncology

International Journal of Oncology is an international journal devoted to oncology research and cancer treatment.

Molecular Medicine Reports

Covers molecular medicine topics such as pharmacology, pathology, genetics, neuroscience, infectious diseases, molecular cardiology, and molecular surgery.

Oncology Reports

Oncology Reports is an international journal devoted to fundamental and applied research in Oncology.

Experimental and Therapeutic Medicine

Experimental and Therapeutic Medicine is an international journal devoted to laboratory and clinical medicine.

Oncology Letters

Oncology Letters is an international journal devoted to Experimental and Clinical Oncology.

Biomedical Reports

Explores a wide range of biological and medical fields, including pharmacology, genetics, microbiology, neuroscience, and molecular cardiology.

Molecular and Clinical Oncology

International journal addressing all aspects of oncology research, from tumorigenesis and oncogenes to chemotherapy and metastasis.

World Academy of Sciences Journal

Multidisciplinary open-access journal spanning biochemistry, genetics, neuroscience, environmental health, and synthetic biology.

International Journal of Functional Nutrition

Open-access journal combining biochemistry, pharmacology, immunology, and genetics to advance health through functional nutrition.

International Journal of Epigenetics

Publishes open-access research on using epigenetics to advance understanding and treatment of human disease.

Medicine International

An International Open Access Journal Devoted to General Medicine.

February 2013 Volume 5 Issue 2

Full Size Image

Article

Regulation of functional corneal endothelial cells isolated from sphere colonies by Rho-associated protein kinase inhibitor

Authors:
- Yan-Long Bi
- Qi Zhou
- Fei Du
- Ming-Feng Wu
- Guo-Tong Xu
- Gui-Qin Sui
View Affiliations / Copyright

Affiliations: Department of Ophthalmology, Tongji Hospital affiliated with Tongji University School of Medicine, Shanghai 200065, P.R. China, Department of Regenerative Medicine and Stem Cell Research Center, Tongji University School of Medicine, Shanghai 200065, P.R. China, Department of Ophthalmology, Jilin University Bethune Second Hospital, Changchun 130041, P.R. China
Pages: 433-437
|
Published online on: November 19, 2012

https://doi.org/10.3892/etm.2012.816
Expand metrics +

Abstract

Human corneal endothelial cells (HCECs) form a monolayer covering the inner side of the cornea. Since HCECs have lower mitotic activity after birth, damage or disease of HCECs will lead to failure of cellular pump functions and inevitably to corneal stroma oedema and loss of vision. Ex vivo cultured HCEC transplantation is the most promising therapy used to avoid donor tissue shortages. However, proliferation of functional adult HCECs is difficult to achieve using standard cell culture techniques. In this study, we isolated sphere colonies from HCECs, which were characterised as HCEC precursor cells. HCECs from these spheres were incubated with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 (30 µM for 1 h at 37˚C) to observe cell pump functions and the levels of several cell markers. Cells from the spheres were immunostained positive for nestin (a marker of immature cells) and for an immature neuronal marker (β3-tubulin). There were no significant differences in the expression of genes associated with cell adhesion or ion transport channels, between the cells with or without Y-27632. However, immunostaining revealed a higher density of Na+/K+-ATPase in cell nuclei from HCECs (sphere-forming) and HCECs (sphere-forming/Y-27632) than normal HCECs. There was a higher resting potential difference and resting short circuit in the HCEC (sphere-forming) and HCEC (sphere-forming/Y-27632) cell lines, compared with normal HCECs. In this study, we successfully cultured functional HCEC cell lines, which possessed several important characteristics required to maintain the transparency and refractive parameters of the human cornea. These included hexagonal cell morphology, higher cell adhesion and proliferation ability, HCEC pump function and positive expression of several cell adhesion markers.

View Figures

View References

1.	Abib FC, Holzchuh R, Schaefer A, Schaefer T and Godois R: The endothelial sample size analysis in corneal specular microscopy clinical examinations. Cornea. 31:546–550. 2012. View Article : Google Scholar : PubMed/NCBI
2.	Koizumi N, Okumura N and Kinoshita S: Development of new therapeutic modalities for corneal endothelial disease focused on the proliferation of corneal endothelial cells using animal models. Exp Eye Res. 95:60–67. 2012. View Article : Google Scholar
3.	Patel SV: Graft survival and endothelial outcomes in the new era of endothelial keratoplasty. Exp Eye Res. 95:40–47. 2012. View Article : Google Scholar : PubMed/NCBI
4.	Peh GS, Beuerman RW, Colman A, Tan DT and Mehta JS: Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview. Transplantation. 91:811–819. 2011. View Article : Google Scholar : PubMed/NCBI
5.	Gao Y, Zhou Q, Qu M, Yang L, Wang Y and Shi W: In vitro culture of human fetal corneal endothelial cells. Graefes Arch Clin Exp Ophthalmol. 249:663–669. 2011. View Article : Google Scholar
6.	Jäckel T, Knels L, Valtink M, Funk RH and Engelmann K: Serum-free corneal organ culture medium (SFM) but not conventional minimal essential organ culture medium (MEM) protects human corneal endothelial cells from apoptotic and necrotic cell death. Br J Ophthalmol. 95:123–130. 2011.
7.	Hitani K, Yokoo S, Honda N, Usui T, Yamagami S and Amano S: Transplantation of a sheet of human corneal endothelial cell in a rabbit model. Mol Vis. 14:1–9. 2008.PubMed/NCBI
8.	Park TY, Park MH, Shin WC, et al: Anti-metastatic potential of ginsenoside Rp1, a novel ginsenoside derivative. Biol Pharm Bull. 31:1802–1805. 2008. View Article : Google Scholar : PubMed/NCBI
9.	Mimura T, Yamagami S, Yokoo S, et al: Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model. Invest Ophthalmol Vis Sci. 45:2992–2997. 2004. View Article : Google Scholar : PubMed/NCBI
10.	Peh GS, Toh KP, Wu FY, Tan DT and Mehta JS: Cultivation of human corneal endothelial cells isolated from paired donor corneas. PLoS One. 6:e283102011. View Article : Google Scholar : PubMed/NCBI
11.	Hatou S, Yamada M, Akune Y, et al: Role of insulin in regulation of Na⁺-/K⁺-dependent ATPase activity and pump function in corneal endothelial cells. Invest Ophthalmol Vis Sci. 51:3935–3942. 2010.PubMed/NCBI
12.	Gallo RM, Khan MA, Shi J, et al: Regulation of the actin cytoskeleton by rho kinase controls antigen presentation by CD1d. J Immunol. July 13–2012.(Epub ahead of print).
13.	Matsuoka T, Yashiro M, Kato Y, Shinto O, Kashiwagi S and Hirakawa K: RhoA/ROCK signaling mediates plasticity of scirrhous gastric carcinoma motility. Clin Exp Metastasis. 28:627–636. 2011. View Article : Google Scholar : PubMed/NCBI
14.	Joyce NC and Harris DL: Decreasing expression of the G1-phase inhibitors, p21Cip1 and p16INK4a, promotes division of corneal endothelial cells from older donors. Mol Vis. 16:897–906. 2010.PubMed/NCBI
15.	Yokoo S, Yamagami S, Yanagi Y, et al: Human corneal endothelial cell precursors isolated by sphere-forming assay. Invest Ophthalmol Vis Sci. 46:1626–1631. 2005. View Article : Google Scholar : PubMed/NCBI

Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Regulation of functional corneal endothelial cells isolated from sphere colonies by Rho-associated protein kinase inhibitor

This article is mentioned in:

Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Related Articles