title: Long-term cultivation of human corneal endothelial cells by telomerase expression.
abstract: The objective of this study was to explore the potential role of human telomerase reverse transcriptase (TERT) in extending the proliferative lifespan of human corneal endothelial cells (HCECs) under long-term cultivation. A primary culture was initiated with a pure population of HCECs in DMEM/F12 media containing 10% fetal bovine serum and other various supplements. TERT gene was successfully transfected into normal HCECs. A stable HCECs cell line (TERT-HCECs) that expressed TERT was established. The cells could be subcultured for 36 passages. Within this line of cells, TERT not only extended proliferative lifespan and inhibited apoptosis but also enhanced the cell line remaining the normal characteristics similar to HCECs. There were no significantly differences in the expression of the pump function related proteins voltage dependent anion channel 3 (VDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), chloride channel protein 3 (CLCN3), Na(+)/K(+)-ATPase α1, and ZO-1 in the cell line TERT-HCECs and primary HCECs. TERT-HCECs formed a monolayer cell sheet, maintained similar cell junction formation and pump function with primary HCECs. Karyotype analysis exhibited normal chromosomal numbers. The soft agar colony assay and tumor formation in nude mice assay showed no malignant alterations in TERT-HCECs. Our findings indicated that we had established a cell line with its similar phenotype and properties to primary HCECs. Further study of the TERT-HCECs may be valuable in studying the function of the cells in vivo.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Zhuang
authors.forename: Chaoyang
authors.initials: C
authors.lastname: Li
authors.forename: Pengxia
authors.initials: P
authors.lastname: Wan
authors.forename: Naiyang
authors.initials: N
authors.lastname: Li
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhou
authors.forename: Chenjing
authors.initials: C
authors.lastname: Zhou
authors.forename: Zheqian
authors.initials: Z
authors.lastname: Huang
authors.forename: Zhichong
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000293:Adolescent; D000368:Aged; D000375:Aging / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002453:Cell Cycle; D049109:Cell Proliferation; D002478:Cells, Cultured; D002648:Child; D018118:Chloride Channels / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D007231:Infant, Newborn; D007621:Karyotyping; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D061251:Primary Cell Culture; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D019098:Telomerase / Q000235:genetics*; D014019:Tissue Donors; D014162:Transfection; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
chemical_list: D018118:Chloride Channels; C115376:ClC-3 channel; D017931:DNA Primers; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D010750:Phosphoproteins; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; C494768:VDAC3 protein, human; D050994:Voltage-Dependent Anion Channels; D062826:Zonula Occludens-1 Protein; C509186:TERT protein, human; D019098:Telomerase; C499076:ATP1A1 protein, human; D000254:Sodium-Potassium-Exchanging ATPase