RESUMEN

PURPOSE: Corneal limbal stem cell (LSC) transplantation has been reported as a potential approach to treat the damaged corneal epithelium. Scaffolds such as human amniotic membrane (hAM) are commonly employed for the in vitro culture and as a carrier during in vivo transplantation. However, they carry the risk of biological contamination and donor to donor variability. To overcome these disadvantages, we herein report the capabilities of a synthetic thermoreversible gelation polymer (TGP) scaffold to serve as an encapsulation support during LSC transplantation and to enable engraftment for corneal regeneration. METHODS: Sixteen discarded human corneas were used to isolate the corneal epithelium which was cultured in TGP and hAM. The cell proliferation and characteristics between TGP and hAM culture methods were evaluated by microscopic observation, 3H Thymidine incorporation assay, immunoperoxidase and immunofluorescence staining. RESULTS: The 3H Thymidine assay's results showed that TGP allowed human-donor cornea-derived LSCs to proliferate well in vitro, compared to hAM and the cells encapsulated in TGP and transplanted ex vivo onto a human cadaver donor cornea denuded of its epithelium, migrated on the ocular surface, and proliferated to form a continuous layer in 25 days. Immunoperoxidase and Immunofluorescence staining of TGP-cultured cells were positive for LSC markers (p63, ABCG2, Connexin 43 and Integrin ß), proving that the TGP helps to preserve the limbal cells' stemness. CONCLUSION: TGP is found to be a multipurpose scaffold for (i) in vitro culture, (ii) ex vivo encapsulation, and in vivo transplantation (iii), enabling engraftment of LSCs in this study, with potentials to extend its application in cell-based therapies in several regenerative medicine approaches.

Asunto(s)

Trasplante de Córnea , Epitelio Corneal , Limbo de la Córnea , Humanos , Córnea , Epitelio Corneal/metabolismo , Células Cultivadas , Proliferación Celular

RESUMEN

Objeetive To explore the influence of extracellular high glucose on the proliferation,migration and biomarkers of corneal limbal stem cells.Methods Establishment of a model of high glucose in cultured human limbal stem cells to observe and investigate the effects of extracellular high glucose on the proliferation and migration of corneal limbal stem cells by immunoflurescence,CCK-8 and Transwell assay,respectively.Totally 16 SPF rats were collected and induced diabetic model by streptozotocin as the high-glucose group,and the normal rats of the same age served as the control group.Corneal epidermises of rats in both groups were scraped to observe the repair of corneal epithelium.And the corneas were treated with HE staining and immunohistochemical staining to detect the expression of biomarkers of corneal limbal stem cells and the modality changes of cells.Results The proliferation rate of human limbal epithelial cells was significantly decreased when exposed to high glucose,and the rate at 24 h,48 h and 72 h was 0.728,0.345 and 0.395,respectively,which was markedly lower than that in the control group,with a significant difference (P ＜ 0.05);meanwhile the cell migration rate of the high-glucose group was 17.6％ at 48 h,which was significantly slower than that of the control group (100％).And the inhibition was accompanied by the decreased expression of β-catenin and vimentin.Furthermore,the expression levels of β-catenin and vimentin mRNA and protein were down-regulated,with abnormal location,in the high-glucose group.And diabetic rats had poor corneal epithelial healing.The epithelial layer became thinner and the structures were disorganized in diabetic rats through HE staining.The immunohistochemical assay revealed the expression of β-catenin and vimentin of cornea limbal stem cells was down-regulated in high-glocose group when compared with the control group.Conclusion High glucose can significantly inhibit the proliferation and migration of cornea limbal stem cells,and its main damage mechanism is correlated with the abnormalities of β-catenin and vimentin.