In vitro transdifferentiation of human skin keratinocytes to corneal epithelial cells

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• 作者：Vidya Gopakumar¹ ; ² ; ³ ; Nivedita Chatterjee¹ ; ² ; Sowmya Parameswaran¹ ; Subramanian Nirmala⁴ ; Subramanian Krishnakumar¹ ; ² ; ^{drkrishnakumar_2000@yahoo.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：bilateral limbal stem cell deficiency ; corneal epithelial cells ; limbal fibroblast conditioned medium ; skin keratinocytes ; transdifferentiation
• 刊名：Cytotherapy
• 出版年：2016
• 出版时间：May 2016
• 年：2016
• 卷：18
• 期：5
• 页码：673-685
• 全文大小：3531 K

文摘

Skin keratinocytes (SKs) share the same surface ectodermal origin as that of corneal epithelium. In this study, the plasticity of epidermal keratinocytes was exploited to generate corneal epithelial–like cells, which might serve as an alternative source of autologous tissue for the treatment of bilateral limbal stem cell deficiency.

Methods

Skin samples were subjected to collagenase digestion to isolate SKs and transdifferentiated to corneal epithelial–like cells using limbal fibroblast conditioned medium (LFCM). SKs and transdifferentiated corneal epithelial cells (TDCECs) were characterized using immunofluorescence and fluorescence-activated cell sorting. The propensity for expression of angiogenic genes in TDCECs was compared with cultured oral mucosal epithelial cells (COMEC) in vitro. RT² quantitative polymerase chain reaction profiler array was performed to study the signaling pathways involved in the transdifferentiation process.

Results

The TDCECs obtained from SKs showed corneal epithelial–like morphology and expressed corneal epithelial markers, CK3 and CK12. Hematoxylin-eosin and immunohistochemistry showed stratified layers of TDCECs expressing CK 3/12, confirming the corneal epithelial phenotype. We found that the expression of several angiogenic and epithelial mesenchymal transition factors were down-regulated in TDCECs compared with COMEC, suggesting a lower capacity to induce angiogenesis in TDCECs. There was considerable difference in the signaling mechanisms between TDCECs and SKs on testing by RT² profiler array, signifying differences at the global gene profile. The comparison of TDCECs and limbal derived corneal epithelial cells showed similar gene expression.

Discussion

Our study shows that SKs have the potential to transdifferentiate into corneal epithelial–like cells using LFCM.