In vitro transdifferentiation of human skin keratinocytes to corneal epithelial cells

Vidya Gopakumar; Nivedita Chatterjee; Sowmya Parameswaran; Subramanian Nirmala; Subramanian Krishnakumar

doi:10.1016/j.jcyt.2016.02.005

In vitro transdifferentiation of human skin keratinocytes to corneal epithelial cells

Cytotherapy. 2016 May;18(5):673-85. doi: 10.1016/j.jcyt.2016.02.005.

Authors

Vidya Gopakumar¹, Nivedita Chatterjee², Sowmya Parameswaran³, Subramanian Nirmala⁴, Subramanian Krishnakumar⁵

Affiliations

¹ Radheshyam Kanoi Stem Cell Laboratory, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, India; Larsen & Toubro Department of Ocular Pathology, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, India; CeNTAB, School of Chemical and Biotechnology, SASTRA University, Tanjore, India.
² Radheshyam Kanoi Stem Cell Laboratory, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, India; Larsen & Toubro Department of Ocular Pathology, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
³ Radheshyam Kanoi Stem Cell Laboratory, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, India.
⁴ Department of Oculoplasty, Medical Research Foundation, Sankara Nethralaya, Chennai, India.
⁵ Radheshyam Kanoi Stem Cell Laboratory, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, India; Larsen & Toubro Department of Ocular Pathology, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, India. Electronic address: drkrishnakumar_2000@yahoo.com.

PMID: 27059204
DOI: 10.1016/j.jcyt.2016.02.005

Abstract

Background aims: 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. RT(2) 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 RT(2) 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.

Keywords: bilateral limbal stem cell deficiency; corneal epithelial cells; limbal fibroblast conditioned medium; skin keratinocytes; transdifferentiation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Proliferation
Cell Transdifferentiation / physiology*
Cells, Cultured
Culture Media, Conditioned / pharmacology*
Epithelial Cells / cytology*
Epithelium, Corneal / cytology*
Fibroblasts / metabolism
Flow Cytometry
Humans
Keratinocytes / cytology*
Middle Aged
Signal Transduction
Skin / cytology

Substances

Culture Media, Conditioned