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Abstract

The surface of the cornea consists of a unique type of non-keratinized epithelial cells arranged in an orderly fashion, and this is essential for vision by maintaining transparency for light transmission. Cornea epithelial cells (CECs) undergo continuous renewal from limbal stem or progenitor cells (LSCs)1,2, and deficiency in LSCs or corneal epithelium—which turns cornea into a non-transparent, keratinized skin-like epithelium—causes corneal surface disease that leads to blindness in millions of people worldwide3. How LSCs are maintained and differentiated into corneal epithelium in healthy individuals and which key molecular events are defective in patients have been largely unknown. Here we report establishment of an in vitro feeder-cell-free LSC expansion and three-dimensional corneal differentiation protocol in which we found that the transcription factors p63 (tumour protein 63) and PAX6 (paired box protein PAX6) act together to specify LSCs, and WNT7A controls corneal epithelium differentiation through PAX6. Loss of WNT7A or PAX6 induces LSCs into skin-like epithelium, a critical defect tightly linked to common human corneal diseases. Notably, transduction of PAX6 in skin epithelial stem cells is sufficient to convert them to LSC-like cells, and upon transplantation onto eyes in a rabbit corneal injury model, these reprogrammed cells are able to replenish CECs and repair damaged corneal surface. These findings suggest a central role of the WNT7A–PAX6 axis in corneal epithelial cell fate determination, and point to a new strategy for treating corneal surface diseases.

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Gene Expression Omnibus

Data deposits

Microarray and RNA sequence information has been submitted to the Gene Expression Omnibus database under accession number GSE32145 and GSE54322.

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Acknowledgements

This study is supported in part by the 973 program (2013CB967504 and 2014CB964900), Project of Fundamental Research Funds (no.2012KF03), State Key laboratory of Ophthalmology, NIH (GM049369), KACST-UCSD Center of Excellence in Nanomedicine, NIH Director’s Transformative RO1 Program (R01 EY021374) and CIRM.

Author information

Author notes

    • Xiaohui Zhang
    •  & Yang Yang

    Present addresses: Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing 100730, China (X.Z.); Department of Ophthalmology, Shengjing Hospital of China Medical University, Shenyang 110004, China (Y.Y.).

Affiliations

  1. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China

    • Hong Ouyang
    • , Ying Lin
    • , Lei Xi
    • , Yizhi Liu
    •  & Kang Zhang
  2. Department of Ophthalmology, and Biomaterial and Tissue Engineering Center of Institute of Engineering in Medicine, University of California San Diego, La Jolla, California 92093, USA

    • Hong Ouyang
    • , Ying Lin
    • , Xiaohui Zhang
    • , Lei Xi
    • , Sherrina Patel
    • , Jing Luo
    • , Yang Yang
    • , Emily Yeh
    • , Jonathan Lin
    • , Michelle Pei
    • , Jin Zhu
    • , Liangfang Zhang
    • , Shaochen Chen
    • , Xiang-Dong Fu
    •  & Kang Zhang
  3. Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA

    • Yuanchao Xue
    • , Hairi Li
    •  & Xiang-Dong Fu
  4. Molecular Medicine Research Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Sichuan 610041, China

    • Huimin Cai
    • , Meixia Zhang
    • , Ming Zhang
    • , Gen Li
    • , Wei Jiang
    • , Guiqun Cao
    •  & Kang Zhang
  5. Guangzhou KangRui Biological Pharmaceutical Technology Company Ltd., Guangzhou 510005, China

    • Huimin Cai
  6. Department of Nanoengineering, University of California San Diego, La Jolla, California 92093, USA

    • Liangfang Zhang
    •  & Shaochen Chen
  7. Department of Medicine, University of California San Diego, La Jolla, California 92093, USA

    • Benjamin Yu
  8. Institute for Genomic Medicine, University of California San Diego, La Jolla, California 92093, USA

    • Benjamin Yu
    • , Xiang-Dong Fu
    •  & Kang Zhang
  9. Veterans Administration Healthcare System, San Diego, California 92093, USA

    • Kang Zhang

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Contributions

H.O., X.-D.F., Yiz.L., and K.Z. designed study, interpreted data and wrote the manuscript. H.O., Y.X., Yin.L., X.Z., L.X., H.C., J.L., Mei.Z., Min.Z., Y.Y., H.L., G.L., E.Y., G.C., J.Z. and B.Y. performed the experiments. Y.L., W.J., J.L. and Yiz.L. obtained human samples. S.C., S.P., M.P. and L.Z. contributed to data analysis and interpretation.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Xiang-Dong Fu or Yizhi Liu or Kang Zhang.

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DOI

https://doi.org/10.1038/nature13465

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