Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs)1,2,3, and LSC deficiency is a major cause of blindness worldwide4. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts5, a gene allowing for prospective LSC enrichment has not been identified so far5. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5)6,7 marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs2 in mice and p63α-positive LSCs8 in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

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Data deposits

The murine Abcb5 messenger RNA sequence has been deposited in GenBank under accession number JQ655148.


  1. 1.

    & Role of the pericorneal papillary structure in renewal of corneal epithelium. Nature 229, 560–561 (1971)

  2. 2.

    , , , & Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 57, 201–209 (1989)

  3. 3.

    , , , & Oligopotent stem cells are distributed throughout the mammalian ocular surface. Nature 456, 250–254 (2008)

  4. 4.

    , , & Stem cell differentiation and the effects of deficiency. Eye (Lond.) 17, 877–885 (2003)

  5. 5.

    et al. Limbal stem-cell therapy and long-term corneal regeneration. N. Engl. J. Med. 363, 147–155 (2010)

  6. 6.

    et al. Regulation of progenitor cell fusion by ABCB5 P-glycoprotein, a novel human ATP-binding cassette transporter. J. Biol. Chem. 278, 47156–47165 (2003)

  7. 7.

    et al. Identification of cells initiating human melanomas. Nature 451, 345–349 (2008)

  8. 8.

    et al. p63 identifies keratinocyte stem cells. Proc. Natl Acad. Sci. USA 98, 3156–3161 (2001)

  9. 9.

    et al. Side population cells from human melanoma tumors reveal diverse mechanisms for chemoresistance. J. Invest. Dermatol. 132, 2440–2450 (2012)

  10. 10.

    et al. ABCB5 identifies a therapy-refractory tumor cell population in colorectal cancer patients. Cancer Res. 71, 5307–5316 (2011)

  11. 11.

    et al. ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma. Cancer Res. 65, 4320–4333 (2005)

  12. 12.

    et al. Genetically determined ABCB5 functionality correlates with pigmentation phenotype and melanoma risk. Biochem. Biophys. Res. Commun. 436, 536–542 (2013)

  13. 13.

    & Biological principals and clinical potentials of limbal epithelial stem cells. Cell Tissue Res. 331, 135–143 (2008)

  14. 14.

    et al. MDR1-P-glycoprotein behaves as an oncofetal protein that promotes cell survival in gastric cancer cells. Lab. Invest. 92, 1407–1418 (2012)

  15. 15.

    , , , & Granulin-epithelin precursor and ATP-dependent binding cassette (ABC)B5 regulate liver cancer cell chemoresistance. Gastroenterology 140, 344–355 (2011)

  16. 16.

    , & A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13, 476–484 (2003)

  17. 17.

    et al. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nature Genet. 25, 139–140 (2000)

  18. 18.

    & Genetic manipulations reveal dynamic cell and gene functions: Cre-ating a new view of myogenesis. Cell Cycle 8, 3675–3678 (2009)

  19. 19.

    et al. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl Acad. Sci. USA 93, 5860–5865 (1996)

  20. 20.

    et al. VEGFR-1 expressed by malignant melanoma-initiating cells is required for tumor growth. Cancer Res. 71, 1474–1485 (2011)

  21. 21.

    et al. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J. Cell Biol. 145, 769–782 (1999)

  22. 22.

    et al. Preservation of the limbal stem cell phenotype by appropriate culture techniques. Invest. Ophthalmol. Vis. Sci. 51, 765–774 (2010)

  23. 23.

    et al. A rapid separation of two distinct populations of mouse corneal epithelial cells with limbal stem cell characteristics by centrifugation on percoll gradient. Invest. Ophthalmol. Vis. Sci. 49, 3903–3908 (2008)

  24. 24.

    , , , & Optical coherence tomography as a rapid, accurate, noncontact method of visualizing the palisades of Vogt. Invest. Ophthalmol. Vis. Sci. 53, 1381–1387 (2012)

  25. 25.

    et al. Keratin 12-deficient mice have fragile corneal epithelia. Invest. Ophthalmol. Vis. Sci. 37, 2572–2584 (1996)

  26. 26.

    , , , & BALB/c and C57BL6 mouse strains vary in their ability to heal corneal epithelial debridement wounds. Exp. Eye Res. 87, 478–486 (2008)

  27. 27.

    , & Examination of the restoration of epithelial barrier function following superficial keratectomy. Exp. Eye Res. 84, 32–38 (2007)

  28. 28.

    et al. Spontaneous bacterial keratitis in CD36 knockout mice. Invest. Ophthalmol. Vis. Sci. 52, 256–263 (2011)

  29. 29.

    et al. The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. Transplantation 68, 868–879 (1999)

  30. 30.

    et al. From hair to cornea: toward the therapeutic use of hair follicle-derived stem cells in the treatment of limbal stem cell deficiency. Stem Cells 29, 57–66 (2011)

  31. 31.

    , , , & In vivo cell tracking with video rate multimodality laser scanning microscopy. IEEE J. Sel. Top. Quant. Electron. 14, 10–18 (2008)

  32. 32.

    et al. Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy. Biomed. Opt. Express 3, 1972–1977 (2012)

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We thank R. Maas for critical reading of the manuscript. We also thank P. Mallen for assistance with graphic illustrations; the Heartland Lions Eye Bank for providing clinical specimens; G. Berg for assistance with mouse colony maintenance; D. Dombkowski, F. Preffer and R. Huang for their assistance with cell sorting and viability studies. This work was supported by National Institutes of Health (NIH)/National Institutes of Neurological Disorders and Stroke grant K08NS051349, VA BLR&D 1I01BX000516 and VA RR&D 1I01RX000989 Merit Review Awards, and a Harvard Stem Cell Institute grant to N.Y.F., NIH/National Cancer Institute grants R01CA113796, R01CA158467 and R01CA138231 to M.H.F., Department of Defense grant PR0332453 to B.R.K., NIH R01EY018624 and P30EY014801 grants to V.L.P., NIH grant R01EY021768 to W.W.K., NIH New Innovator Award DP2OD007483 and a Corley Research Foundation grant to B.A.T., NIH R01CA138231 to G.F.M., Western Pennsylvania Medical Eye Bank Core Grant for Vision Research (EY08098) to K.L.L., and NIH grants R01 EB017274, U01HL100402 and P41EB015903 to C.P.L. F.C.G. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation.

Author information

Author notes

    • Bruce R. Ksander
    • , Paraskevi E. Kolovou
    • , Markus H. Frank
    •  & Natasha Y. Frank

    These authors contributed equally to this work.


  1. Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts 02114, USA

    • Bruce R. Ksander
    • , Paraskevi E. Kolovou
    • , Sean P. McGuire
    • , Meredith S. Gregory
    • , William J. B. Vincent
    •  & James D. Zieske
  2. Transplant Research Program, Division of Nephrology, Boston Children’s Hospital, Boston, Massachusetts 02115, USA

    • Brian J. Wilson
    • , Karim R. Saab
    • , Qin Guo
    • , Jie Ma
    • , Markus H. Frank
    •  & Natasha Y. Frank
  3. Department of Dermatology, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA

    • Brian J. Wilson
    • , Karim R. Saab
    • , Qin Guo
    • , Jie Ma
    •  & Markus H. Frank
  4. Department of Medicine, VA Boston Healthcare System, Boston, Massachusetts 02130, USA

    • Brian J. Wilson
    • , Qin Guo
    •  & Natasha Y. Frank
  5. Bascom Palmer Eye Institute and the Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA

    • Victor L. Perez
    •  & Fernando Cruz-Guilloty
  6. Department of Ophthalmology, University of Cincinnati Medical Center, Cincinnati, Ohio 45229, USA

    • Winston W. Y. Kao
    •  & Mindy K. Call
  7. Stephen A Wynn Institute for Vision Research, Carver College of Medicine, Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa 52242, USA

    • Budd A. Tucker
  8. Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA

    • Qian Zhan
    •  & George F. Murphy
  9. Department of Ophthalmology, University of Pittsburgh School of Medicine & Department of Bioengineering, University of Pittsburgh Swanson School of Engineering, Pittsburgh, Pennsylvania 15213, USA

    • Kira L. Lathrop
  10. Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA

    • Clemens Alt
    • , Luke J. Mortensen
    •  & Charles P. Lin
  11. Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts 02138, USA

    • Markus H. Frank
    •  & Natasha Y. Frank
  12. Division of Genetics, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA

    • Natasha Y. Frank


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N.Y.F., M.H.F. and B.R.K. designed the study. P.E.K., B.R.K., N.Y.F., M.H.F., B.J.W., K.R.S., Q.G., J.M., S.P.M., M.S.G., W.J.B.V., Q.Z., K.L.L., C.P.L., C.A. and L.J.M. performed experiments. V.L.P., F.C.-G. and B.A.T. provided reagents and specimens. W.W.Y.K. and M.K.C. provided technical assistance. P.E.K., B.J.W., Q.G., J.M., S.P.M., M.S.G., W.J.B.V., J.D.Z., G.F.M., B.R.K., M.H.F. and N.Y.F. analysed the data. N.Y.F., M.H.F. and B.R.K. wrote the manuscript. M.H.F. and N.Y.F. are co-senior investigators.

Competing interests

M.H.F. is co-inventor of the ABCB5-related US patent 6,846,883 (Gene encoding a multidrug resistance human P-glycoprotein homologue on chromosome 7p15-21 and uses thereof) assigned to Brigham and Women’s Hospital, Boston, Massachusetts, and licensed to Ticeba GmbH (Heidelberg, Germany) and Rheacell GmbH & Co. KG (Heidelberg, Germany). M.H.F. serves as scientific advisor to Ticeba GmbH and Rheacell GmbH & Co. KG.

Corresponding authors

Correspondence to Markus H. Frank or Natasha Y. Frank.

Extended data

Supplementary information


  1. 1.

    Limbal epithelial cell Abcb5/BrdU co-expression.

    Immunofluorescent staining of limbal epithelial cells for Abcb5 (red) and BrdU (green) co-expression counterstained with DAPI (blue), analyzed by confocal microscopy in the limbal epithelium of mouse corneal whole mounts. This supplementary video consists of sequential confocal images depicting a cluster of ABCB5(+)/BrdU(+) label-retaining cells surrounded by double-negative ABCB5(-)/BrdU(-) cells and relatively rare, single-positive ABCB5(+)/BrdU(-) cells. Magnification: 60x.

  2. 2.

    Identification of the limbal epithelium.

    Identification of the limbal epithelium within corneal whole mounts via identification of posteriorly localized limbal vessels within the underlying stroma. Cell nuclei are counterstained with DAPI (blue) and sequential confocal images are displayed beginning from the apical corneal epithelial layer and moving down into the stroma of the cornea and limbus. Appearance of the limbal vessels in the stroma is used to identify the overlaying limbal epithelial layer. This technique was used for immunostaining analyses. Magnification: 20x.

  3. 3.

    ABCB5(+) cells within palisades of Vogt.

    Sections through the palisades of Vogt in the anterior limbus were stained with anti-ABCB5 mAb (red) with nuclear counterstaining (blue), and analyzed by confocal microscopy. This supplementary video consists of sequential confocal images depicting the location of ABCB5(+) cells within palisades. Magnification: 20x.

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