Abstract

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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Accessions

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

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

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Acknowledgements

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.

Affiliations

  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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Contributions

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

Videos

  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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DOI

https://doi.org/10.1038/nature13426

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