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