Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs)1–3, 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.
Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species.
Lens regeneration in adult newts is a classic example of faithfully regenerating an entire organ via transdifferentiation1-6. After lentectomy, intriguing regulation allows the pigment epithelial cells (PECs) of the dorsal iris, but not the ventral, to dedifferentiate and then differentiate to form a new lens. This regulation might provide clues to the lack of lens regeneration in higher vertebrates. Six-3 and pax-6 known for their ability to induce ectopic lenses during embryogenesis7,8 and members of the BMP pathway, which are regulators of the dorsal/ventral axis establishment in embryos9 were examined for their role in induction of lens regeneration. Here we show that lens regeneration from the ventral iris is possible by inhibiting the BMP pathway or by transfecting ventral iris cells with six-3 and concomitant treatment with retinoic acid. In intact irises six-3 is expressed higher in the ventral iris. During regeneration, however, only levels in the dorsal iris are significantly increased. Such an increase is seen in ventral irises only when they are induced to transdifferentiate by six-3/RA or BMP inhibitors. Therefore, transcriptional regulation associated with competency for lens regeneration, aims to increase levels over established thresholds and not to merely render a regulatory gene as dorsal-specific. Lack of induction in the axolotl, a salamander incapable of lens regeneration seems to be associated with repression of six-3 expression.
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