Corneal regeneration using autologous ABCB5+ skin stem cells for bilateral limbal stem cell deficiency

Abstract

Purpose : Patients with bilateral LSCD (limbal stem cell deficiency) have no source of autologous LSCs (limbal stem cells) and therefore must use either allogeneic LSCs that are targets of immune rejection or an alternative source of autologous adult stem cells that possess the ability to transdifferentiate into Krt 12⁺ corneal epithelial cells. Previously, we demonstrated that the cell surface ABCB5 protein identifies LSCs and anti-ABCB5 antibodies can purify LSCs for transplantation. Since ABCB5 also identifies adult stem cells in the skin dermis, we now determined whether purified autologous ABCB5⁺ skin stem cells can regenerate the corneal epithelium as a treatment for bilateral LSCD.

Methods : ABCB5⁺ skin stem cells were recovered from human donor skin biopsies, expanded in vitro and purified by cell sorting with magnetic beads coated with anti-ABCB5 monoclonal antibodies. Purified cells were >90% ABCB5⁺ by FACS analysis. Immunofluorescent staining and FACS was used to identify cell surface markers. Donor ABCB5⁺ skin stem cells were placed into fibrin gels (500 ABCB5⁺ cells/graft) and transplanted onto recipient immunodeficient NSG mice with an induced LSCD, as described in our previous publication. These human to mouse xenografts were examined via slit lamp and H&E.

Results : The reticular skin dermis contains 1-4% ABCB5⁺ cells that are negative for markers of dendritic, endothelial, and hematopoietic cells (CD45^-, CD31^-, CD34^-) and positive for mesenchymal stem cell markers (CD29⁺, CD44⁺, CD49e⁺, CD73⁺, CD105⁺, CD166⁺). In vitro expanded ABCB5⁺ cells displayed no karyotypic abnormalities and retained a non-transformed phenotype. A small percentage (<30%) of in vitro expanded ABCB5⁺ cells expressed Pax6 and Krt12 by FACS analysis, which increased to 85% and 78%, respectively after culturing for 14 days in CntT-20 corneal differentiation medium. Donor transplants containing no cells (negative control) resulted in conjunctivalization, while ABCB5⁺ skin cell transplants produced clear avascular corneas with no evidence of keratinization at 8 weeks post transplantation.

Conclusions : ABCB5⁺ dermal skin stem cells transplanted to the ocular surface can transdifferentiate into Krt12⁺ corneal epithelial cells, indicating a new source of autologous stem cells for bilateral LSCD patients.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.