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Bruce Ksander, Ekaterina Korobkina, Christoph Ganss, Andreas Kluth, Natasha Frank, Markus Hermann Frank; In vitro expanded human purified ABCB5-positive limbal stem cells for treatment of limbal stem cell deficiency.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2287.
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© ARVO (1962-2015); The Authors (2016-present)
Limbal stem cell deficiency (LSCD) is a potentially blinding condition that occurs when limbal stem cells (LSC) are lost due to disease or injury. We previously demonstrated that freshly isolated and purified human and mouse ABCB5+ LSC possessed long-term (13 months) epithelial regenerative capacity in a xenogeneic and autologous mouse model of induced LSCD. However, using freshly isolated LSC limits the number of cells available for transplantation due to the small percentage of LSC. Importantly, long-term clinical success of transplants in unilateral LSCD patients was shown by P. Rama et al. NEJM 2010 to depend on the number of LSC in the donor graft, creating the impetus to expand LSC in vitro. In this study we determined whether we could expand human purified ABCB5+ LSC that retained their regenerative capacity.
Human ABCB5+ LSC were purified from cadaveric donor rims via Ab-mediated magnetic beads and viability (DAPI exclusion), ABCB5 expression (flow cytometry), bead residues, and capacity to differentiate into KRT12+ cells was determined. A xenogeneic human to immunodeficient NSG mouse model of induced LSCD was used to determine corneal epithelial regenerative capacity.
LSC were expanded in cultures resulting in purification of ABCB5+ LSC that were >95% ABCB5+ by flow cytometry (mean 98.6% n=19 donors), 98.9% viable, and <0.05% remaining bead residues. Cultures were negative for endotoxin and mycoplasma. On average, 4.2 x 106 ABCB5+ LSC were expanded from n=45 donors. Purified KRT12 negative ABCB5+ LSC co-expressed ΔNp63α and could be induced to differentiate into KRT12+ epithelial cells. ABCB5+ LSC regenerated a clear epithelium in LSCD-induced NSG mice (0.8 ± 0.62 opacity score ± SD; p < 0.05 vs negative controls 2.5 ± 0.67 by t-test).
Human purified ABCB5+ LSC can be consistently and reproducibly recovered and expanded in vitro to generate large numbers of donor LSC with regenerative capacity, indicating the therapeutic potential of these cells.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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