Correction of the Skin Defect in Murine Recessive Dystrophic Epidermolysis Bullosa by Bone Marrow Derived SLAM Family Receptor Enriched Cells.
Authors: Tolar Jakub , McElmurry Ron T. , Bazzi Hisham , Xia Lily , Ishida-Yamamoto Akemi , Christiano Angela M. , Blazar Bruce R. 
Published: October 2019

Abstract
Dystrophic epidermolysis bullosa (EB) is a disorder of incurable skin fragility, blistering and perturbations in anchoring fibrils caused by mutations in the type VII collagen gene (COL7A1). Since bone marrow (BM) includes cells with capacity to differentiate into diverse cell types and can seed various organs, including skin, we hypothesized that if type VII collagen producing cells are present in BM, BM infusions could be a beneficial therapy for EB. To test this hypothesis this in vivo, we assessed multiple candidate populations for cellular therapy of murine recessive dystrophic EB (RDEB, col7a1 −/−). As the col7a1 −/− newborn mice develop blisters and do not survive after two weeks of life, we can readily identify the mutant pups and the lethality provided a robust readout for the cellular interventions. The following cell populations were tested: epidermal stem cells and transit amplifying cells (from epidermis); total BM; BM derived multipotent adult progenitor cells and mesenchymal stem cells; fetal liver cells; and lineage positive and lineage negative BM fractions. None of these cell populations, however, provided any correction in col7a1 −/− recipients (N=223), even when the infusion regimen was varied by cell dose, age of donor, in utero and postnatal age at the time of infusion, and intravenous versus intrahepatic infusion. In a search for alternatives, we used CD150+ and CD48− BM cells, since signaling molecule SLAM family receptor positive populations have been shown to have significant pluripotentiality. We used several doses and infused them early postnatally into unconditioned col7a1 −/− mutant animals. One of these conditions, namely CD150+/CD48− BM population from C57Bl/6 GFP transgenic mice at a dose of 8 million cells per animal administered on day 3 or 4 after birth intravenously, resulted in survival of 3 out of 13 (23%) animals in two independent experiments. One of them was harvested at postnatal day 55; two of them were electively harvested at postnatal day 70. The animals were confirmed to be mutants by genotyping using PCR. The weights of these three animals were lower than that of their normal litter mates. Donor engraftment was up to 3% in peripheral blood and up to 12% in BM. Strikingly, the skin blisters characteristic of EB healed. Tissue analyses showed that the adoptive transfer of donor SLAM CD150+ selected BM resulted in skin engraftment of GFP+ donor cells and in the production of VII collagen mRNA in the skin, as assessed by RT- PCR. BM cells explanted from the 3 surviving recipients showed collagen VII producing cells. Ultrastructurally, we were able to identify anchoring fibrils, structures in the skin that are comprised exclusively of type VII collagen protein. This is consistent with the hypothesis that adoptive transfer of enriched wild-type BM cells resulted in, at least partial, functional correction of RDEB. Experiments are ongoing to identify the type VII collagen producing cells in CD150+ and CD48− BM population, and, in parallel, to identify immature BM cells that could be induced into becoming a skin stem cell. Collectively, these data demonstrate proof-of-principle of BM transfer for correction of the basement membrane zone defect in col7a1 −/− animals that may offer a valuable approach for treatment of human RDEB.