Rescue of the Mucocutaneous Manifestations in a Mouse Model of Recessive Dystrophic Epidermolysis Bullosa (RDEB) By Human Cord Blood Derived Unrestricted Somatic Stem Cells (USSCs)

Background:

RDEB is a severe inherited skin blistering disease caused by mutations in the COL7A1 gene that encodes type VII collagen (C7), a major component in the anchoring fibrils at the basement membrane. Allogeneic stem cell transplantation in both experimental animals and human subjects with RDEB resulted in increased C7 deposition and alleviation of the blistering phenotype in some recipients (Kiuru/Cairo/Christiano etal 2011). However, there was no distinct anchoring fibril formation in the recipient skin and the effective cell types that contribute to the regeneration in RDEB skin remain to be identified. Our previous studies demonstrated that CB-derived USSCs express C7 and promote epithelialization and wound closure in a murine wound healing model (Liao/Cairo etal Cell Transplant 2014).

Methods:

CB-derived USSCs were labeled with GFP/Luc reporter gene and injected in newborn col7a1-/- (RDEB) mice without immunosuppression. The migration of USSCs was determined by bioluminescent imaging (BLI) of RDEB mice as well as excised internal organs.

Results:

A single intra-hepatic injection of USSCs (0.2 x 10⁶) in newborn RDEB mice arrested the blistering phenotype and significantly enhanced the median survival to 8 days (n=21), as compared to 2 days in PBS injected animals (n=18) (P<0.0001) (Figure 1). About 30% of the USSC- treated mice lived beyond 2 weeks of life span and one mouse lived for four weeks. Histological analysis on the footpad skin of the RDEB mice demonstrated that the percentage of the skin with intact epidermis-dermis attachment was significantly improved from 20% ± 6% on D0 to 70% ± 12% on day 5, afterwards after a single intra-hepatic USSC injection. A second USSC intra- hepatic injection further elongated the median life span (16 days, n = 18, P<0.0001) (Figure 1). Remarkably, two mice that received repeated injections survived more than 12weeks. BLI of the RDEB mice following USSC injection further demonstrated that USSCs rapidly went into circulation after intra-hepatic injection and did not result in detectable retention in heart, kidney, and spleen. Immunocytochemical analysis revealed specific engraftment of USSCs in gastrointestinal tract and the dermis and hair follicles of the skin, suggesting a specific migration of USSCs toward the sites of blisters.  USSC injection also enhanced the expression of C7 in the recipient skin and electron microscopy revealed a partial restoration of anchoring fibrils in skin biopsies from the USSC-treated RDEB mice. We also demonstrated that USSCs treatment induced an infiltration of macrophages with a regenerative “M2” phenotype.

Conclusion:

Our data suggest that HUCB- derived USSCs improved the RDEB mucocutaneous manifestations in col7a1^-/- mice through multiple mechanisms.  This study warrants future clinical investigation of USSCs as a novel and universal allogeneic stem cell donor source in selected patients with RDEB.