Preconditioning of mesenchymal stem cells for improved transplantation efficacy in recessive dystrophic epidermolysis bullosa

Abstract

Introduction: The use of hematopoietic cell transplantation (HCT) has previously been shown to ameliorate cutaneous blistering in pediatric patients with recessive dystrophic epidermolysis bullosa (RDEB), an inherited skin disorder that results from loss-of-function mutations in COL7A1 and manifests as deficient or absent type VII collagen protein (C7) within the epidermal basement membrane. Mesenchymal stem cells (MSCs) found within the HCT graft are believed to be partially responsible for this amelioration, in part due to their intrinsic immunomodulatory and trophic properties and also because they have been shown to restore C7 protein following intradermal injections in models of RDEB. However, MSCs have not yet been demonstrated to improve disease severity as a stand-alone systemic infusion therapy. Improving the efficacy and functional utility of MSCs via a pre-transplant conditioning regimen may bring systemic MSC infusions closer to clinical practice.

Methods: MSCs were isolated from 2- to 4-week-old mice and treated with varying concentrations of transforming growth factor-β (TGFβ; 5-20 ng/mL), tumor necrosis factor- α (TNFα; 10-40 ng/mL), and stromal cell-derived factor 1-α (SDF-1α; 30 ng/mL) for 24-72 hours.

Results: We demonstrate that treating murine MSCs with exogenous TGFβ (15 ng/mL) and TNFα (30 ng/mL) for 48 hours induces an 8-fold increase in Col7a1 expression and a significant increase in secretion of C7 protein, and that the effects of these cytokines are both time and concentration dependent. This cytokine treatment also promotes a 4-fold increase in Tsg-6 expression, a gene whose product is associated with improved wound-healing and immunosuppressive features. Finally, the addition of exogenous SDF-1α to this regimen induces a simultaneous upregulation of Col7a1, Tsg-6, and Cxcr4 expression.

Conclusions: These data suggest that preconditioning represents a feasible method for improving the functional utility of MSCs in the context of RDEB stem cell transplantation, and also highlight the applicability of preconditioning principles toward other cell-based therapies aimed at treating RDEB patients.

Figure 1

Cytokine preconditioning induces simultaneous upregulation of Col7a1 and Tsg-6 mRNA expression in mesenchymal stem cells. (a) Untreated mesenchymal stem cells (MSCs) exhibit detectable baseline expression of Col7a1, Tsg-6, and Cxcr4. (b) MSCs were treated with 10 ng/ml transforming growth factor beta (TGFβ) +20 ng/ml tumor necrosis factor alpha (TNFα) for 24, 48, or 72 hours. Quantitative polymerase chain reaction (qPCR) was performed for Col7a1 and Tsg-6 expression in treated groups relative to untreated MSCs. (c) MSCs were treated across concentration gradients of TGFβ and TNFα for 48 hours. qPCR was performed for Col7a1 and Tsg-6 expression in treated groups relative to untreated MSCs. (d) MSCs were treated with 15 ng/ml TGFβ +30 ng/ml TNFα for 48 hours. Cells were transferred to an alpha minimum essential medium-only environment for a subsequent 48 hours, after which qPCR was performed for Col7a1 and Tsg-6 expression in treated groups relative to untreated MSCs. All qPCR values in (b) to (d) were normalized against endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. All qPCR experiments were run in triplicate and across two experimental groups per condition. Data presented as mean ± standard deviation. *P <0.05 by Student’s t test.

Figure 2

Cytokine preconditioning results in increased type VII collagen protein secretion. Mesenchymal stem cells (MSCs) were treated with 15 ng/ml transforming growth factor beta +30 ng/ml tumor necrosis factor alpha for 48 hours. Following this incubation period, culture medium was extracted and subjected to sandwich enzyme-linked immunosorbent assay (ELISA) analysis. Culture medium from treated groups was compared with that of untreated MSCs. ELISA experiments were carried out across two biological groups per condition (10⁵ cells per group). Data presented as mean ± standard deviation. *P <0.005 by Student’s t test. C7, type VII collagen protein.

Figure 3

Addition of SDF-1α to the preconditioning protocol induces simultaneous upregulation of Col7a1, Tsg-6, and Cxcr4 mRNA expression. Mesenchymal stem cells (MSCs) were treated with 15 ng/ml transforming growth factor beta +30 ng/ml tumor necrosis factor alpha for 48 hours. At 47 hours, cells were exposed to 30 ng/ml stromal cell-derived factor 1-alpha (SDF-1α) for 1 hour. (a) Quantitative polymerase chain reaction (qPCR) was performed for Col7a1, Tsg-6, and Cxcr4 expression in treated cells relative to untreated MSCs. qPCR values were normalized against endogenous glyceraldehyde 3-phosphate dehydrogenase expression, and experiments were run in triplicate and across two experimental groups. (b) Flow cytometry was performed to assess cell surface CXCR4 expression in treated versus untreated cells. (c) Chemotaxis assay results of treated versus untreated cells: 50,000 GFP-expressing cells were placed in each top well, while increasing SDF-1α gradients were used in the bottom wells. For blocking controls, treated and untreated cells were incubated in presence of 100 μg/ml AMD3100 for 1 hour and exposed to a 90 ng/ml SDF-1α concentration gradient during the assay. Experiments were run in duplicate. (d) Representative fluorescent microscopy images of the chemotaxis membrane following the assay. Data presented as mean ± standard deviation. *P <0.05 by Student’s t test.