Abstract 123: Functional Characterization of Fibroblasts Differentiated from Adipose-Derived Stem Cells

PURPOSE: Several dermal wound healing applications have been used to test the therapeutic potential of Adipose-derived stem cells (ASCs); however, we have yet to see dramatic improvement in healing time or scar preventing in a large scale clinical trial. Here, we characterize the fibroblastic differentiation capacity of these cells and extracellular matrix (ECM) production compared to primary cutaneous fibroblasts as recent literature suggests the wound healing capabilities and ECM production of fibroblasts differentiated from ASCs (dFib cells) may be superior. METHODS: ASCs and primary fibroblasts were isolated from healthy female patients undergoing abdominoplasty (n=8, 45.14 ± 14.16 years old). ASCs underwent fibroblastic differentiation via incubation with Connective Tissue Growth Factor and Ascorbic Acid for 3 weeks. Fibroblasts and dFib cells were then allowed to reach confluence and produce ECM. Proliferating fibroblasts and dFib cells were assayed for ASC and fibroblast markers to confirm differentiation. Further, mRNA and protein were harvested on Days 1, 7 and/or 21 of ECM production to quantify healthy and scar ECM marker expression via qPCR. Additionally, cells underwent in vitro scratch test migration analysis and subsequent Masson’s trichrome stain to evaluate migration and defect closure. RESULTS: Differentiated cells showed increased RNA expression of the fibroblast marker Ephb3 (6.94 ± 1.98-Fold, p<0.05) and decreased expression in the stem cell markers CD34 (0.35 ± 0.23-Fold, p<0.01) and CD105 (0.65 ± 0.18-Fold, p<0.01) when normalized to ASC expression which parallels primary fibroblast expression levels (4.61 ± 1.8-Fold, 0.027 ± 0.03-Fold, and 0.22 ± 0.17-Fold respectively, p<0.05). dFib cells also showed increased RNA expression of healthy ECM marker genes Fibronectin (0.93 ± 0.28-Fold, p<0.05) and Collagen 1 (4.67 ± 1.4-Fold, p<0.05), and Elastin (0.93 ± 0.63-Fold, p<0.01) compared to primary fibroblasts (0.62 ± 0.16-Fold, 2.21 ± 0.86-Fold, and vs. 0.27 ± 0.08-Fold respectively). Proliferating dFib cells further showed differential expression compared to fibroblasts for the scar tissue markers αSMA (0.011 ± 0.006-Fold vs. 0.024 ± 0.012-Fold, p<0.05), Collagen III (0.72 ± 0.2-Fold vs. 0.26 ± 0.11-Fold, p<0.001), and TIMP-1 (2.33 ± 0.63-Fold vs. 0.6 ± 0.18-Fold, p<0.001). Scratch test assays revealed dFib cells maintain smaller defects throughout the healing time course with more cells migrating into the defect. Finally, dFib cells closed the defects significantly faster than primary fibroblasts (32 ± 12.85 hours vs. 64 ± 13.85 hours, p<0.01). Similarly, Masson’s Trichrome staining demonstrates smaller defects after 3 weeks of recovery using dFib cells compared to primary fibroblast (1.04 ± 0.13mm2 vs. 1.29 ± 0.39 mm2) however, this difference did not reach significance (p=0.16). CONCLUSION: ASCs can be differentiated into fibroblast-like cells. These cells produce a robust ECM more similar to healthy skin as opposed to the scar tissue produced by primary cutaneous fibroblasts. These cells migrate into and close in vitro scratch defects more quickly than primary cutaneous fibroblasts and trend toward smaller long-term wounds. ASC differentiated fibroblasts show initial promise for regenerative medicine applications and should be investigated further for optimization in cutaneous wound healing and other therapeutic applications. I. Percec: None. R. Gersch: None.