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Fibroblast Depletion Enhance Stem Cells Differentiation In Pre-plate Technique: Study On Porcine Muscle Derived Stem Cells
Mohamed Awad, Anil Chaturvedi, Arvin Smith, MD, Anand Kumar, MD.
Case Western Reserve University, Cleveland, OH, USA.

Background: Muscle-derived stem cells (MDSCs) are multipotent cells with both immuno-regulatory and regenerative capacity. Our lab has previously determined, in mice, that fibroblast depletion, using a modified preplate technique, results in greater isolation of progenitor cells. Our study aim was to evaluate the differentiation potential of MDSCs isolated from a porcine model using the modified preplate technique.
Methods: Muscle samples were harvested from the hindlimb of an adult female pig. MDSCs were isolated using a modified preplate technique and cells in preplates 2, 4, and 6 were grown in osteogenic, adipogenic and chrondrogenic media over 21 days; at 21 days, assays were fixed, and imaged. Images were analyzed for stain coverage using ImageJ, statistical analysis was performed using Mathematica.
Results: Alizarin Red stain area, verifying osteogenesis, was significantly different in preplate 2 vs. preplate 6 (27% vs. 61%) (p<0.01) and between preplates 2 vs. 4 (27% vs. 74%)(p<0.01) but was not significantly different between preplate 4 vs. preplate 6 (74% vs. 61%)(p=0.18). Alcian Blue stain area, verifying chondrogenesis, was significantly different between preplates 2 vs. 6 (3% vs. 21%)(p<0.01), between preplates 2 vs. 4 (3% vs. 13%)(p<0.01), and between preplates 4 vs. 6 (13% vs. 27%)(p=0.03). Oil Red O stain area, verifying adipogenesis, was significantly different preplates 2 and 6 (9% vs. 23%)(p=0.04) but not significantly different between preplates 2 vs. 4 (9% vs. 18%)(p=0.16) or between preplates 4 vs. 6 (18% vs. 23%)(p=0.47).
Conclusion: Fibroblast depletion appears to significantly bolster the trilineage differentiation potential of porcine MDSCs, just as in murine MDSCs. Across all three differentiation assays, the earliest preplates (2) exhibited the lowest stain coverage. The differences in stain coverage between intermediate (4) and later preplates (6) were less significant, except in our chondrogenesis assay, which can reinforce intermediary preplate populations as viable candidates in MDSC-seeded allografts. Further studies will investigate scaffold growth and growth factor modulation.

Mean Stain Areas (%)
PP2PP4PP6
Osteogenesis277461
Adipogenesis91823
Chondrogenesis31327


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