Fibroblast Depletion Bolsters Tri-lineage Differentiation in Porcine Muscle-Derived Stem Cells
Anil Chaturvedi, MS1, Mohamed Awad, MD1, Samuel Boas, BS1, Arvin Smith, BS1, James Reynolds, PhD2, Anand Kumar, MD1.
1University Hospitals Cleveland, Cleveland, OH, USA, 2Case Western Reserve University, Cleveland, OH, USA.
Background: The modified preplate technique is a method of isolating muscle-derived stem cells (MDSCs), using collagen-coated flasks, by selective adherence of rapidly adhering cells (fibroblasts), in the early stages of the process (preplates 1-2), and isolation of slowly adhering MDSCs later in the process (preplates 4-6). Our lab has previously shown that later preplates (4-6) of murine hind-limb muscle have greater differentiation capacity compared with cells isolated in earlier preplates. The aim of our study was to evaluate the differentiation potential of fibroblast-depleted, porcine MDSC populations using the modified preplate technique.
Methods: Muscle samples were harvested from the hindlimb of an adult female pig. MDSCs were isolated using the modified collagen adherence preplate technique. Cells in preplates 2, 4, and 6 were grown in osteogenic, adipogenic and chrondrogenic media over 21 days. At 21 days, assays were fixed, stained, and imaged. Alizarin Red stain was used to determine osteogenic differentiation, Alcian Blue was used to determine chondrogenic differentiation, and Oil Red O stain was used to determine adipogenic differentiation. Images were analyzed for stain coverage using ImageJ, statistical analysis was performed using Mathematica.
Results: Alizarin Red stain area was significantly different between preplate 2 vs. preplate 6 (p<0.01) and between preplates 2 vs. 4 (p<0.01) but was not significantly different between preplate 4 vs. preplate 6 (p=0.18). Alcian Blue stain area was significantly different between preplates 2 vs. 6 (p<0.01), between preplates 2 vs. 4 (p<0.01), and between preplates 4 vs. 6 (p=0.03). Oil Red O stain area was significantly different between preplates 2 and 6 (p=0.04) but not significantly different between preplates 2 vs. 4 (p=0.16) or between preplates 4 vs. 6 (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), which are rich in fibroblasts, exhibited the lowest stain coverage. Other than the chondrogenesis assay, differences in stain coverage between mixed stem, and support, cell intermediate (4) and later stem cell rich preplates (6) were not significant. Our study offers additional data to suggest the use of intermediary preplate populations as viable candidates in MDSC-seeded allografts. Further studies will investigate three-dimensional growth of porcine MDSCs on collagen scaffolds.
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