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A Modified Approach to Induce Differentiation of Muscle-Derived Stem Cells into Schwann Cell-Like Cells: Implications for Remyelination Therapy
Joseph Lopez, MD MBA, Leila Musavi, BS, Jose A. Escalante, MD, Amy Quan, MPH, Markus Tammia, PhD, Howard D. Wang, MD, Qiongyu Gup, PhD, Gerald Brandacher, MD, W.P. Andrew Lee, MD, Ahmet Hoke, MD PhD, Anand Kumar, MD.
Johns Hopkins Hospital, Baltimore, MD, USA.

Purpose: Muscle-derived stem cells (MDSCs) are a distinct population of cells with pronounce pluripotent potential. Previous findings from our laboratory have demonstrated that MDSCs have special osteogenic, vascular, and endothelial potential. However, no study has explored the schwann-cell differentiation potential of MDSCs in-vitro. The purpose of this study was to characterize the transformation potential of MDSCs to transform into cells with Schwann Cell-like phenotypes.
Methods: MDSCs were isolated from 4-8 weeks-old C57BL/6J mice using a previously described pre-plate technique based on the selective adhesion potential of various cell types to type I collagen coated surfaces. MDSCs were grown for 12 days in: a) basal media or b) Schwann Cell (SC) transformation media composed of various glial-growth factors. Immunocytochemistry and flow cytometry were performed on transformed and non-transformed MDSCs to assess the expression of schwann-cell markers including S-100, GFAP, and p75. A Schwannoma cell line (S16) and primary SCs derived from the sciatic nerve of young Sprague Dawley rats were used as a positive control for all experiments. Cell morphology was also assessed via electron microscopy (EM) and bright field imaging. The myelinating capabilities of transformed and non-transformed MDSCs was evaluated in vitro by co-culturing MDSCs with dorsal root ganglion (DRG) neurons isolated from E13 pregnant Sprague Dawley rats.
Results:SC-transformation media upregulated the expression of SC-intracellular maker, S-100, in MDSCs when compared S16 cells and MDSCs cultured in basal media via immunocytochemistry ((CTCF 4.9 vs 0.5 vs 0.2, p = 0.002). Flow cytometry revealed that culturing MDSCs in SC transformation media for twelve days led to an upregulation in the fraction of cells expressing p75 compared to untreated MDSCs controls (21.0 ± 1.6% vs 6.63 ± 2.4%, p < 0.001). Furthermore, transformed MDSCs displayed similar but distinct cell morphology to non-transformed MDSCs and rat-derived SCs via bright field and EM (p < 0.01). Transformed MDSCs aggregate at 80%V confluence similarly to how rat-derived SC aggregate in-vitro. Lastly, non-transformed and transformed MDSCs demonstrated different myelination capabilities in vitro when co-cultured with DRG neurons (p < 0.014).
Conclusion:MDSCs can be transform into cells with SC-like properties and functional capabilities in vitro. These findings suggest that MDSCs may be utilized to augment peripheral nerve regeneration.


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