Molecular and Genetic Characterization of Functional Schwann-Cell Like Cells (iMDSCs) Transformed from Muscle Derived Stem Cells
Pooja S. Yesantharao, MS, Helen Xun, BS, Leila Musavi, BS, Howard Wang, MD, Amy Quan, MD, Markus Tammia, PhD, Aysel Cetinkaya-Fisgin, PhD, Ahmet Hoke, MD, PhD, Gerald Brandacher, MD, W P Andrew Lee, MD, Joseph Lopez, MD.
Johns Hopkins Medicine, Baltimore, MD, USA.
PURPOSE: Current microsurgical techniques do not address the atrophy and loss of Schwann cells (SCs) after peripheral nerve trauma. SCs are the principal glial cells that are known to facilitate axonal regeneration through cytokine signaling and spatial cues for directed axonal sprouting. To enhance peripheral nerve regeneration, SC replacement therapies using mesenchymal stem cells transformed into SC-like phenotypes have been investigated, but a robust source of mesenchymal stem cells is yet to be identified. Our lab has previously demonstrated that muscle-derived stem cells (MDSCs) induced to express SC-like phenotypes reduce denervation muscle atrophy, improve neuromuscular re-innervation, and restore muscle function after upper extremity nerve trauma in rodents, given their myelination capabilities in vivo. This study further characterizes these MDSC-derived SC-like cells and confirms the myelination capacity of the SC-like cells through molecular and genetic profiling.
METHODS: SC-like cells were derived from GFP+ mouse MDSCs (GFP+ iMDSCs) using a glial growth factor-based induction protocol developed by our laboratory. Flow cytometry using the FACScan System and FlowJo data analysis software was used to quantify expression of well-established SC markers in the SC-like iMDSCs. The SC-like iMDSCs were then further characterized using RT-PCR and microarray pathway analyses, with mouse Schwann cells (ScienCell) serving as positive controls and uninduced MDSCs as negative controls. The 2-ΔΔCr method was used to analyze relative changes in gene expression between these cell lines.
RESULTS: Flow cytometry revealed that iMDSCs cultured in the SC transformation media for 12 days demonstrated increased expression of SC-defining marker P75 compared to uninduced MDSC controls (21.0±1.6% vs. 6.63±2.4%; p < 0.001). Upon a gene-cluster analysis comparing SC-like iMDSCs to mouse SCs, there were differences in the overall gene expression profiles of these two cell lines. However, microarray analysis identified the axonal guidance pathway as a top canonical pathway in both iMDSCs and mouse SCs. Unlike uninduced MDSCs, iMDSCs were found to upregulate expression of many genes (confirmed via RT-PCR) that are known to be involved in promoting myelination, such as Lgi4 (10-fold vs 2-fold; p<0.001), and Cadm4 (8-fold vs 2-fold; p<0.01). Also, iMDSCs and mouse SCs both demonstrated upregulated expression of Gldn (necessary for the formation of nodes of Ranvier; 15-fold vs 3-fold; p<0.01) and Sema3b (plays a role in growth cone guidance during neurogenesis; 25-fold vs 2-fold; p<0.001).
CONCLUSION: Schwann cell-like cells derived from MDSCs (iMDSCs) show differences in overall gene expression profiles when compared to true SCs, indicating that they are SC-like cells that express key transcription factors and proteins involved in myelination. These transcriptome and genetic changes are believed to play a role in promoting the functional capabilities of these SC-like cells.
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