Transgenic Schwann cells expressing c-Jun promote an “immature” SC phenotype and support robust PC12 neurite outgrowth
Xueping Ee, MD; Lauren Schellhardt, BS; Susan E. Mackinnon, MD; Matthew Wood, PhD
Washington University School of Medicine, St Louis, MO
Background: Cell transplantation therapies that utilize Schwann cells (SCs) or Schwann cell-like cells seek to capitalize on the beneficial properties of dedifferentiated Schwann cells in promoting regeneration. However, expansion-culture of SCs leads to genetic drift and loss of their native, in vivo phenotype, which may in turn impact the abilities of these cells to promote nerve regeneration. The in vivo, dedifferentiated, "repair" SC phenotype requires expression of the transcription factor c-Jun, which mediates the repair program in SCs to promote nerve regeneration. Therefore, we developed transgenic SCs with inducible expression of c-Jun to promote a more "repair" SC phenotype and assessed whether these "c-Jun SCs" had advantages over expansion-culture SCs in their ability to promote axon growth.
Methods: Naive Schwann cells (NSCs) from rat sciatic nerve were isolated and transduced with lentivirus encoding transcription of c-Jun to generate "c-Jun SCs." All SCs were grown in conditions consisting of Modified Eagle's Medium (MEM) with D-Valine containing 10% fetal bovine serum (FBS), supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, 20 µg/mL bovine pituitary extract, and 5 µM forskolin. For experiments, SCs were seeded to culture plates coated with poly-L-Lysine and collagen and cultured with similar medium minus bovine pituitary extract and forskolin. Forty eight (48) hours after seeding, the SC medium was collected, and RNA was isolated from the SCs for gene analysis. The conditioned SC medium was added to cultured PC12 cells, which were previously differentiated to neurons via 1% horse serum and 100 ng/ml nerve growth factor. Resulting neurite outgrowth was analyzed using Image J software.
Results: C-Jun SCs expressed genes in a pattern that demonstrated a more "immature" phenotype with an up-regulation of genes involved in regeneration, proliferation, migration and epithelium mesenchymal transition compared to NSCs. Furthermore, conditioned medium collected from c-Jun SCs was able to promote robust neurite outgrowth from PC12 cells compared to medium collected from NSCs. C-Jun SC medium increased PC12 cell longest neurite length by ~63% (p<0.001), increased average neurite branching by 162% (p<0.001), and increased the number of PC12 cells possessing neurites by ~62% (p<0.001) compared to NSC medium.
Conclusions: Based on the potential of c-Jun SCs to provide a favorable environment to PC12 cells and their neurite outgrowth in vitro, we will investigate the future potential of these induced SCs to promote nerve regeneration in vivo.
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