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Determining the Roles of the Components of Schwann Cell Differentiation Medium
Heath Charvet, MD1; Hakan Orbay, MD, PhD1; Chris K. Gold, MD2; Christopher Little, BS1; David Sahar, MD1
1Department of Surgery, Division of Plastic Surgery, University of California Davis, Sacramento, CA; 2Division of Plastic Surgery, Travis AFB - David Grant Medical Center, Travis, CA
Introduction Adipose-derived stem cells (ASCs) differentiated into Schwann Cells (SC) may potentially facilitate peripheral nerve regeneration. However, the role of the components of ASC-SC differentiation medium has not been well-studied. In this study, we removed the components of ASC-SC differentiation medium one at a time from the medium and observed the changes in the expression of SC specific genes.
Materials & Methods: ASCs isolated from rat inguinal fat pads were characterized with flow cytometry and adipogenic, osteogenic, and chondrogenic differentiation. Cells in culture flasks were divided into 6 groups. In the control group, cells were supplemented with cell growth medium. In experimental groups cells were initially treated with ?-mercaptoethanol and all-trans-retinoic acid. In group I cells were fed with SC differentiation medium [cell growth medium supplemented with platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), forskolin, and glial growth factor (GGF)]. In each of the other groups, one of the above components was removed from differentiation medium: GGF in group II, forskolin in group III, bFGF in group IV, and PGDF in group V. Cells were fed with corresponding differentiation medium for two weeks. The fold changes in expression levels of the genes S100, integrin?4, and nerve growth factor receptor (NGFR) were detected with qRT-PCR. Results were confirmed with immunofluorescence (IF) staining.
Results: ASCs were negative for endothelial and white blood cell markers CD 31 and CD 45, but were positive for mesenchymal stem cell markers CD44 and CD90. Adipogenic, osteogenic, and chondrogenic differentiation of ASCs were documented by oil O red, alizarin red, and alcian blue staining respectively. The results of qRT-PCR revealed that undifferentiated ASCs expressed S100 gene at a certain level but did not express integrin?4, and NGFR. Groups III and IV-that lacked forskolin and bFGF respectively-had the highest expression levels of integrin?4, and NGFR. The cells treated with complete differentiation medium showed a 3.2 fold increase in the expression of S100 but the expression of integrin?4 and NGFR was significantly lower in comparison to groups III and IV. Group II-that lacked GGF-showed no significant levels of SC specific gene expression. In comparison to other groups, the gene expression profile in group IV was the closest to the SC differentiation group. IF staining confirmed the results obtained with qRT-PCR.
Conclusions: Our results showed that GGF is a vital component of SC differentiation medium, while bFGF played no significant role in a murine model.
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