Human Schwann Cell Derivation From Small Skin Biopsies
Tak Ho Chu, PhD1; Tanmay Mishra, BSc1; Jo Stratton, PhD2; Jeff Biernaskie, PhD3; Rajiv Midha, MD, MSc, FRCS(C)4
1University of Calgary, Calgary, AB, Canada, 2Neuroscience, University of Calgary, Calgary, AB, Canada, 3Hotchkiss Brain Institute, and Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada, 4Department of Clinical Neuroscience and Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada
Introduction
Skin is an easily accessible tissue, which can be harvested with minimally invasive approaches from patients. We have previously shown that human Schwann cells (SCs) can be selected and expanded in culture from autopsy-derived adult skin cell. Towards clinical application of autologous SC therapies, we aim to improve the specificity and reliability of our protocol to obtain SCs from small skin biopsies.
Methods
Biopsy face skin samples were provided by plastic surgery clinic approved by our Health Research Ethics Board. Skin tissue (~1 cm2) was washed and epithelium removed by incubating with dispase (5 U/ml) for 1.5 hr. Samples were then cut into small cubes and incubated with DMEM with 10% fetal bovine serum (FBS), forskolin (Fsk, 5 µM), neuregulin (NRG, 50 ng/ml) and plasmocin (0.02%) for 2 weeks. At the end of 2 weeks, tissue explants were digested overnight at 37 °C with enzymatic cocktail containing dispase (1.25 U/ml) and collagenase IV (0.125%). The cell suspension was filtered through 40 µm filter and seeded onto poly-D-lysine and laminin coated cell culture dish, supplemented with DMEM with 2% FBS, Fsk and NRG. After 1 week, SCs were enriched using immunopanning against primate-specific p75 antibody. Identical steps, but without a 2 week explant incubation was performed to compare the effects of explant-processing to immediate digestion-processing of skin biopsy samples for SC yield.
Results
When the culture was analyzed one day after seeding onto poly-D-lysine and laminin, the explant model increased the proportion of SCs compared to immediately dissociated condition (14.2% (n=3) vs 0.02% (n=1)), as identified by p75 labeling. These p75+ve cells also co-expressed SC markers such as S100, Sox10, nestin and c-Jun, and maintained the human nuclear antigen. Further incubation for 1 week in SC growth factors Fsk and NRG increased the proportion of SCs to 21.4% compared to only 0.5% for the immediately dissociated condition. Immunopanning further increased the purity of SCs to 80-90% and we were able to harvest 2-3 million cells by 4 weeks. In preliminary proof of principle experiments, these human SCs demonstrate survival and ensheathment of regenerating mouse axons following nerve crush injury in an immunodeficient rodent model.
Conclusions
We have identified a 2-week explant incubation as a critical step in reliably isolating human SCs from a small biopsy skin sample. SCs of increased purity can be obtained. Their effect in neural remyelination and regeneration studies are ongoing.
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