Illumina HiSeq 4000 paired end sequencing; Graphene Oxide Substrate Promotes Neurotrophic Factor Secretion and Survival of Human Schwann-Like Adipose Mesenchymal Stromal Cells - RNAseq
Graphene Oxide Substrate Promotes Neurotrophic Factor Secretion and Survival of Human Schwann-Like Adipose Mesenchymal Stromal Cells - RNAseq
SAD-MSCs were isolated from human tissue biopsies, following procedures approved by the National Research Ethics Committee, UK (NRES 13/SC/0499). Biopsies were harvested from the abdominal site of female patients undergoing reconstructive breast surgery (Age range: 32-64 yrs).  Adipose tissue was separated from skin and fibrous regions, then dissociated mechanically with a razorblade. The tissue was treated with collagenase (0.2% w/v) in Hank's Balanced Salt Solution (HBSS) with 1% v/v Penicillin-Streptomycin (P-S), under constant agitation in a water bath for 45 minutes (280 revs/min, 37°C). Tissue was filtered through a nylon mesh (100 μm) and supplemented with 20 ml Minimum Essential Medium Eagle alpha (αMEM), with 10% foetal bovine serum (FBS, Labtech, UK), 1% P-S, and 200 mM Glutamate. This is referred as growth media (GM). Tissue was centrifuged (300 x g, 10 minutes, Instrument; U-32 R, Boeco, Germany) to separate stromal vascular fraction (SVF). The SVF pellet was suspended in 1ml Red Lysis Buffer for 1 minute and neutralised with GM exposure (15 ml). The suspension was centrifuged (300 x g, 10 minutes), then suspended in GM. Isolated SVF were cultured in T75 flasks, at 37°C, 5% CO2. Three medium changes were applied weekly. Passaging occurred at 80% confluency. Following initial passaging, cells were classified as AD-MSCs. For 'Schwann-like' AD-MSC (sAD-MSC) stimulation, cells were seeded at 2x105 cells per T75 at passage 2. Populations were exposed to 1 mM β-Mercaptoethanol for 24 hours and washed twice with HBSS. Cells were primed with 35 ng/ml all-trans-retinoic acid in GM over 72 hours. Stimulation was induced using GM supplemented with 5 ng/ml platelet-derived growth factor (Peprotech, UK), 10 ng/ml basic fibroblast growth factor (Peprotech, UK), 14 mM Forskolin and 192 ng/ml glial growth factor (Acorda Therapeutics, Ardsley, NY, USA). This was classified as maintenance media (MM). Stimulation occurred over 14 days. Three MM changes were applied weekly, with passaging at 80% confluency. Following 14 days, cells were classified sAD-MSC. Experiments were carried out using sAD-MSC passage 4-6. Stimulation was confirmed using morphological assessment and immunostaining Schwann markers. sAD-MSCs from four patient-samples (N=4) were used for transcriptome sequencing. 5x10^4 cells were plated per glass or GO substrate, in triplicate (n=3).  RNA was isolated after 3 days incubation, using RNeasy kit, following manufacturer instructions (Qiagen, UK). Total RNA was submitted to the University of Manchester Genomic Technologies Core Facility (GTCF). Quality and integrity of the RNA samples were assessed using a 2200 TapeStation (Agilent Technologies). Illumina TruSeq Stranded mRNA kit, according to the manufacturer's protocol.