One of the promising approaches to slow down or treat neurodegenerative diseases or spinal cord injuries represent cell therapies. The grafted cells could either integrate into the damaged tissue to replace dead or damaged cells or by secretion of different factors modulate inflammatory reaction, reduce tissue damage and support neuronal survival. However, due to the heterogeneity of in vitro cultured cells, comprehensive characterization of such cells is absolutely crucial to prevent safety risks. Here, we performed SWATH-MS analysis to characterize changes in proteome of human neural stem cells (NSCs) during their differentiation either spontaneously by withdrawal of EGF and FGF2 in cell culture media or by trophic support of BDNF/GDNF growth factors. We quantified about 2800 proteins over the 28 days of differentiation and showed that changes in cellular proteomes are caused mostly by differentiation time course, rather than type of differentiation itself and that the major changes in protein expression occurred between day 0 and day 7 of both differentiations.

Human neural stem cells (NSCs) derived from NIH approved H9 human embryonic stem cells were cultivated on polyornithine/laminine coated dishes in the KnockOut Dulbecco’s modified Eagle’s medium/F-12, 2 mM GlutaMA, 1% penicillin-streptomycin, 2% StemPro Neural Supplement, and supplemented with 20 ng/mL human recombinant EGF and 20 ng/mL human recombinant FGF2 and differentiated either spontaneously (S) by withdrawal of EGF and FGF2 growth factors from the cell culture media or by trophic support of BDNF and GDNF growth factors (B/G). The cells were differentiated for 0, 7, 14, 21 and 28 days, harvested manually and SWATH-MS analysis was performed to reveal changes in cellular proteome during both differentiation protocols.

Proteomic sample preparation: Cells on culture dishes were washed with PBS and harvested mechanically. Cells were then lysed with 8 M urea and 5 mM EDTA in 50 mM ammonium bicarbonate and homogenized. Protein concentration was determined by Pierce 660 nm protein assay. Each protein lysate sample was then treated with ProteaseMAX surfactant, reduced and alkylated, followed by enzymatic digestion with endoproteinase LysC and trypsin. The digestion was stopped with formic acid (FA); the resulting peptide mixtures were desalted on C18 spin columns. The eluted peptides were vacuum centrifuged to dryness and resuspended in aqueous solution with 2% ACN and 0.5% FA. The peptide concentration was determined by measuring absorbance at 280 nm and the peptide samples were diluted to a final concentration of 0.5 µg/µl with 1:30 (v/v) of spiked-in indexed Retention Time Biognosys peptides. The peptide mixtures in randomized order were loaded and separated on C18 columns in a trap-elute mode using the Eksigent nano-LC 425 (Sciex) on-line connected to 5600+ TripleTOF (Sciex) on a linear gradient of 5-35% acetonitrile in 0.1% FA over 120 min and 35-50% acetonitrile in 0.1% FA over 10 minutes at a flow rate of 200 nL/min. For DDA, the top 30 precursors with accumulation time 300 ms and mass range 400-1250 in high sensitivity mode were fragmented (MS/MS accumulation time 150 ms and mass range 170-1500 Da) in each cycle. For SWATH-MS were monitored 35 variable windows calculated with SWATH Variable window calculator (Sciex) with 150 ms accumulation time in MS and 100 ms accumulation tome in MS/MS with mass range 400-1250 Da, respectively 170-2000 Da, cycle time 3.5 s.