Day 97-105 astrocytes were seeded at 300,000 cells/well in a 6-well plate coated with 20 µg/ml P/O (Sigma-Aldrich) and 5 µg/ml mouse laminin for 2 days and treated with either PBS (referred to as non-treated in the study), 100 ng/ml TNFα, or 10 µM αSYN (monomers or fibrils) for 6 days.
Growth protocol
On day one of differentiation, human iPSC colonies were harvested, dissociated, filtered (100 µm), and added to an ultra-low-adherent flask (Sigma-Aldrich) with 0.1 µM LDN (Stemgent), 10 µM SB (Selleckchem), 200 ng/ml SHH-C25II (Thermo Fisher Scientific), 0.8 µM CHIR (Stemgent), and 1 µM SAG (Millipore) for 0-4 days in neural induction medium [NIM; advanced DMEM-F12 with 1% L-Glu, 1% NEAA, 1% N2, and 1% P/S (100 U/mL)]. From day 6-8 NIM with 0.1 µM LDN, 0.8 µM CHIR (Stemgent), and 2 µM SAG (Millipore) was used. On day 10, NIM with 0.8 µM CHIR (Stemgent) and 2 µM SAG (Millipore) were used. From days 12-30, NIM with 100 ng/ml FGF8b (Thermo Fisher Scientific) and 2 µM SAG (Millipore) was used. On day 30 EBs were dissociated, filter strained (100 µm), and transferred to a new ultra-low-adherent flask containing neural expansion medium [(NEM; DMEM-F12 with 1% L-Glu, 1% NEAA, 2% B27 without vitamin A, 1% P/S, and 0.2 µg/ml heparin (Sigma-Aldrich)] with 20 ng/ml FGF2 (Thermo Fisher Scientific) and 20 ng/ml EGF (Peprotech) for 30 days. On day 60, EBs were washed and dissociated with 0.05% trypsin/1X-EDTA and seeded to adherent culture flasks coated with 20 µg/ml Poly-L-ornithine hydrobromide (P/O; Sigma-Aldrich) and mouse laminin (15 µg/ml for initial seeding, then 5 µg/ml for subsequently passages) containing NEM and 20 ng/ml CNTF (R&D Systems) for 60-80 days in NDM. Finally, from days 80-100, astrocytes were cultured in neural differentiation medium [NDM; (NB with 1% L-Glu, 1% NEAA, 1% N2, and 1% P/S] with and 20 ng/ml CNTF (R&D Systems). Cells were passaged at confluency. Media was changed every 2 days from day 0-30 and every 3-4 days from day 30-100.
Extracted molecule
total RNA
Extraction protocol
Astrocytes were then washed twice with PBS and frozen at -80oC before shipping to Kompetenzzentrum Fluoreszente Bioanalytik (Germany) for gene expression analysis using the Clariom S array (Affymetrix). Total RNA was extracted from dry cells according to the “Purification of total RNA from animal cells using spin technology” protocol of the RNeasy Micro Kit (QIAGEN, Hilden, Germany). In brief, cells were grown in 6-well plates. After removing the cell-culture medium, the plates were frozen and shipped on dry ice. After thawing, 250 µl of lysis buffer RLT was added to each well and the cells were harvested with a cell scraper. The lysates were pipeted into QIAshredder spin columns and homogenized by centrifugation. Next 1 volume of 70% ethanol was added and the samples were applied to RNeasy MinElute spin columns followed by an on-column DNase digestion and several wash steps. Finally total RNA was eluted in 14 μl of nuclease free water. Purity and integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip reagent set (Agilent, Palo Alto, CA, USA).
Label
biotin
Label protocol
Sample preparation for microarray hybridization was carried out as described in the Affymetrix GeneChip WT PLUS Reagent Kit User Manual (Affymetrix, Inc., Santa Clara, CA, USA). In brief, 200 ng of total RNA was used to generate double-stranded cDNA. 12 µg of subsequently synthesized cRNA was purified and reverse transcribed into sense-strand (ss) cDNA, whereat unnatural dUTP residues were incorporated. Purified ss cDNA was fragmented using a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) followed by a terminal labeling with biotin. 3,8 µg fragmented and labeled ss cDNA were hybridized to Affymetrix Clariom S human arrays for 16 h at 45 ° C in a GeneChip hybridization oven 640. Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluidics and scan functions were controlled by Affymetrix GeneChip Command Console v4.1.3 software. Sample processing was performed at an Affymetrix Service Provider and Core Facility, “KFB - Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany; www.kfb-regensburg.de).
Hybridization protocol
Sample preparation for microarray hybridization was carried out as described in the Affymetrix GeneChip WT PLUS Reagent Kit User Manual (Affymetrix, Inc., Santa Clara, CA, USA). In brief, 200 ng of total RNA was used to generate double-stranded cDNA. 12 µg of subsequently synthesized cRNA was purified and reverse transcribed into sense-strand (ss) cDNA, whereat unnatural dUTP residues were incorporated. Purified ss cDNA was fragmented using a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) followed by a terminal labeling with biotin. 3,8 µg fragmented and labeled ss cDNA were hybridized to Affymetrix Clariom S human arrays for 16 h at 45 ° C in a GeneChip hybridization oven 640. Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluidics and scan functions were controlled by Affymetrix GeneChip Command Console v4.1.3 software. Sample processing was performed at an Affymetrix Service Provider and Core Facility, “KFB - Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany; www.kfb-regensburg.de).
Scan protocol
Sample preparation for microarray hybridization was carried out as described in the Affymetrix GeneChip WT PLUS Reagent Kit User Manual (Affymetrix, Inc., Santa Clara, CA, USA). In brief, 200 ng of total RNA was used to generate double-stranded cDNA. 12 µg of subsequently synthesized cRNA was purified and reverse transcribed into sense-strand (ss) cDNA, whereat unnatural dUTP residues were incorporated. Purified ss cDNA was fragmented using a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) followed by a terminal labeling with biotin. 3,8 µg fragmented and labeled ss cDNA were hybridized to Affymetrix Clariom S human arrays for 16 h at 45 ° C in a GeneChip hybridization oven 640. Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluidics and scan functions were controlled by Affymetrix GeneChip Command Console v4.1.3 software. Sample processing was performed at an Affymetrix Service Provider and Core Facility, “KFB - Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany; www.kfb-regensburg.de).
Data processing
Summarized probe set signals in log2 scale were calculated by using the GCCN-SST-RMA algorithm with the Affymetrix GeneChip Expression Console v1.4 Software. After exporting into Microsoft Excel, average signal values, comparison fold changes and significance P values were calculated. Probe sets with a fold change above 2.0 fold and a student’s t test P value lower than 0.05 were considered as significantly regulated.
