Illumina NovaSeq 6000 paired end sequencing; Reactive astrocytes transduce blood-brain barrier inflammation through a TNFα-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin
Reactive astrocytes transduce blood-brain barrier inflammation through a TNFα-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin
All samples were collected at the day 5 time point, regardless of monoculture or coculture. For astrocyte, to reduce variability in astrocyte generation, manually isolated neural progenitors were used as the starting population. iPSCs were dissociated with Versene and seeded into 6-well low-attachment plates to form embryoid bodies (EBs) at a density of 4x105 cells/well in E6 medium supplemented with 100x N2 (Invitrogen; E6N2) and 10 μM Y27632 (Tocris). Neural differentiation in the EBs was achieved by dual inhibition of SMAD signaling for 7 days. EBs were then seeded onto growth factor reduced Matrigel-coated plates in E6N2 containing 1 μg/ml laminin (Sigma Aldrich). After another 7 days (Day 0), neural progenitor cells were manually isolated from rosettes and expanded as neurospheres in a suspension culture for 7 days in E6N2 supplemented with 50x B27 lacking retinoic acid (Invitrogen) and 20 ng/ml FGF2 (Peprotech). Then, the neurospheres were dissociated into single cells with TrypLETM (Thermo Fisher Scientific) and seeded onto growth factor reduced Matrigel-coated 6-well plates in E6N2 supplemented with 50x B27 lacking retinoic acid, 10 ng/ml BMP4 (Peprotech) and 20 ng/ml FGF2 for directed astroglial differentiation. Medium changes were performed every 48 hours, and cells were passaged upon reaching approximately 80% confluency. Astrocytes were seeded for coculture at a ratio of 1 well of a 6-well plate to 6 wells of a 12-well plate and maintained in E6 medium containing 10 ng/ml CNTF (Peprotech) and 10 ng/ml EGF (Peprotech) until initiation of coculture. For BMEC-like cells, iPSCs were dissociated with Accutase and seeded onto growth factor reduced Matrigel-coated plates in E8 medium containing 10 μM Y27632 at a density of 15,000 cells/cm2. Differentiation was initiated 24 hours after seeding by changing to E6 medium, with daily medium changes for 4 days. Next, cells were expanded with serum-free basal endothelial cell medium (EC medium) supplemented with 50x B27 (Thermo Fisher Scientific), GlutaMAXTM (Thermo Fisher Scientific), 10 μM retinoic acid (Sigma Aldrich), and 20 μg/ml FGF2 for 2 days without media change. Following this treatment, cells were collected by a 20-minute incubation in Accutase and seeded onto Transwell filters (1.1 cm2 polyethylene terephthalate membranes with 0.4 μm pores; Fisher Scientific) coated with a mixture of 400 μg/ml collagen IV (Sigma Aldrich) and 100 μg/ml fibronectin (Sigma Aldrich). The following day, cells were switched to EC medium lacking FGF2 and RA. If coculture was being initiated, filters were transferred to 12-well plates containing astrocytes and the same medium was utilized. For TNFα conditions, cells were treated with 10 ng/ml of TNFα for 4 days before extraction of RNA.  Total RNA was isolated using TRIzol (Sigma Aldrich). RNA was further purified with a Qiagen RNeasy Plus mini kit (Qiagen)