Co-stimulation with IL-1β and TNF-α induces an inflammatory reactive astrocyte phenotype with neurosupportive characteristics in a human pluripotent stem cell model system

Astrocyte reactivation has been discovered to be an important contributor to several neurological diseases. In vitro models involving human astrocytes have the potential to reveal disease-specific mechanisms of these cells and to advance research on neuropathological conditions. Here, we induced a reactive phenotype in human induced pluripotent stem cell (hiPSC)-derived astrocytes and studied the inflammatory natures and effects of these cells on human neurons. Astrocytes responded to interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) treatment with a typical transition to polygonal morphology and a shift to an inflammatory phenotype characterized by altered gene and protein expression profiles. Astrocyte-secreted factors did not exert neurotoxic effects, whereas they transiently promoted the functional activity of neurons. Importantly, we engineered a novel microfluidic platform designed for investigating interactions between neuronal axons and reactive astrocytes that also enables the implementation of a controlled inflammatory environment. In this platform, selective stimulation of astrocytes resulted in an inflammatory niche that sustained axonal growth, further suggesting that treatment induces a reactive astrocyte phenotype with neurosupportive characteristics. Our findings show that hiPSC-derived astrocytes are suitable for modeling astrogliosis, and the developed in vitro platform provides promising novel tools for studying neuron-astrocyte crosstalk and human brain disease in a dish.

The secretion profiles of reactive astrocytes were confirmed at the 7-, 8-and 9-day time points. The astrocytes were first treated with IL-1β and TNF-α for 7 days; thereafter, the cytokines were removed from the astrocytes, which were cultured without cytokines for an additional 2 days (the 8 d and 9 d time points) before collection of astrocyte conditioned medium. The blots are from one experiment. The most upregulated proteins are marked with rectangles in the images, and the corresponding analytes are listed on the right. The red rectangles show the positive controls of the arrays. The data are shown as the mean ± s.e.m. Statistical analysis within groups was performed with the Friedman test (shown in the images for d and e) followed by the Wilcoxon signed-rank test (shown in the tables for d and e). (f-g) To highlight significant differences between treatment groups, the data from panels d-e were reused and are presented as the changes in spike and burst rates compared to the baseline measurements. The data in (f) and (g) are shown as Tukey boxplots, and statistical significance was tested with the Mann-Whitney U-test. For the MEA results, n=9-10 networks derived from one differentiation.  Fig. S8. Secretion of proteins from reactive and control astrocytes during culture on the microfluidic device. Secretion of inflammatory factors by control astrocytes and reactive astrocytes was analyzed at 72 h. To analyze the factors secreted from astrocytes, medium was collected from the astrocyte compartment, and the inflammatory factor levels were analyzed with a cytokine array. The most upregulated proteins are marked with rectangles in the image, and the names of the analytes are listed on the right.
Sequential coating with 100 µg/ml poly-L-ornithine (PO, Sigma) and 15 µg/ml LN521 was performed for plastic well plates and glass coverslips, and 0.1% polyethyleneimine (PEI, Sigma) and 50 µg/ml LN521 were used for MEAs. The cells were passaged with Accutase (Thermo Fisher Scientific) and plated in medium containing 10 µM ROCK Inhibitor (Sigma). For neural induction, the neural maintenance medium was supplemented with 100 nM LDN193189 and 10 µM SB431542 (both from Sigma) for 12 days. Neural progenitor cells were expanded in maintenance medium supplemented with 20 ng/ml fibroblast growth factor-2 (FGF2, R&D Systems) until day 25. Final maturation was achieved by supplementing the maintenance medium with 20 ng/ml brain-derived neurotrophic factor (BDNF, R&D Systems), 10 ng/ml glial-derived neurotrophic factor (GDNF, R&D Systems), 500 µM dibutyryl-cyclic AMP (db-cAMP, Sigma) and 200 µM ascorbic acid (AA, Sigma). At day 32, the cells were plated for experiments. The cells were plated on plastic well plates at a density of 50000 cells/cm 2 except in the cell viability and apoptosis assays (100000 cells/cm 2 ) and on MEAs at a density of 1×10 6 cells/cm 2 . The medium was changed every two to three days.

Western blot analysis
Cells were washed with ice-cold PBS, lysed in Laemmli sample buffer (Bio-Rad) and heated for 5 min at 95°C before storage at -80°C. The total protein concentrations were measured using a Pierce × the estimate of the noise standard deviation. Burst analysis was performed utilizing the R package meaRtools 6 , and for burst detection, the logISI algorithm was integrated into the analysis code 7 with a minor modification. The minimum number of spikes in a burst was set to five. Short bursts were merged when the ISIth was lower than 100 ms. A cutoff of 100 ms was applied as the minimum time required between bursts.

Fabrication of the microfluidics device
The device was produced from two PDMS parts: 1) a microfluidic device containing the cell compartments and microtunnels and 2) a medium reservoir part ( Supplementary Fig. S7). The microfluidic device part was fabricated from PDMS (10:1 mass ratio with curing agent, SYLGARD 184, Dow Corning) using replica molding [8][9][10] . For the fabrication of the mold, multilayer SU-8 rapid prototyping methods were used: SU-8 5 (MicroChem) photoresist was used to produce the 3.5 µm high microtunnels, and SU-8 3050 photoresist was used to produce the 100 µm high cell compartments. The microfluidic device was separated from the replica using a ø 32 mm punch. To connect the cell compartments to the medium reservoirs and to enable cell plating, three inlets and one outlet (Supplementary Fig. S7) were punched in the device using a ø 3 mm punch. The medium reservoir part was fabricated from an 8 mm thick PDMS sheet using a ø 32 mm punch. The medium reservoirs for each cell compartment were created with a ø 6 mm punch.

Fluidic isolation between the cell compartments
To demonstrate fluidic isolation between the cell compartments in the device, FITC-conjugated dextran particles (15-25 kDa) (TdB Consultancy, Uppsala, Sweden) were used. The particles (50 µM) were added to the astrocyte compartment, and their diffusion was evaluated for 1 h and 24 h at 37°C.
The diffusion of the dextran particles into the axonal and neuronal compartments was visualized with an Olympus IX51 microscope equipped with an Olympus DP30BW camera (Olympus Corporation).
To quantify the amounts of dextran particles in the neuronal soma and astrocyte compartments after 24 h, the absorbance of medium samples at 490 nm was measured using a NanoDrop 1000 (Thermo Fisher Scientific).

Assembly of the microfluidic platform and cell plating
The microfluidic devices and medium reservoirs were attached together and treated with oxygen plasma in a PICO plasma system (Diener Electronic, Germany) for 4 min at 30 W and 0.3 mbar pressure to make the cell compartments and microtunnels hydrophilic and to facilitate the supply of laminin in the microscale features. Thereafter, the devices were reversibly bonded on 250 µg/ml POcoated coverslips (ø 30 mm), and the cell compartments and microtunnels were filled with 20 µg/ml LN521 via the inlets in the neuron and astrocyte compartments. Coating was performed overnight at 4°C. Neurons were seeded into the inlets of the neuronal compartments at a density of 150000 cells/cm 2 , while 50000 astrocytes were seeded into the inlet of the astrocyte compartment. Cell plating on the astrocyte compartment is based on fluid flow, which enables astrocytes to settle in the cell area next to the microtunnels ( Supplementary Fig. S7). The fluid flow is a result of differences in width along the astrocyte compartment: the inlet side of the astrocyte compartment is wider than the outlet side of the astrocyte compartment.