Fig 2: Differentiation of WTC-11 induced pluripotent stem cells (iPSC) into motor neurons (MN). a Schematic diagram of the overall experimental design. Shown are the small molecule stimuli for each developmental stage and the corresponding morphological changes in the course of iPSC conversion into MN. The scale bar for the phase contrast images is 400 µm; (b) Representative immunofluorescence images of key pluripotency (Oct4) and pan-neuronal (Nestin, Pax6, and ß-III tubulin) biomarkers indicated at efficient iPSC differentiation into neuronal stem cells (NSC) at day 7th post-induction (accumulation of Nestin is shown in orange). Upregulation of Pax6 and ß-III tubulin (shown in green) at day 13 of iPSC induction marked the formation of motor neuron progenitor (MNP) cells. c Immunostaining of neuronal structural proteins (beta-III-tubulin in green, MAP2 in orange), motor neuron specific markers (HB9 and ChAT in green), and synaptic vesicles protein (Synaptophysin in green) showed a pure population of mature MN at day 28 post-stimulation of iPSC. d Image quantification of Pax6- and HB9-labeled cells exemplified the percentage of cells that transitioned from NSC to MN. e Immunocytochemistry with anti-ISL1 antibody and quantitative analysis of biomarker positive cells at D28 of the differentiation protocol. The graphs show average of 6 cultures with > 300 cells in random fields for each culture. Scale bar: 100 µm

Fig 3: Comparative analysis of differential gene expression of key tissue development markers by RT-qPCR (gray) and RNA-seq (black). Pluripotency and NSC markers validated include (a) Oct4, transcription factor that maintains self-renewal and pluripotency; (b) Nestin, a filament protein marker of neural stem cells; (c) Pax6, a transcription factor that drives neurogenesis; and (d) NgN2, a neuronal-specific transcription factor. Motor neuron specification markers validated include (e) Isl1, a transcription factor required for motor neuron generation; (f) Map2, a neuron-specific cytoskeletal protein; (g) HB9, an early marker of cholinergic neurons; and (h) ChAT, an enzyme required for acetylcholine synthesis. Shown are the averages and standard error from three independent biological replicas from the iPSC to MN differentiation trajectory. The RNA-Seq transcripts were normalized to the total read per analyzed sample (in FPKM: fragments per kilobase per million mapped fragments) and the transcript levels determined by RT-qPCR were normalized to GAPDH as the endogenous sample control. Fold change was calculated for each developmental stage relative to transcript levels in iPSC (D0). Statistical significance (p < 1.5e-6) was determined with Student t-test

Fig 4: Reconstitution of the human substantia nigra Substantia Nigra Brain-Chip.a Schematic depiction of the Substantia Nigra Brain-Chip of a two-channel microengineered chip with iPSC-derived brain endothelial cells cultured on all surfaces of the lower vascular channel, and iPSC-derived dopaminergic neurons, primary human brain astrocytes, microglia, and pericytes on the upper surface of the central horizontal membrane in the upper brain channel. b 3D reconstruction of a confocal z-stack showing the organization of all five cell types in the Substantia Nigra (SN) Brain-Chip. c Representative image of iPSC-derived dopaminergic neurons that are stained with DAPI (blue), microtubule-associated protein 2 (green, MAP2), tyrosine hydroxylase (red, TH), and a merged image on day 8. Scale bars: 100 µm. d Immunofluorescence micrographs of the human brain endothelium cultured on the vascular channel of Brain-Chip for 7 days post-seeding (D8) labeled with Claudin-1 (red), Claudin-5 (cyan), Occludin (yellow), and CD31 (white). Scale bars: 100 µm. e Immunofluorescence micrographs demonstrate high levels of expression of zonula occludens-1 (red, ZO-1) across the entire endothelial monolayer. Scale bars: 100 µm. f Quantitative barrier function analysis via apparent permeability to 3 kDa fluorescent dextran, and 0.5 kDa lucifer yellow (LucY) crossing through the vascular to the neuronal channel on day 5 and 8. Statistical analysis is two-way ANOVA with Tukey’s multiple comparisons test (n = 18 independent chips, NS not significant). Error bars represent mean ± SEM.

Fig 5: aSyn-induced caspase-3 activation and neuroinflammation.a Representative merged images showing immunostaining for microtubule-associated protein 2 (gray, MAP2), tyrosine hydroxylase (red, TH), and Cleaved Caspase-3 (green, CC3) in the brain channel at 6-days post-exposure to aSyn fibrils or aSyn monomers. Scale bars: 100 µm. b Quantitation of the number of CC3 and MAP2- or TH-positive neurons. Statistical analysis by two-way ANOVA with Tukey’s multiple comparisons test (3–4 randomly selected different areas per chip, n = 3 independent chips/experimental group, **P = 0.0033, ****P < 0.0001, NS not significant). Error bars represent mean ± SEM. Scale bars: 100 µm. c Immunostaining of the astrocyte marker glial fibrillary acidic protein (magenta, GFAP) demonstrated activation of astrocytes (white arrow) at day 6 post exposure to aSyn fibrils compared to monomeric aSyn. Scale bar, 100 µm. d Immunostaining of the microglial CD68 (red) demonstrated activation of microglia (white arrow) at day 6 post exposure to aSyn fibrils compared to monomeric aSyn. Scale bar, 100 µm. e The secreted levels of tumor necrosis factor-alpha (TNF-a) in the aSyn fibril model. Statistical analysis is two-sided unpaired t-test (n = 6–7 independent chips, **P = 0.0023). Error bars represent mean ± SEM. f The secreted levels of proinflammatory cytokine interleukin-6 (IL-6) in the aSyn fibril model. Statistical analysis is two-sided unpaired t-test (n = 4–7 independent chips, ****P < 0.0001). Error bars represent mean ± SEM.