Engineered 3D immuno-glial-neurovascular human miBrain model

Abstract

Patient-specific, human-based cellular models integrating a biomimetic blood-brain barrier, immune, and myelinated neuron components are critically needed to enable accelerated, translationally relevant discovery of neurological disease mechanisms and interventions. To construct a human cell-based model that includes these features and all six major brain cell types needed to mimic disease and dissect pathological mechanisms, we have constructed, characterized, and utilized a multicellular integrated brain (miBrain) immuno-glial-neurovascular model by engineering a brain-inspired 3D hydrogel and identifying conditions to coculture these six brain cell types, all differentiated from patient induced pluripotent stem cells. miBrains recapitulate in vivo-like hallmarks inclusive of neuronal activity, functional connectivity, barrier function, myelin-producing oligodendrocyte engagement with neurons, multicellular interactions, and transcriptomic profiles. We implemented the model to study Alzheimer's Disease pathologies associated with APOE4 genetic risk. APOE4 miBrains differentially exhibit amyloid aggregation, tau phosphorylation, and astrocytic glial fibrillary acidic protein. Unlike the coemergent fate specification of glia and neurons in other organoid approaches, miBrains integrate independently differentiated cell types, a feature we harnessed to identify that APOE4 in astrocytes promotes neuronal tau pathogenesis and dysregulation through crosstalk with microglia.

Keywords: biomaterials; brain organoid; microphysiological system; neuro-immune; neurovascular.

Fig. 1.

Human integrated 3D-immuno-glial-neurovascular miBrain model. (A) Schematic of miBrain formation (B) macroscopic view of miBrains from (i) Top and (ii) side view with a dime for reference, (C) distribution of (Left to Right) iPSC-derived pericytes (cyan: mCherry-pericytes), astrocytes (cyan: mCherry-astrocytes), iPSC-derived BMECs (cyan: mCherry-BMECs), neurons (cyan: tubulin), oligodendroglia (cyan: tdTomato pretransfected oligodendroglia), and iPSC-derived microglia (iMG) (cyan: membrane prelabeled iMG), (D) neuronal phenotypes in miBrains cultured in dextran-based hydrogels fabricated with various brain ECM proteins (cyan: neurofilament, blue: Hoechst); (Scale bar, 50 µm), (E) quantification of neurofilament immunoreactivity (averages of n = 8 samples, n = 3 FOV per sample, ordinary one-way ANOVA statistical test), (F) neuronal firing as assessed on an MEA system (n = 3 wells per group, mean, SEM, ordinary one-way ANOVA statistical test), (G) persistence of neurovascular unit phenotypes in miBrains cultured in Neuromatrix Hydrogel versus Matrigel after 5 wk in culture [red: PECAM, cyan: tubulin beta III (TUBB3), blue: Hoechst]; (Scale bar, 50 µm), (H) macroscopic view of gel structural integrity for miBrains cultured in versican (VCN)-incorporated engineered dextran-based hydrogel named Neuromatrix Hydrogel versus Matrigel after 4 wk, (I) example raster plots from MEA recordings of miBrains in Neuromatrix Hydrogel compared to Matrigel; miBrains recapitulate key hallmarks of human brain tissue, inclusive of (J) neurovascular units: (i) 3D BMEC and neuronal networks (red: mCherry-BMEC-surfaces, cyan: tubulin neuron label); (Scale bar, 500 µm), (ii) higher magnification (red: mCherry-BMEC-surfaces, cyan: siR-tubulin); (Scale bar, 500 µm), (iii) 3D integrated astrocyte and neuronal networks (green: mCherry-astrocyte-surfaces, cyan: siR-tubulin); (Scale bar, 500 µm), (iv) higher magnification (green: mCherry-astrocytes, cyan: tubulin); (Scale bar, 100 µm), and (v) miBrain 3D rendering (red: PECAM, cyan: neurofilament, blue: Hoechst); (Scale bar, 50 µm), (K) microglia: (i) 3D iMG distributed throughout BMEC networks (Imaris surfaces from red: mCherry-BMECs, green: membrane prelabeled iMG); (Scale bar, 500 µm), (ii) higher magnification (Imaris surfaces from red: mCherry-BMECs, green: membrane prelabeled iMG); (Scale bar, 100 µm), (iii) 3D iMG distributed throughout neuronal networks (cyan: tubulin, green: membrane prelabeled iMG-surfaces); (Scale bar, 500 µm), (iv) higher magnification (Imaris reconstruction of cyan: tubulin, green: membrane prelabeled iMG); (Scale bar, 100 µm), and (v) distribution of iMG with BMEC and neuronal networks (cyan: tubulin; Imaris surfaces from red: mCherry-BMECs, green: membrane prelabeled iMG); (Scale bar, 100 µm), (L) BBB: (i) 3D astrocytes distributed throughout BMEC networks (red: Imaris surfaces from ZO1-BMECs, green: mCherry-astrocytes); (Scale bar, 500 µm), (ii) higher magnification [Imaris surfaces from red: zona occludens-1 (ZO-1)-BMECs, green: mCherry-astrocytes]; (Scale bar, 100 µm), (iii) magnified (Scale bar, 50 µm), (iv) ZO-1 tight junctions (green: ZO-1, red: PECAM, blue: Hoechst); (Scale bar, 30 µm), (v) astrocytes at the vasculature [green: GFAP, gray: aquaporin-4 (AQP4), red: PECAM, blue: Hoechst; (Scale bar, 30 µm); insert, gray: AQP4], and (vi) pericytes at the vasculature [green: neuron-glial antigen 2 (NG2), red: PECAM, blue: Hoechst]; (Scale bar, 30 µm), and (M) myelinated neuronal networks: (i) 3D oligodendroglia distributed throughout neuronal networks (cyan: tubulin, green: tdTomato pretransfected oligodendroglia-surfaces); (Scale bar, 500 µm), (ii) higher magnification (cyan: tubulin, green: tdTomato pretransfected oligodendroglia); (Scale bar, 100 µm), (iii) myelin dye-labeled neurons and oligodendroglia (red: FluoroMyelin, cyan: tubulin, green: tdTomato pretransfected oligodendroglia), (Scale bar, 100 µm), (iv) visualizing via Imaris reconstructions (cyan: tubulin, Imaris surfaces of red: FluoroMyelin, green: tdTomato pretransfected oligodendroglia); (Scale bar, 100 µm), and (v) myelin and oligodendroglia alone (Imaris surfaces of red: FluoroMyelin, green: tdTomato pretransfected oligodendroglia); (Scale bar, 100 µm), and (vi) myelination of neuronal projections (green: MBP, cyan: neurofilament, blue: Hoechst); (Scale bar, 10 µm).

Fig. 2.

Neuronal and vascular phenotypes enabled by the miBrain. (A) Characterization of neurons integrated into miBrain immunohistochemistry [green: microtubule-associated protein 2 (MAP2), red: TUBB3, blue: Hoechst]; (Scale bar, 30 µm), (B) synapses in miBrain (Top) versus neuronal monocultures (Bottom) [red: vesicular glutamate transporter 1 (vGlut1), green: postsynaptic density protein 95 (PSD95), cyan: neurofilament, blue: Hoechst]; (Scale bar, 5 µm), (C) quantification of synapses (number of PSD95/synapsin colocalized puncta per volume neurofilament; n = 4, Student’s t test P = 0.0185), (D) macroscopic image of miBrain on MEA, (E) example raster plot miBrain at week 10, (F) characterization of spontaneous activity: (Left) weighted mean firing rate, (Right) burst frequency (mean, SEM; statistical analysis as unpaired t test), (G) evoked activity: (Left) latency after stimulation, (Right) number of evoked spikes per trial (mean, SEM; statistical analysis as unpaired t test), (H) assessing neuronal connectivity between two neuronal populations in miBrain for: (Top) channelrhodopsin-2 (ChR2)-mCherry for optogenetic stimulation (green: ChR2-mCherry-neurons), (Bottom) control tdTomato for the no stimulation condition (green: tdTomato-neurons) and (Right) sequential images of calcium transients imaged under blue light (cyan: GCaMP6f-neurons), (Scale bar, 30 µm), (I) corresponding example spike traces (J) quantification of calcium events (mean dF/F per calcium trace per neuron recorded (n = 3 miBrains/group, repeated in three independent trials; statistical analysis via Student t test P = 0.0010), (K) lumenized 3D BMEC vessels (red: PECAM, green: ZO-1, blue: Hoechst); (Scale bar, 15 µm), and (L) claudin-5 (CLDN-5) tight junctions along vessels (green: CLDN-5, cyan: ZO-1, red: PECAM, blue: Hoechst); (Scale bar, 100 µm).

Fig. 3.

Glial phenotypes enabled by the miBrain. (A) immunohistochemistry for astrocyte marker S100 calcium-binding protein B (S100β) (green: S100β, red: mCherry-BMEC, blue: Hoechst); (Scale bar, 50 µm), (B) expression of key in vivo genes in RNAseq from astrocytes isolated from miBrains versus monoculture (TMM-normalized, scaled expression), (C) gene ontology analysis of biological pathways upregulated in miBrain-cultured astrocytes based on significantly upregulated and downregulated DEGs (FDR P-value < 0.05), (D) calcium imaging of pericytes (Left, blue) before and (Right, red) during a signaling event; (Scale bar, 10 µm), (E) example traces of calcium transients, (F) spike traces (Top, blue) with and (Bottom, red) without application of vasoconstrictor ET-1, (G) quantification of rate of calcium events (mean, SEM; statistical analysis via paired t test), (H) miBrain-iMG immunoreactivity to purinergic receptor P2RY12 (cyan: P2RY12, green: neurofilament, red: PECAM, blue: Hoechst); (Scale bar, 50 µm), (I) expression of key in vivo genes in RNAseq of miBrain-iMG versus monoculture (TMM-normalized, scaled expression; *P < 0.05, **P < 0.01, ***P < 0.001), (J) gene ontology analysis for biological pathways significantly altered in miBrain-cultured iMG, (K) characterization of myelination via immunohistochemistry (red: MBP, green: neurofilament, blue: Hoechst); (Scale bar, 10 µm), (L) 3D reconstructions of MBP-positive regions around neuronal projections with (i) Top and (ii) side views (red: MBP-Surface, green: neurofilament-Surface, blue: Hoechst); (Scale bar, 10 µm), (M) expression of key myelination genes in RNAseq of miBrain-oligodendroglia versus monoculture (TMM-normalized, scaled expression), (N) gene ontology analysis of biological pathways significantly altered in miBrain-cultured oligodendroglia based on significantly upregulated and downregulated DEGs, and (O) expression of key genes associated with OPC development in RNAseq of miBrain-oligodendroglia versus monoculture (TMM-normalized, scaled expression).

Fig. 4.

miBrain reveals APOE4 astrocytes promote tau phosphorylation via crosstalk with microglia. (A) GFAP immunoreactivity in APOE3 (Left) versus APOE4 (Right) miBrains (green: GFAP, blue: Hoechst); (Scale bar, 50 µm), (B) quantification of GFAP (mean intensity normalized by nuclei area; n = 3 samples, images analyzed from n = 6 max projections; statistical analysis by the t test), (C) S100β immunoreactivity in APOE3 (Left) versus APOE4 (Right) miBrains (green: S100b, blue: Hoechst); (Scale bar, 50 µm), (D) quantification of S100β (mean intensity normalized by nuclei area; n = 3 samples, images analyzed from n = 6 max projections; statistical analysis by the t test), (E) DEGs in human astrocytes from snRNAseq dataset (22) of APOE3/4 and APOE4/4 versus APOE3/3 individuals (positive log[FC] is upregulated in APOE4) for reactive astrocyte genes, (F) hydrogen peroxide (H2O2) in APOE3 (Left) versus APOE4 (Right) astrocytes incorporated into otherwise-APOE3 miBrains (green: H2O2, red: tubulin, cyan: RFP-astrocytes, blue: Hoechst); (Scale bar, 50 µm), (G) quantification of (i) mean H2O2 intensity colocalized with astrocytes, (ii) mean H2O2 intensity in positive regions, and (iii) mean H2O2 intensity colocalized with tubulin-positive regions (n = 3 replicates, images from n = 6 max projections; statistical analysis via t test; conducted in three independent trials), (H) NO in miBrains with APOE3 (Left) versus APOE4 (Right) astrocytes incorporated into otherwise-APOE3 miBrains (green: NO, red: RFP-astrocytes, blue: Hoechst); (Scale bar, 50 µm), (I) quantification of: (i) NO droplets colocalized with astrocytes normalized by astrocyte area, (ii) number of NO droplets normalized by nuclei area, and (iii) total area of NO segmented particles normalized by nuclei area (n = 3 replicates, images from n = 9 max projections; statistical analysis via t test; conducted in three independent trials), (J) tau phosphorylation in APOE3 (Left), APOE4 (Middle), and APOE3 miBrains with APOE4 astrocytes (Right) treated with 20 nM exogenous Aβ 1-42 (red: AT8, green: TUBB3, blue: Hoechst); (Scale bar, 30 µm), (K) quantification of somatic tau phosphorylation in TUBB3-positive neurons (mean AT8 intensity per cell; n = 3 replicates, images from n = 12 fields of view; statistical analysis via ANOVA; repeated for three independent trials), (L) tau phosphorylation in APOE4 miBrains with iMG (Left) versus without iMG (Right) treated with exogenous Aβ 1-42 (red: AT8, green: TUBB3, blue: Hoechst); (Scale bar, 50 µm), (M) quantification of somatic tau phosphorylation in TUBB3-positive neurons (mean AT8 intensity per cell; n = 3 replicates, images from n = 12 fields of view; statistical analysis via t test; repeated for three independent trials), (N) schematic illustrating experiments testing the effect of APOE4 microglia-astrocyte crosstalk by applying CM from astrocyte monocultures and microglia monocultures versus microglia-astrocyte coculture, (O) tau phosphorylation in APOE4 miBrains treated with (Left) media only, (Middle) monoculture CM, (Right) coculture CM, coadministered with exogenous Aβ 1-42 (red: AT8, green: TUBB3, blue: Hoechst); (Scale bar, 50 µm), and (P) quantification of somatic tau phosphorylation in TUBB3-positive neurons (mean AT8 intensity normalized by mean TUBB3 intensity per cell; n = 3 replicates, images from n = 12 fields of view; statistical analysis via ANOVA).