(91e) 3D Human iPSC-Derived Immuno-Glial-Neurovascular Mibrain Tissue Model for Enhanced Modeling and Therapeutic Discovery and Development

Up to 1 in 6 people worldwide, 1 billion people, are estimated to have a neurological disorder, costing over $500B in the US alone. For most neurological diseases there is still no pharmacologic treatment available that can slow or stop neuronal damage. To advance the discovery and development of effective treatments for neurological disease, better preclinical models are critically needed. We sought to establish a human-based, patient-specific model inclusive of integrated vascular networks and blood-brain barrier, myelinated neuronal networks, and microglia, with in vivo-like functionalities for enhanced preclinical studies, that can provide novel insights into disease mechanisms and effects of interventions (Fig. 1A,B). By engineering a novel brain-mimicking 3D hydrogel and enabling the integral co-culture of all six major brain cell types derived from patient iPSCs, we have established a 3D immuno-glial-neurovascular brain model (miBrain) (Fig. 1C). Taking inspiration from the human brain, we developed the Neuromatrix hydrogel to mimic the composition of brain extracellular matrix, the majority of which is comprised of glycosaminoglycans interspersed with proteoglycans surrounding neurons and glia in the interstitial space, within which are basement membrane proteins lining the vascular networks. Correspondingly, we developed a glycosaminoglycan dextran-based hydrogel with mechanical properties tuned to support the miBrain structurally, while enabling both neuronal and vascular network formation and the integral inclusion of the other four cell components, screened proteoglycan components to promote neuronal maturation and activity, and incorporated basement membrane protein mimics to facilitate vascular network formation (Fig. 1D-I). Differentiating each of the six major brain cell types from patient-specific iPSCs, we optimized cell protocols and developed a method for combining the cells together in Neuromatrix Hydrogel in a mixed media. This method and its underlying technologies enable a 3D immuno-glial-neurovascular co-culture inclusive of all six major brain cell types with enriched cellular phenotypes and tissue-scale phenotypes. miBrains mimic in vivo-like hallmarks of human brain with integrated neurovascular unit (Fig. 1J), enhanced in vivo-like microglia signatures (Fig. 1K), a blood-brain barrier with lumenized microvessels with tight junctions and surrounded by pericytes and astrocytic endfeet (Fig. 1L), myelinated neuronal networks (Fig. 1M), neuron maturation with robust activity and electrophysiological properties, enrichment of important in vivo-like transcriptional signatures, and functional interactions between cell types. This is the first brain model to our knowledge that integrates all six major brain cell types, and one which does so with biomimetic 3D tissue architecture and with all iPSC-derived cells. To model Alzheimer’s Disease (AD) pathogenesis associated with the strongest genetic risk factor for sporadic AD, we constructed miBrains from cell lines of APOE4 genetic background versus those of APOE3. We found that APOE4 miBrains differentially exhibit amyloid aggregation, tau phosphorylation, and astrocytic GFAP. Harnessing the decoupled-genotype feature of our platforms, we probed the effect of APOE4 astrocytes, identifying that APOE4 astrocytes promote tau pathogenesis and neuronal dysregulation through crosstalk with microglia, proposing a mechanism that could be investigated for therapeutic potential. This work has established an enhanced brain tissue mimic that recapitulates human brain physiology and pathology with unprecedented biomimicry. The miBrain holds promise for enabling a new approach to target discovery for neurological disease and a valuable tool for therapeutic screening and testing of drug efficacy.

Fig. 1: Human Integrated 3D-Immuno-Glial-Neurovascular miBrain Model. (A) Schematic of miBrain formation harnessing patient-specific iPSCs differentiated into each of the resident brain cell types, encapsulated in Neuromatrix Hydrogel, and co-cultured for integral cell network self-assembly and microglia-like cell integration, (B) macroscopic view of miBrains from (i) top and (ii) side angle pictured with a dime for reference, (C) distribution of (from left to right) iPSC-derived pericytes (cyan: mCherry-pericytes), astrocytes (cyan: mCherry-astrocytes), BMECs (cyan: mCherry-BMECs), neurons (cyan: tubulin neuron label), oligodendroglia (cyan: tdTomato pre-transfected oligodendroglia), and iMG (cyan: membrane pre-labeled iMG) throughout the full 3D miBrain, (D) neuronal phenotypes in miBrains cultured in dextran-based hydrogels fabricated with various brain ECM proteins (cyan: neurofilament, blue: Hoechst; scale bars, 50 µm), (E) quantification of neurofilament immunoreactivity (averages of n = 8 samples from n = 3 fields of view per sample), (F) neuronal firing as assessed on an MEA system across conditions (n = 3 wells per group, displayed as mean and S.E.M.), (G) persistence of neurovascular unit phenotypes in miBrains cultured in Neuromatrix Hydrogel versus Matrigel after 5 weeks in culture (red: PECAM, cyan: TUBB3, blue: Hoechst; scale bar, 50 µm), (H) macroscopic view of gel structural integrity for miBrains cultured in VCN-incorporated engineered dextran-based hydrogel named Neuromatrix Hydrogel versus Matrigel after 4 weeks, (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 integrated BMEC and neuronal networks throughout the miBrain (red: Imaris reconstruction of mCherry-BMECs, cyan: tubulin neuron label, scale bar, 500 µm), (ii) visualized at higher magnification (red: Imaris surfaces of mCherry-BMECs, cyan: siR-tubulin neuron label; scale bar, 500 µm), (iii) 3D integrated astrocyte and neuronal networks throughout the miBrain (green: Imaris surfaces of mCherry-astrocytes, cyan: siR-tubulin neuron label; scale bar, 500 µm), (iv) visualized at higher magnification (green: mCherry-astrocytes, cyan: tubulin neuron label, scale bar, 100 µm), and (v) anterior view of a miBrain 3D rendering (red: PECAM, cyan: neurofilament, blue: Hoechst; scale bar, 50 µm), (K) microglia: (i) 3D iMG distributed throughout BMEC networks throughout the miBrain (red: Imaris surfaces from mCherry-BMECs, green: Imaris surfaces from membrane pre-labeled iMG; scale bar, 500 µm), (ii) visualized at higher magnification (red: Imaris surfaces from mCherry-BMECs, green: Imaris surfaces from membrane pre-labeled iMG; scale bar, 100 µm), (iii) 3D iMG distributed throughout neuronal networks throughout the miBrain (cyan: tubulin neuron label, green: Imaris surfaces from membrane pre-labeled iMG; scale bar, 500 µm), (iv) visualized at higher magnification (cyan: Imaris reconstruction of tubulin neuron label, green: Imaris surfaces from membrane pre-labeled iMG; scale bar, 100 µm), and (v) distribution of iMG with BMEC and neuronal networks together (cyan: tubulin neuron label, red: Imaris surfaces from mCherry-BMECs, green: Imaris surfaces from membrane pre-labeled iMG; scale bar, 100 µm), (L) blood-brain barrier: (i) 3D astrocytes distributed throughout BMEC networks throughout the miBrain (red: Imaris surfaces from ZO1-BMECs, green: mCherry-astrocytes; scale bar, 500 µm), (ii) visualized at higher magnification (red: Imaris surfaces from ZO1-BMECs, green: Imaris surfaces from mCherry-astrocytes; scale bar, 100 µm), (iii) further magnified (scale bar, 50 µm), (iv) ZO-1 tight junctions along vessels (green: ZO-1, red: PECAM, blue: Hoechst; scale bar, 30 µm), (v) astrocytes with end-feet extending to vessels expressing canonical aquaporin-4 transporter (green: GFAP, gray: AQP4, red: PECAM, blue: Hoechst; scale bar, 30 µm; insert, gray: AQP4), and (vi) pericytes localized to the vessels (green: NG2, red: PECAM, blue: Hoechst; scale bar, 30 µm), and (M) myelinated neuronal networks: (i) 3D oligodendroglia distributed throughout neuronal networks throughout the miBrain (cyan: tubulin neuron label, green: Imaris surfaces from tdTomato pre-transfected oligodendroglia; scale bar, 500 µm), (ii) visualized at higher magnification (cyan: tubulin neuron label, green: tdTomato pre-transfected oligodendroglia; scale bar, 100 µm), (iii) myelin dye-labeled neurons and oligodendroglia (red: FluoroMyelin, cyan: tubulin neuron label, green: tdTomato pre-transfected oligodendroglia, scale bar, 100 µm), (iv) visualized also via Imaris reconstructions for myelin along with neurons and oligodendroglia (red: Imaris surfaces of FluoroMyelin, cyan: tubulin neuron label, green: Imaris surfaces of tdTomato pre-transfected oligodendroglia; scale bar, 100 µm), and (v) myelin and oligodendroglia alone (red: Imaris surfaces of FluoroMyelin, green: Imaris surfaces of tdTomato pre-transfected oligodendroglia; scale bar, 100 µm), and (vi) myelination of neuronal projections (green: MBP, cyan: neurofilament, blue: Hoechst; scale bar, 10 µm).