Human iPSC-Based Modeling of Central Nerve System Disorders for Drug Discovery

Abstract

A high-throughput drug screen identifies potentially promising therapeutics for clinical trials. However, limitations that persist in current disease modeling with limited physiological relevancy of human patients skew drug responses, hamper translation of clinical efficacy, and contribute to high clinical attritions. The emergence of induced pluripotent stem cell (iPSC) technology revolutionizes the paradigm of drug discovery. In particular, iPSC-based three-dimensional (3D) tissue engineering that appears as a promising vehicle of in vitro disease modeling provides more sophisticated tissue architectures and micro-environmental cues than a traditional two-dimensional (2D) culture. Here we discuss 3D based organoids/spheroids that construct the advanced modeling with evolved structural complexity, which propels drug discovery by exhibiting more human specific and diverse pathologies that are not perceived in 2D or animal models. We will then focus on various central nerve system (CNS) disease modeling using human iPSCs, leading to uncovering disease pathogenesis that guides the development of therapeutic strategies. Finally, we will address new opportunities of iPSC-assisted drug discovery with multi-disciplinary approaches from bioengineering to Omics technology. Despite technological challenges, iPSC-derived cytoarchitectures through interactions of diverse cell types mimic patients' CNS and serve as a platform for therapeutic development and personalized precision medicine.

Keywords: Alzheimer’s disease; CNS disease modeling; COVID-19; astrocytes; drug discovery; induced pluripotent stem cells (iPSC); neurons; three-dimensional cerebral organoids; toxicity screening.

Figure 1

3D induced pluripotent stem cell (iPSC)-based vascularized organoids for drug perfusion modeling. (A) iPSC-derived blood-brain-barrier model. Astrocytes, pericytes and endothelial cells are generated from human iPSCs and mixed in suspension for self-aggregation. The resulted spheroid comprises an inner layer of astrocytes, intermediate layer of pericytes, and an outer layer of endothelial cells. (B) Scaffold-dependent vascularization of organoids through sacrificial writing into functional tissues (SWIFT). Cerebral organoids are mixed and compacted into a solid-like construct in an appropriate extracellular matrix medium. The sacrificial gelatin ink is 3D typed into the tissue construct. The tissue construct solidifies with increased temperature, while the sacrificial ink melts away, leaving tubular structures embedded within. Perfusion of endothelial cells endothelializes the interior of the channels and form vascular beds. (C) External revascularization of human iPSC-cerebral organoids within mouse brain cavity. Non-vascularized organoids are seeded onto endothelial containing matrigel droplets DIV 34 and transplanted onto raw edges of the resection cavity in the mouse brain. (D) ETV2-facilitated endogenous vascularization of cerebral organoids in vitro. A mixture of BC4 hECs and ETV2-infected BC4 hECs is prepared with a 20% cell infection rate. On day 18 of organoid differentiation, viral induction is initiated. Endothelial tubes are formed along the organoid differentiation and continuously evolve with increased structural complexity. (E) A dual-channeled microifluidic organ/organ-on-a chip (OoC) to model neural-vascular interactions. Human iPSC-derived spinal neural progenitors (hiPSC-spNPs) are seeded on the top channel and iPSC-derived brain endothelial cells (BECs) on the bottom. Vasculature develops as the neural progenitors differentiate into spinal neurons with mediated signaling between two different cell types.

Figure 2

iPSC modeling of diverse CNS diseases for phenotype studies and drug discovery. (A) A proof-of-concept experiment to conformationally correct APOE4 in Alzheimer’s disease (AD) with the compound PH002. PH002 improves cytopathologies of APOE 44 human iPSC-GABAergic neurons with reduced p-tau accumulation, Aβ40/Aβ42 production/secretion, and ApoE4 fragments. (B) A co-culture model of astrocytes and ventral midbrain dopaminergic neurons (vmDAns) derived from Parkinson’s disease (PD) patient-specific iPSCs carrying LRRK2 G2019S mutation. Co-culture of LRRK2 G2019S astrocytes with healthy vmDAns induces α-syn toxicity and vmDAns neuron degeneration as observed in PD patient brains. (C) Model of de novo TDP-43 proteinopathy and motor neuron (MN) degeneration in sporadic amyotrophic lateral sclerosis (ALS) patient iPSC-MNs. Automated robotics is applied on a high-content assay to screen against 1757 bioactive compounds. Based on the evaluation of reduced TDP-43 aggregation, 38 hit compounds are identified to effectively reduce the percent cells containing TDP-43 aggregates. (D) Utilize Huntington’s disease (HD) iPSCs as a model to study reprogramming-associated anti-mHTT aggregation compared to aggregate-free HD iPSCs. Treatment of the proteasomal inhibitor MG-132 increases mHTT aggregates in HD iPSCs, indicating a correlation between anti-mHTT aggregation and proteasomal homeostasis. Genome-wide association studies (GWAS) analysis shows upregulation of UBR5 particularly at the iPSC stage, which rejuvenates the proteasomal function of HD iPSCs to actively degrade mHTT aggregates. UBR5 thus serves as a potential therapeutic target to remove mHTT aggregation in HD patient cells. (E) Generation of neural progenitors and immature neurons from isogenic Rett syndrome (RTT) patient iPSCs to model MeCP2 mutation-associated neurogenic deficiency during the early brain development. In comparison with the healthy control, MeCP2 mutant neural progenitors and neurons show upregulated microRNA miR-199 and miR-214 in microRNA screening. Molecularly, immunoblots show decreased ERK activation and increased AKT activation, implicating a possibility of delayed neuron differentiation. (F) Treatment of recombinant human IGF-1 to improve neural performance in RTT. RTT iPSC-astrocytes are found to reduce the amount of terminal ends of RTT iPSC-GABAergic interneurons in co-culture. Short-term administration of IGF-1 modestly improves the neurite length of RTT-neurons in a diseased environment. (G) Model familial dysautonomia (FD) of various disease severities with FD iPSC-rosette neural crests (rNCs). In comparison to a healthy control, mild FD rNCs do not display differences in cell migration or the expression level of ASCL1, a proneural transcriptional factor for early neurogenesis and progenitor differentiation. In contrast, severe FD rNCs show compromised cell motility and decreased ASCL1 expression, indicating a disease severity-dependent functional defect that might shape the accuracy of disease modeling. (H) Compound screen flow chart for ZIKV therapy development. Three different cell types are used as a platform for drug screen: iPSC-neural progenitor, iPSC-astrocytes, and glioblastoma cell (SNB-19). SNB-19 and neural progenitors are exposed to ZIKV infection and screened against compounds that reduce apoptotic caspase-3 activity. The derived primary hits are re-evaluated on all cell types along with cytotoxicity assay. ATP assay is used as secondary confirmation of post-infection cell survival. (I) Utilize human iPSC-Choroid plexus (ChP) and cortical organoids to investigate neurotropism in SARS-CoV-2 infection. GFP-encoded pseudovirion is made to intracellularly visualize viral invasion. When cortical organoids mixed with ChP cells are exposed to the virus, only ChP cells but not neural progenitors or neurons show positive infection signals; infection does not spread to nearby cortical regions but infected ChP cells lead to global transcriptomic dysregulations in multiple cellular functions (top). When air–liquid-interface cerebral organoids (ALI-COs) are introduced with greater neuronal maturity to examine the viral infection in neurons, signal is undetectable relative to the positive control, evidencing a preferred attack on ChP cells and a lack of viral entry in neural cells (bottom).