A High-Throughput Screen for Wnt/β-Catenin Signaling Pathway Modulators in Human iPSC-Derived Neural Progenitors

Abstract

Wnt/β-catenin signaling has emerged as a central player in pathways implicated in the pathophysiology and treatment of neuropsychiatric disorders. To identify potential novel therapeutics for these disorders, high-throughput screening (HTS) assays reporting on Wnt/β-catenin signaling in disease relevant contexts are needed. The use of human patient-derived induced pluripotent stem cell (iPSC) models provides ideal disease relevant context if these stem cell cultures can be adapted for HTS-compatible formats. Here, we describe a sensitive, HTS-compatible Wnt/β-catenin signaling reporter system generated in homogeneous, expandable neural progenitor cells (NPCs) derived from human iPSCs. We validated this system by demonstrating dose responsive stimulation by several known Wnt/β-catenin signaling pathway modulators, including Wnt3a, a glycogen synthase kinase-3 (GSK3) inhibitor, and the bipolar disorder therapeutic lithium. These responses were robust and reproducible over time across many repeated assays. We then conducted a screen of ~1,500 compounds from a library of FDA-approved drugs and known bioactives, and confirmed HTS hits, revealing multiple chemical and biological classes of novel small molecule probes of Wnt/β-catenin signaling. Generating this type of pathway-selective, cell-based phenotypic assays in human iPSC-derived neural cells will advance the field of human experimental neurobiology toward the goal of identifying and validating targets for neuropsychiatric disorder therapeutics.

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

“…Another advantage of adherent culture systems is that more uniform exposure to morphogens and growth/differentiation factors is achieved. 12 Neural rosettes (which represent a distinct class of NSCs) 17 generated in these adherent culture systems are isolated mechanically, then transferred and cultured into low attachment plates, where they form spherical cell aggregates called neurospheres that can be propagated as 3-dimensional structures, 18 or expanded as monolayer cultures of NSCs/ NPCs 11 However, neurospheres are not ideal for large-scale production of neurons in multi-well plates for high-throughput screening because of technical difficulties in loading uniform numbers of spheres with uniform size into individual wells. Thus, monolayer cultures of NSCs/NPCs would be advantageous.…”

Phenotypic Screens Targeting Neurodegenerative Diseases

“…This system was validated in 384-well format, and an FDAapproved drug library of 1500 compounds was screened with the identification of several activators for the Wnt/β-catenin pathway. 60 As activation of the Wnt/β-catenin pathway plays an important role in neurogenesis, the screen demonstrated the feasibility of establishing a pathway-specific phenotypic assay for neural regeneration.…”

Induced Pluripotent Stem Cell Models to Enable In Vitro Models for Screening in the Central Nervous System

“…Zhao et al provide a noteworthy example supporting iPSC-based screens for novel drug discovery [27]. They developed a high-throughput compatible Wnt/beta-catenin signaling reporter system using neural progenitor cells derived from human iPSCs.…”

“…Control, disease-specific, and engineered iPSCs and their secondary derivatives could be used in numerous Tumor line; lack patient and disease specificity; therefore, difficult to determine biological significance; difficult to maintain characteristics in culture Rodent cells Abundant supply; difficult to obtain functional data of neurological functions; models may lack human-specific response Zebrafish Abundant supply; lack patient and disease specificity; difficult to culture cells, immature cells; wider species differences and differing biological response types of screens, including large-scale small molecules, mechanism of action, repurposing of FDA-approved drugs, toxicity-related, high-content, and genomic types [30,31]. Each of these screens utilizes distinct screening agents, including siRNAs, natural compounds, biologics, small molecules, and even FDA-approved drug libraries [27]. These can further include measurement approaches ( Fig.…”