A Simple Procedure for Creating Scalable Phenotypic Screening Assays in Human Neurons

Abstract

Neurons created from human induced pluripotent stem cells (hiPSCs) provide the capability of identifying biological mechanisms that underlie brain disorders. IPSC-derived human neurons, or iNs, hold promise for advancing precision medicine through drug screening, though it remains unclear to what extent iNs can support early-stage drug discovery efforts in industrial-scale screening centers. Despite several reported approaches to generate iNs from iPSCs, each suffer from technological limitations that challenge their scalability and reproducibility, both requirements for successful screening assays. We addressed these challenges by initially removing the roadblocks related to scaling of iNs for high throughput screening (HTS)-ready assays. We accomplished this by simplifying the production and plating of iNs and adapting them to a freezer-ready format. We then tested the performance of freezer-ready iNs in an HTS-amenable phenotypic assay that measured neurite outgrowth. This assay successfully identified small molecule inhibitors of neurite outgrowth. Importantly, we provide evidence that this scalable iN-based assay was both robust and highly reproducible across different laboratories. These streamlined approaches are compatible with any iPSC line that can produce iNs. Thus, our findings indicate that current methods for producing iPSCs are appropriate for large-scale drug-discovery campaigns (i.e. >10e 5 compounds) that read out simple neuronal phenotypes. However, due to the inherent limitations of currently available iN differentiation protocols, technological advances are required to achieve similar scalability for screens that require more complex phenotypes related to neuronal function.

Human in vitro models for understanding mechanisms of autism spectrum disorder

“…These large repositories will allow researchers to use larger sample sizes to study the effects of genetic background on ASD and will allow them to stratify their studies based on both symptoms and genetic background. Efforts are also being made to increase the throughput of these models to reduce variability and make them more amenable for drug and genetic screens [84,86,184].…”

Application of CRISPR/Cas9 to human-induced pluripotent stem cells: from gene editing to drug discovery

“…One of the strengths of the hiPSCs is based on their use not only for target-based screening but also for phenotypic screening. In the last few years, the phenotypic screenings are undergoing a reassessment to which hiPSCs have contributed, above all because they provide an easy access to multiple cell types disease-involved, especially those hard to obtain [31,32]. For example, phenotypic screening for pain research has always been hampered by the lack of recruit enough neuronal cell types [33].…”

Unprecedented Potential for Neural Drug Discovery Based on Self-Organizing hiPSC Platforms

“…They simplified the overall process to increase scalability of iNs, including the generation of large batches of cryopreserved neurons. Post-thaw these iNs were tested in a phenotypic toxicity assay with the LOPAC library (1280 bioactive small molecules) identifying 14 compounds that targeted neurite outgrowth [35]. This work is a prominent example of generating robust and efficient protocols for HTS assays of hiPSCs-derived neurons.…”

“…To overcome this Zhang et al developed a method to generate nearly 100% purity of excitatory neurons which were relatively mature within two weeks of culture via the forced overexpression of the pro-neural gene Neurogenin-2 (NGN2) from both hESCs and hiPSCs [59]. Kondo et al used the same approach to obtain highly yields (nearly 100%) of cortical neurons with hiPSCs derived from familial and sporadic Alzheimer's patients, providing a novel platform for new drug development [35]. As mentioned above, Sridharan et al used the same protocol for rapid glutamatergic induced-neurons (iNs) production with high purity [35].…”

“…Kondo et al used the same approach to obtain highly yields (nearly 100%) of cortical neurons with hiPSCs derived from familial and sporadic Alzheimer's patients, providing a novel platform for new drug development [35]. As mentioned above, Sridharan et al used the same protocol for rapid glutamatergic induced-neurons (iNs) production with high purity [35]. These and other approaches, including the evaluation of the transcriptional identity markers through fluorescent reporters during differentiation protocols and Fluorescent Activate Cell-Sorting (FACS) may significantly improve homogeneity of hiPSCs cultures [60].…”