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

Area of Science:

• Neuroscience
• Stem Cell Biology
• Drug Discovery

Background:

• Human induced pluripotent stem cell-derived neurons (iNs) offer potential for understanding brain disorders and advancing precision medicine.
• Current iN generation methods face scalability and reproducibility challenges, hindering their use in industrial drug screening.
• High-throughput screening (HTS) requires scalable and reproducible cellular assays for effective early-stage drug discovery.

Purpose of the Study:

• To develop scalable and reproducible methods for generating iNs suitable for HTS drug discovery assays.
• To validate the performance of these scalable iNs in a phenotypic assay for neurite outgrowth inhibition.
• To assess the robustness and inter-laboratory reproducibility of the developed iN-based screening platform.

Main Methods:

• Simplified iN production and plating protocols were implemented.
• iNs were adapted into a freezer-ready format to enhance usability and scalability.
• A high-throughput screening (HTS)-amenable phenotypic assay measuring neurite outgrowth was developed and utilized.
• The assay's performance, robustness, and reproducibility were evaluated across multiple laboratories.

Main Results:

• Streamlined protocols enabled scalable production of iNs compatible with HTS.
• Freezer-ready iNs supported a robust and reproducible neurite outgrowth inhibition assay.
• The assay successfully identified small molecule inhibitors of neurite outgrowth.
• The developed iN-based platform demonstrated high reproducibility across different laboratory settings.

Conclusions:

• Scalable production methods for iNs are now available for large-scale drug discovery campaigns targeting simple neuronal phenotypes.
• Current iPSC-derived neuron technologies are suitable for HTS campaigns screening over 100,000 compounds.
• Further technological advancements are needed to achieve similar scalability for screens requiring complex neuronal function phenotypes.