Automated Large-Scale Culture and Medium-Throughput Chemical Screen for Modulators of Proliferation and Viability of Human Induced Pluripotent Stem Cell–Derived Neuroepithelial-like Stem Cells

Abstract

The aim of this study was to demonstrate proof-of-concept feasibility for the use of human neural stem cells (NSCs) for highthroughput screening (HTS) applications. For this study, an adherent human induced pluripotent stem (iPS) cell-derived long-term, self-renewing, neuroepithelial-like stem (lt-NES) cell line was selected as a representative NSC. Here, we describe the automated large-scale serum-free culture ("scale-up") of human lt-NES cells on the CompacT SelecT cell culture robotic platform, followed by their subsequent automated "scale-out" into a microwell plate format. We also report a medium-throughput screen of 1000 compounds to identify modulators of neural stem cell proliferation and/or survival. The screen was performed on two independent occasions using a cell viability assay with end-point reading resulting in the identification of 24 potential hit compounds, 5 of which were found to increase the proliferation and/or survival of human lt-NES on both occasions. Follow-up studies confirmed a dosedependent effect of one of the hit compounds, which was a Cdk-2 modulator. This approach could be further developed as part of a strategy to screen compounds to either improve the procedures for the in vitro expansion of neural stem cells or to potentially modulate endogenous neural stem cell behavior in the diseased nervous system.

Phenotypic screens as a renewed approach for drug discovery

“…While the methods for stem cell differentiation including the differentiation efficiency, scale-up, reproducibility and cost effectiveness are still being improved, several pilot compound screens using stem cell differentiated progenitor cells have been recently reported. High throughput screens with smaller compound collections have been performed in the precursor cells derived patient or normal stem cells, including neural crest stem cells (from iPSCs with familial dysautonomia, IKBKAP expression) [63], neural progenitor cells (from normal iPS cells, Wnt/β-catenin signaling) [64], neuroepithelial-like stem cells (from normal iPS cells, cell proliferation and viability) [65], and neurons (from ES cells, AMPA glutamate receptor) [66]. Additionally, several other types of human cells derived from stem cells have also been used to assess drug efficacy and evaluate compound toxicity for a small set of compounds [67,68].…”

HTS/HCS to Screen Molecules Able to Maintain Embryonic Stem Cell Self-Renewal or to Induce Differentiation: Overview of Protocols

“…In a medium-throughput screening of 1,000 compounds, 24 potential hit compounds, 5 of which increased the proliferation and/or survival of human lt-NES, have been identified. In particular, these data suggest that GSK-3 inhibition, cell cycle modulation, p38 MAP kinase inhibition, and α-adrenoceptor agonism need further investigation, because the identification of multiple hits for each mode appears to confirm their involvement in modulating cell proliferation and survival [65].…”

“…Indeed, the possibility to reprogram somatic cells into induces pluripotent cells has the potential to supply large numbers of autologous cells that are capable of differentiation into all cell lineages and may be used for transplantation. Moreover, the capacity to derive disease-specific stem cells and panels of stem cells of different genetic backgrounds opens an avenue in the field of drug discovery [65]. Considering the current inefficiency and non-specificity of reprogramming, the discovery of small molecules that modulate this phenomenon could represent an important advance in the field of regenerative medicine.…”

Use of Neural Models of Proliferation and Neurite Outgrowth to Screen Environmental Chemicals in the ToxCast Phase I Library

“…6,7 These assays for proliferation in neuroprogenitor cells and neurite outgrowth in NS-1 cells provide information on biological processes that were not covered in the initial ToxCast bioassay portfolio. While HCI assays for proliferation 8 and neurite outgrowth 9,10 have been used for pharmaceutical screening, they have not been widely applied to environmental chemicals. Thus, we tested the ToxCast Phase I library of compounds using these assays, and present here the initial outcomes of screening these compounds and a preliminary analysis of the data.…”