Abstract
BackgroundWe have previously described fundamental differences in the biology of stem cells as compared to other dividing cell populations. We reasoned therefore that a differential screen using US Food and Drug Administration (FDA)-approved compounds may identify either selective survival factors or specific toxins and may be useful for the therapeutically-driven manufacturing of cells in vitro and possibly in vivo.Methodology/Principal FindingsIn this study we report on optimized methods for feeder-free culture of hESCs and hESC-derived neural stem cells (NSCs) to facilitate automated screening. We show that we are able to measure ATP as an indicator of metabolic activity in an automated screening assay. With this optimized platform we screened a collection of FDA-approved drugs to identify compounds that have differential toxicity to hESCs and their neural derivatives. Nine compounds were identified to be specifically toxic for NSCs to a greater extent than for hESCs. Six of these initial hits were retested and verified by large-scale cell culture to determine dose-responsive NSC toxicity. One of the compounds retested, amiodarone HCL, was further tested for possible effects on postmitotic neurons, a likely target for transplant therapy. Amiodarone HCL was found to be selectively toxic to NSCs but not to differentiated neurons or glial cells. Treated and untreated NSCs and neurons were then interrogated with global gene expression analysis to explore the mechanisms of action of amiodarone HCl. The gene expression analysis suggests that activation of cell-type specific cationic channels may underlie the toxicity of the drug.Conclusions/SignificanceIn conclusion, we have developed a screening strategy that allows us to rapidly identify clinically approved drugs for use in a Chemistry, Manufacture and Control protocol that can be safely used to deplete unwanted contaminating precursor cells from a differentiated cell product. Our results also suggest that such a strategy is rich in the potential of identifying lineage specific reagents and provides additional evidence for the utility of stem cells in screening and discovery paradigms.
Highlights
The ability to expand human embryonic stem cells unlimitedly in culture and to differentiate them into specific somatic cell types [1] make them a useful tool in the development of hESC-based automated screening platforms for drug discovery
We have previously shown that neural stem cells (NSCs) can be generated from multiple hESC lines and can be cultured for prolonged periods without losing their ability to differentiate into neurons, astrocytes and oligodendrocytes [11]
Gene Set Enrichment Analysis (GSEA) was conducted to identify pathways, biological process and molecular functions that are enriched in genes differentially expressed by NSCs or dopaminergic neurons treated with amiodarone HC
Summary
The ability to expand human embryonic stem cells (hESCs) unlimitedly in culture and to differentiate them into specific somatic cell types [1] make them a useful tool in the development of hESC-based automated screening platforms for drug discovery. This possibility has not yet attracted as much attention as the ideas of cell replacement, personalized medicine and other more direct clinical applications, hESCs are expected to be superior to most commonly used cell-culture models of drug discovery which employ tumor-derived or immortalized cell lines or primary cell culture. We found amiodarone HCL to be toxic to NSCs but not to postmitotic neurons, indicating its potential use for depleting proliferating NSCs in hESC-derived cell populations for possible neural transplantation
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