Quantification of Etoposide Hypersensitivity: A Sensitive, Functional Method for Assessing Pluripotent Stem Cell Quality

Abstract

Human induced pluripotent stem cells (hiPSC) hold great promise in diagnostic and therapeutic applications. However, translation of hiPSC technology depends upon a means of assessing hiPSC quality that is quantitative, high-throughput, and can decipher malignant teratocarcinoma clones from normal cell lines. These attributes are lacking in current approaches such as detection of cell surface makers, RNA profiling, and/or teratoma formation assays. The latter remains the gold standard for assessing clone quality in hiPSCs, but is expensive, time-consuming, and incompatible with high-throughput platforms. Herein, we describe a novel method for determining hiPSC quality that exploits pluripotent cells' documented hypersensitivity to the topoisomerase inhibitor etoposide (CAS No. 33419-42-0). Based on a study of 115 unique hiPSC clones, we established that a half maximal effective concentration (EC50) value of <300 nM following 24 hours of exposure to etoposide demonstrated a positive correlation with RNA profiles and colony morphology metrics associated with high quality hiPSC clones. Moreover, our etoposide sensitivity assay (ESA) detected differences associated with culture maintenance, and successfully distinguished malignant from normal pluripotent clones independent of cellular morphology. Overall, the ESA provides a simple, straightforward method to establish hiPSC quality in a quantitative and functional assay capable of being incorporated into a generalized method for establishing a quality control standard for all types of pluripotent stem cells. STEM CELLS TRANSLATIONAL MEDICINE 2017;6:1829-1839 SIGNIFICANCE STATEMENTA quantitative measure of human induced pluripotent stem cell (hiPSC) pluripotency has yet to be standardized, an issue that must be remedied if widespread translational application of this technology is to occur. Secreto et al. provide an innovative method for quantifying hiPSC pluripotency based upon exploiting the hypersensitivity pluripotent cells exhibit to the DNA damaging agent etoposide. Their etoposide sensitivity assay determines hiPSC suitability by quantifying the amount of etoposide required to kill the treated cells, and comparing that amount to a standard value known to be associated with high quality clones.

Clinical Translation of Pluripotent Stem Cell Therapies: Challenges and Considerations

“…Conversely, if cells are still at an early-differentiated progenitor state, it is mandatory, from a safety viewpoint, to eliminate those that have retained their pluripotency. This objective can be achieved by a variety of techniques including chemical inhibitors (such as etoposide), which exploit the sensitivity of PSCs to genotoxic agents (Secreto et al, 2017), small molecule-based induction of PSC apoptosis (Ben-David et al, 2013), recombinant lectin-toxin fusion proteins selectively binding to PSCs and delivering a cytotoxic cargo following their internalization (Tateno et al, 2015), or, in the case of the heart, culture under glucose depletion and lactate enrichment to enhance the exclusive survival of differentiated cardiomyocytes (Tohyama et al, 2013) (for a more extensive review of the current methods of cardiomyocyte enrichment, see Ban et al, 2017). In the clinics, however, enrichment for a pure population of progenitor cells rather relies on antibody-based sorting targeting lineage-specific surface markers such as CD142 for pancreatic endoderm cells (Kelly et al, 2011), LR-TMP1 for midbrain dopaminergic neurons (Samata et al, 2016), or SSEA-1 for cardiovascular progenitors (Menasché et al, 2015).…”