Kinase inhibitor-induced cardiotoxicity assessed in vitro with human pluripotent stem cell derived cardiomyocytes.

Many small molecule kinase inhibitors (SMKIs), used predominantly in cancer therapy, have been implicated in serious clinical cardiac adverse events, which means that traditional preclinical drug development assays were not sufficient for identifying these cardiac liabilities. To improve clinical cardiac safety predictions, the effects of SMKIs targeting many different signaling pathways were studied using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) in combined assays designed for the detection of both electrophysiological (proarrhythmic) and non-electrophysiological (non-proarrhythmic) drug-induced cardiotoxicity. Several microplate-based assays were used to quantitate cell death, apoptosis, mitochondrial damage, energy depletion, and oxidative stress as mechanism-based non-electrophysiological cardiomyocyte toxicities. Microelectrode arrays (MEA) were used to quantitate in vitro arrhythmic events (iAEs), field potential duration (FPD) prolongation, and spike amplitude suppression (SAS) as electrophysiological effects. To enhance the clinical relevance, SMKI-induced cardiotoxicities were compared by converting drug concentrations into multiples of reported clinical maximum therapeutic plasma concentration, "FoldCmax", for each assay. The results support the conclusion that the combination of the hPSC-CM based electrophysiological and non-electrophysiological assays have significantly more predictive value than either assay alone and significantly more than the current FDA-recommended hERG assay. In addition, the combination of these assays provided mechanistic information relevant to cardiomyocyte toxicities, thus providing valuable information on potential drug-induced cardiotoxicities early in drug development prior to animal and clinical testing. We believe that this early information will be helpful to guide the development of safer and more cost-effective drugs.

Promoter analysis in ES cell-derived neural cells.

Neural cells derived from ES cells in cell culture (ESNCs) have many of the properties of normal neural cells and provide a model of "neurogenesis-in-a-dish." Here we show that ESNCs provide a powerful system for analyzing neural gene transcription. ES cells are transfected with bacterial artificial chromosomes (BACs) containing Olig2, a gene with a key role in neural fate choice. One BAC is modified by recombineering to insert a reporter gene and a gene for selecting stably transfected clones. Another BAC contains a deletion of a suspected Olig2 promoter. Stable transgenic clones of ES cells are isolated, differentiated in culture, and the expression of transgenes is assayed. Differentiated cells dramatically up-regulate transgene expression and a deletion analysis reveals a basal promoter for Olig2. The combination of ESNCs and BAC recombineering will have broad application for analyzing gene transcription in the nervous system and will be applicable to human ES cells. The general approach should also be applicable to the many other cell lineages that can now be derived from mouse and human ES cells in culture.

Double lox targeting for neural cell transgenesis.

ES cells differentiated along the neural lineage in vitro are an attractive model system. Here we have developed ES cell lines that are suitable for inserting transgenes at a single chromosomal site. ES cell line CE1 (for Cassette Exchange) contains one "acceptor" module (CE1) that allows for efficient double lox targeting. The site is also permissive for gene expression in neural progenitor cells, as well as differentiated neurons and glia. Line CE2 was derived by swapping a puromycin resistance cassette into CE1. Neural progenitors derived from this line are puromycin-resistant. A beta-actin/GFP expression cassette was inserted into the CE1 site to create CE3. The CE3 cell line was differentiated into neural cells and displayed strong EGFP expression in neural progenitors, differentiated neurons and glia. Differentiated CE3 ES cells (4-/4+ RA) were transplanted into the injured rat somatosensory cortex where many of the transplanted cells survived and differentiated into neurons expressing GFP. This strategy for creating sets of transgenic lines with multiple cassettes inserted into a single chromosomal site provides a powerful tool for studying development and function of ES cell-derived neural cells. Many of these will be useful in transplantation research.

A subset of ES-cell-derived neural cells marked by gene targeting.

Embryonic stem cells differentiate efficiently in culture into neural progenitors, neurons, oligodendrocytes, and astrocytes. An embryonic stem (ES) cell line with green fluorescent protein (GFP) inserted into the gene for Olig2, a lineage-specific transcription factor, permits visualization and physical separation of a subset of living ES-cell-derived neural cells. GFP-expressing cells have morphological and antigenic properties of the oligodendrocyte lineage. The differentiation of living GFP-expressing cells can be followed in cultures, and they can be separated by fluorescence-activated cell sorting and cultured as pure populations. This system will allow detailed biochemical and molecular analysis of a neural differentiation pathway at a level not previously feasible. The strategy may have general applicability, since other neural lineages can be marked in an analogous manner.