While the potential applications for induced pluripotent stem cells range from cell replacement to research to understand basic cellular biology, the symposium entitled “Induced Human Pluripotent Stem Cells and Their Differentiated Progeny Cells: Implementation in Toxicity Testing” focused on applications for in vitro toxicity evaluation. Presenters discussed applications ranging, including hepatocyte toxicity testing, neuronal cells, and cardiomyocytes, illustrating the potential range of utility. Dr. Blake Anson introduced the symposium with an overview of the topics to be presented.
Dr. Salman Khetani of Colorado State University engineers liver tissues for a broad variety of in vitro screening, cell-based therapies, and structure-function studies. Issues he mentioned as critically important to evaluate are: 1) the stability of cell phenotypes stable over time; 2) the microenvironment; and 3) cell-cell interaction and shape (architecture). Dr. Khetani uses micropatterned co-cultures with added collagen under the patterns and then floods remaining areas with a second cell type, 3T3-J2 murine embryonic fibroblasts. The format and density allow for excellent 3-D imaging. Cell functionality is stable for about a month in vitro, and enzyme expression is similar, but not identical to in vivo metabolism (Phase I, II).
Dr. Khetani compared and contrasted the iPS cells to both primary hepatocytes, as well as the iMPCC. His lab profiled 47 drugs that included several true positives and negatives, as well as false negatives. Overall, the iPS cells have properties similar to primary hepatocytecs, but are not identical to in vivo or even primary hepatocytes. Dr. Khetani is working to understand and find culture conditions to make the iPS hepatoytes closer to primary hepatocytes and, more importantly, closer to in vivo in terms of metabolism and sensitivity to toxicants. At this time, the iPS cells provide higher liver functions and maturation. The longevity of iHLC in iMPCC compared to primary hepatocytes is superior, and they are nearly as sensitive for toxicity screening as primary hepatocytes.
Dr. Paul Watkins of the Hamner Institutes for Health Sciences in Research Triangle Park, North Carolina, discussed the utility of iPS cells for evaluation of idiosyncratic hepatotoxicity. There has been limited progress in the detection or understanding of idiosyncratic hepatotoxicity. The DILI injury network provides a very valuable research tool as they has enrolled more than 1,500 subjects, 1,100 subjects for whom GWAS has been conducted and exome chipped. Some of the patients have undergone whole exome sequencing. The value of the injury network is they maintain links to individuals to maintain IRB access to prepare iPS cells.
Dr. Watkins' presentation focused on evaluation of isoniazid-related DILI. This is one of the most frequent causes of DILI with an incidence of 1:1,000, and the toxicity appears to be related to the adaptive immune response. Dr. Watkins explored the hypothesis that circulating t-cells cause an immune response that is related to the subsequent liver injury, and there is some evidence in the literature supportive of that idea. Genome-wide studies have revealed only HLA-risk alleles without any liver specific or hepatocyte specific alleles. As isoniazid forms hydrazine and can cross link proteins, Dr. Watkins group evaluated the formation of adducts and found two, a mitochondrial protein and macrophage migration inhibitory factor. However, this does not answer the question as to how immune function would lead to liver injury. One possibility is that microvesicles are formed at subtoxic concentrations so that that the antigens from liver cells can be presented to the immune system, resulting in subsequent toxicity. He was able to demonstrate that iPS cells do release microvesicles when incubated with subtoxic concentrations of compounds. Another cofactor in the liver injury may be the presence of so-called “danger signals,” cytokines, chemokines, and damage associated molecular patterns within kuppfer cells.
Dr. Watson wants to evaluate iPS cells generated from numerous individuals in order to increase the understanding of DILI. Dr. Watkins also encouraged viewing of the US DILIM network poster tomorrow (#527).
Dr. Ingrid Druwe is trying to develop HTS in vitro assays that reliably detect alterations in processes relevant to CNS development. Her hypothesis is that chemicals that perturb neurodevelopmental process in vitro have the potential to perturb these processes in vivo. Dr. Druwe used iPS cells to try to understand the toxicity reducing neurite outgrowth. Current neuronal models—clonal cell lines, primary cultures, and progenitor cells all have limitations. The iPS cells were set up and imaged with high-content imaging. Controlled studies over time confirmed that the cells could be used to evaluate neurite outgrowth. Although some neuronal markers differed from primary cultures, overall, there was adequate characterization of the cells to use for evaluating parameters for appropriate cell growth and testing of toxicants. Dr. Druwe set up a high throughput assay to evaluate neurite outgrowth and found little death. A positive toxicant control (LiCl) inhibited outgrowth. While the Z prime score was 0.5, additional controls will be used to improve the screening assay.
Dr. Druwe used a chemical training set and compared to primary rat cultures. The results show that the iPSC neurons had more neurites. This is because primary cells were not pure neurons. It isn’t clear if that is the reason the results were different between the rat primary cells and the iPS cells. Even when the results showed the same general response, the dose response differed.
Dr. Hong Shi’s talk focused on human iPSC myocytes screening to improve the predictability of cardiotoxicity compared to currently used methods. After setting up the screening method and comparing and contrasting it to that of currently used methods, Dr. Shi described several examples in which standard preclinical assessment, including cardiovascular safety pharmacology studies had not elucidated any obvious toxicity. However, all of these compounds ended up causing cardiotoxicity in human clinical studies. The iPS cell myocytes showed toxicity that CHO hERG patch clamp did not.
In all cases, the human cardiomyocytes were more sensitive predictors than conventional historic cell lines being tested. Of all the different iPS cell assays that were discussed today, the cardiomyocytes appear to be the most advanced and validated.