Automation and scale-up of human induced pluripotent stem cell models of cardiovascular disease for drug screening

Abstract

The global cost of heart failure is USD$45 billion and set to double in the next 15 years. The only method of treatment is heart transplant but demand far exceeds supply and is projected to increase. Meanwhile, global pharmaceutical development has been hindered by poor drug development success rates. Of the drugs that make it to phase I clinical trials, only 8 % pass phase III and existing drug screens do not always accurately predict or detect adverse cardiac events. Cardiotoxicity is the underlying reason for 26 % of safety related drug withdrawals between 1990-2006. Therefore, a source of human cardiomyocytes (CMs) is required to fill the need for regenerative medicine and drug screening applications. Differentiation of human pluripotent stem cells (hPSCs) to CMs is a viable solution to this bottleneck but the number of cells required is staggering; up to 5 million novel compounds are registered annually by pharmaceutical and academic institutions, while cell replacement studies in primates suggest that 10 billion CMs will be required per patient to repair the damaged myocardium post infarction. The objective of this thesis was to evaluate whether automated high throughput manufacture of hPSCs and CMs was possible, and to demonstrate that hPSC-CMs could be used in automated high throughput drug screening by carrying out assays in 384-well plates. This thesis started by carrying out three manual differentiation methods; an embryoid body (EB) based method and two monolayer methods. Batch variability in mouse embryonic fibroblast conditioned medium (MEF-CM) led to erratic and variable differentiation outcomes (as high as 94+/-0.3 % to as low as 25.6+/-39.7 % beating EBs per 96 well plate). Two monolayer methods, using defined media (mTeSR and E8) increased cell yields by up to 12-fold and 65-fold respectively and simplified the process technically. When these methods were automated, EB differentiation failed to generate spontaneously beating EBs, whereas both monolayer methods succeeded in generating spontaneously beating cardiomyocytes of purities >90 %. Finally, cryopreserved stocks of hiPSC-CMs produced by automation were used to evaluate whether cardiotoxicity from the anticancer drug doxorubicin could be decreased by co-treating with dexrazoxane (an existing doxorubicin cardio-protectant), carvedilol (a β-blocker), sildenafil (a vasoactive agent) and isoprenaline (a β-adrenoreceptor agonist). This was carried out in a real-time, fully automated assay setup to monitor induction of apoptosis by the marker propidium iodide using the Operetta confocal plate reader. The concentration of doxorubicin that led to 50 % hiPSC-CM death (TD50) was significantly reduced by co-treatment with dexrazoxane, carvedilol and sildenafil. Carvedilol showed the highest level of cardioprotection by increasing TD50 of doxorubicin by 7.5-fold. In contrast, isoprenaline reduced TD50 of doxorubicin, suggesting that isoprenaline would be contraindicated in patients undergoing doxorubicin treatment. Thus, this thesis demonstrated that automated differentiation of cardiomyocytes was technically feasible with capability of generating high yields (up to 39 million cells per flask) and high purity (>90 %) cardiomyocytes. Furthermore, this system was compatible with high content assays in 384-well plates for evaluating drug toxicity