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Date of Award
Doctor of Philosophy (PhD)
Drug-induced cardiotoxicity is a critical challenge in the development of new drugs. Since the advent of human pluripotent stem cell-derived cardiomyocytes (CMs), researchers have explored ways to utilize these cells for in vitro preclinical drug screening applications. One area of interest is microphysiological systems (i.e. organ-on-a-chip), which aims to create more complex in vitro models of human organ systems, thus improving drug response predictions. In this dissertation, we investigated novel analysis methods and model platforms for detecting drug-induced cardiotoxicity using human induced pluripotent stem cell (iPSC)-derived cardiovascular cells.
First, we utilized human iPSC-derived CMs (iPS-CMs) to establish optical methods of detecting cardioactive compounds. We utilized optical flow to assess the iPS-CM contractions captured using brightfield microscopy. The parameters were then analyzed using a machine learning algorithm to determine the accuracy of detection that can be obtained by the model for a given drug concentration. This result was compared to the analysis of the calcium transients measured using a genetically encoded calcium indicator (GCaMP6). The brightfield contraction analysis matched the detection sensitivity of fluorescent calcium transient analysis, while also being able to detect the effects of excitation-contraction decoupler (blebbistatin), which was not detected using calcium transient analysis.
Second, we utilize iPS-CMs to model trastuzumab-related cardiotoxicity. Trastuzumab, a monoclonal antibody against ErbB2 (Her2), is used to treat Her2+ breast cancer and has known clinical cardiotoxicity. We demonstrated that an active ErbB2 signaling via binding of neuregulin-1 (NRG-1) to ErbB4 is necessary to detect the cardiotoxic effects of trastuzumab. Activation of ErbB2/4 pathway via NRG-1 is cardioprotective, and we also demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) similarly activates the ErbB2/4 pathway. Finally, we established a co-culture platform of iPS-CMs and endothelial cells (ECs), which recapitulated the physiological phenomenon of EC-secreted NRG-1 activating the ErbB2/4 pathway on the CMs.
Third, we demonstrated the use of human iPSC-derived ECs (iPS-ECs) for creating 3-dimensionial vascular networks inside microfluidic devices. The iPS-ECs were characterized for EC markers and physiological functions. We utilized a CDH5-mCherry iPSC line to create iPS-ECs that expressed VE-cadherin fused to mCherry. The vascular networks formed by the iPS-ECs were patent and perfusable, retaining 70 kDa dextran within the lumen of the vessels. The vasculature responded to small molecule inhibitors, showing increased vessel formation in response to TGF-β inhibitor SB431542 and decreased vessel formation in response to multi-targeted receptor tyrosine kinase inhibitor sunitinib.
Taken together, our findings advance the current understanding and utility of iPS-CMs for drug screening applications, while establishing platforms for creating microphysiological systems that incorporate iPS-EC co-culture. The use of iPSC-derived cells opens possibilities for disease-specific and patient-specific drug screening applications in the future.
Steven Jonathan C. George Silva
Lilianna Solnica-Krezel, Stacey L. Rentschler, Jessica E. Wagenseil,
Available for download on Wednesday, December 19, 2018