Reproducibility of drug-induced effects on the contractility of an engineered heart tissue derived from human pluripotent stem cells.

Introduction: Engineered heart tissues (EHTs) are three-dimensional culture platforms with cardiomyocytes differentiated from human pluripotent stem cells (hPSCs) and were designed for assaying cardiac contractility. For drug development applications, EHTs must have a stable function and provide reproducible results. We investigated these properties with EHTs made with different tissue casting batches and lines of differentiated hPSC-cardiomyocytes and analyzed them at different times after being fabricated. Methods: A video-optical assay was used for measuring EHT contractile outputs, and these results were compared with results from motion traction analysis of beating hPSC-cardiomyocytes cultured as monolayers in two-dimensional cultures. The reproducibility of induced contractile variations was tested using compounds with known mechanistic cardiac effects (isoproterenol, EMD-57033, omecamtiv mecarbil, verapamil, ranolazine, and mavacamten), or known to be clinically cardiotoxic (doxorubicin, sunitinib). These drug-induced variations were characterized at different electrical pacing rates and variations in intracellular calcium transients were also assessed in EHTs. Results: To ensure reproducibility in experiments, we established EHT quality control criteria based on excitation-contraction coupling and contractile sensitivity to extracellular calcium concentration. In summary, a baseline contractile force of 0.2 mN and excitation-contraction coupling of EHTs were used as quality control criteria to select suitable EHTs for analysis. Overall, drug-induced contractile responses were similar between monolayers and EHTs, where a close relationship was observed between contractile output and calcium kinetics. Contractile variations at multiple time points after adding cardiotoxic compounds were also detectable in EHTs. Discussion: Reproducibility of drug-induced effects in EHTs between experiments and relative to published work on these cellular models was generally observed. Future applications for EHTs may require additional mechanistic criteria related to drug effects and cardiac functional outputs to be measured in regard to specific contexts of use.

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects.

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.

Characterizing the reproducibility in using a liver microphysiological system for assaying drug toxicity, metabolism, and accumulation.

Liver microphysiological systems (MPSs) are promising models for predicting hepatic drug effects. Yet, after a decade since their introduction, MPSs are not routinely used in drug development due to lack of criteria for ensuring reproducibility of results. We characterized the feasibility of a liver MPS to yield reproducible outcomes of experiments assaying drug toxicity, metabolism, and intracellular accumulation. The ability of the liver MPS to reproduce hepatotoxic effects was assessed using trovafloxacin, which increased lactate dehydrogenase (LDH) release and reduced cytochrome P450 3A4 (CYP3A4) activity. These observations were made in two test sites and with different batches of Kupffer cells. Upon culturing equivalent hepatocytes in the MPS, spheroids, and sandwich cultures, differences between culture formats were detected in CYP3A4 activity and albumin production. Cells in all culture formats exhibited different sensitivities to hepatotoxicant exposure. Hepatocytes in the MPS were more functionally stable than those of other culture platforms, as CYP3A4 activity and albumin secretion remained prominent for greater than 18 days in culture, whereas functional decline occurred earlier in spheroids (12 days) and sandwich cultures (7 days). The MPS was also demonstrated to be suitable for metabolism studies, where CYP3A4 activity, troglitazone metabolites, diclofenac clearance, and intracellular accumulation of chloroquine were quantified. To ensure reproducibility between studies with the MPS, the combined use of LDH and CYP3A4 assays were implemented as quality control metrics. Overall results indicated that the liver MPS can be used reproducibly in general drug evaluation applications. Study outcomes led to general considerations and recommendations for using liver MPSs. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Microphysiological systems (MPSs) have been designed to recreate organ- or tissue-specific characteristics of extracellular microenvironments that enhance the physiological relevance of cells in culture. Liver MPSs enable long-lasting and stable culture of hepatic cells by culturing them in three-dimensions and exposing them to fluid flow. WHAT QUESTION DID THIS STUDY ADDRESS? What is the functional performance relative to other cell culture platforms and the reproducibility of a liver MPS for assessing drug development and evaluation questions, such as toxicity, metabolism, and pharmacokinetics? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? The liver MPS systematically detected the toxicity of trovafloxacin. When compared with spheroids and sandwich cultures, this system had a more stable function and different sensitivity to troglitazone, tamoxifen, and digoxin. Quantifying phase II metabolism of troglitazone and intracellular accumulation of chloroquine demonstrated the potential use of the liver MPS for studying drug metabolism and pharmacokinetics. Quality control criteria for assessing chip function were key for reliably using the liver MPS. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Due to its functional robustness and physiological relevance (3D culture, cells expose to fluid flow and co-culture of different cell types), the liver MPS can, in a reproducible manner: (i) detect inflammatory-induced drug toxicity, as demonstrated with trovafloxacin, (ii) detect the toxicity of other drugs, such as troglitazone, tamoxifen, and digoxin, with different effects than those detected in spheroids and sandwich cultures, (iii) enable studies of hepatic function that rely on prolonged cellular activity, and (iv) detect phase II metabolites and drug accumulation to potentially support the interpretation of clinical data. The integration of MPSs in drug development will be facilitated by careful evaluation of performance and reproducibility as performed in this study.

Quantifying drug-induced structural toxicity in hepatocytes and cardiomyocytes derived from hiPSCs using a deep learning method.

Cardiac and hepatic toxicity result from induced disruption of the functioning of cardiomyocytes and hepatocytes, respectively, which is tightly related to the organization of their subcellular structures. Cellular structure can be analyzed from microscopy imaging data. However, subtle or complex structural changes that are not easily perceived may be missed by conventional image-analysis techniques. Here we report the evaluation of PhenoTox, an image-based deep-learning method of quantifying drug-induced structural changes using human hepatocytes and cardiomyocytes derived from human induced pluripotent stem cells. We assessed the ability of the deep learning method to detect variations in the organization of cellular structures from images of fixed or live cells. We also evaluated the power and sensitivity of the method for detecting toxic effects of drugs by conducting a set of experiments using known toxicants and other methods of screening for cytotoxic effects. Moreover, we used PhenoTox to characterize the effects of tamoxifen and doxorubicin-which cause liver toxicity-on hepatocytes. PhenoTox revealed differences related to loss of cytochrome P450 3A4 activity, for which it showed greater sensitivity than a caspase 3/7 assay. Finally, PhenoTox detected structural toxicity in cardiomyocytes, which was correlated with contractility defects induced by doxorubicin, erlotinib, and sorafenib. Taken together, the results demonstrated that PhenoTox can capture the subtle morphological changes that are early signs of toxicity in both hepatocytes and cardiomyocytes.

The in vivo genetic program of murine primordial lung epithelial progenitors.

Multipotent Nkx2-1-positive lung epithelial primordial progenitors of the foregut endoderm are thought to be the developmental precursors to all adult lung epithelial lineages. However, little is known about the global transcriptomic programs or gene networks that regulate these gateway progenitors in vivo. Here we use bulk RNA-sequencing to describe the unique genetic program of in vivo murine lung primordial progenitors and computationally identify signaling pathways, such as Wnt and Tgf-ß superfamily pathways, that are involved in their cell-fate determination from pre-specified embryonic foregut. We integrate this information in computational models to generate in vitro engineered lung primordial progenitors from mouse pluripotent stem cells, improving the fidelity of the resulting cells through unbiased, easy-to-interpret similarity scores and modulation of cell culture conditions, including substratum elastic modulus and extracellular matrix composition. The methodology proposed here can have wide applicability to the in vitro derivation of bona fide tissue progenitors of all germ layers.

Thyroid Progenitors Are Robustly Derived from Embryonic Stem Cells through Transient, Developmental Stage-Specific Overexpression of Nkx2-1.

The clinical importance of anterior foregut endoderm (AFE) derivatives, such as thyrocytes, has led to intense research efforts for their derivation through directed differentiation of pluripotent stem cells (PSCs). Here, we identify transient overexpression of the transcription factor (TF) NKX2-1 as a powerful inductive signal for the robust derivation of thyrocyte-like cells from mouse PSC-derived AFE. This effect is highly developmental stage specific and dependent on FOXA2 expression levels and precise modulation of BMP and FGF signaling. The majority of the resulting cells express thyroid TFs (Nkx2-1, Pax8, Foxe1, Hhex) and thyroid hormone synthesis-related genes (Tg, Tpo, Nis, Iyd) at levels similar to adult mouse thyroid and give rise to functional follicle-like epithelial structures in Matrigel culture. Our findings demonstrate that NKX2-1 overexpression converts AFE to thyroid epithelium in a developmental time-sensitive manner and suggest a general methodology for manipulation of cell-fate decisions of developmental intermediates.