Aldehyde Dehydrogenase 2 (ALDH2): A novel sorafenib target in hepatocellular carcinoma unraveled by the proteome-wide cellular thermal shift assay.

Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale. Among the potential sorafenib targets, we identified aldehyde dehydrogenase 2 (ALDH2), an enzyme that plays a major role in alcohol metabolism. We validated the interaction of sorafenib with ALDH2 by orthogonal methods using pure recombinant protein, proving that this interaction is not mediated by other cellular components. Moreover, we showed that sorafenib inhibits ALDH2 activity, supporting a functional role for this interaction. Finally, we were able to demonstrate that both ALDH2 protein expression and activity were reduced in sorafenib-resistant cells compared to the parental cell line. Overall, our study allowed the identification of ALDH2 as a novel sorafenib target and sheds light on its potential role in both hepatocellular carcinoma and sorafenib resistance condition.

Online monitoring of hiPSC expansion and hepatic differentiation in 3D culture by dielectric spectroscopy.

Hepatocyte-like cells derived from human-induced pluripotent stem cells (hiPSC-HLC) are expected to have important applications in drug screening and regenerative medicine. However, hiPSC-HLC are difficult to produce on a large-scale to obtain relevant numbers for such applications. The aim of this study was to implement a novel integrated strategy for scalable production of hiPSC-HLC and demonstrate the applicability of dielectric spectroscopy to monitor hiPSC expansion/differentiation processes. We cultured hiPSC as three-dimensional (3D) aggregates in stirred-tank bioreactors (STB) operated in perfusion with an in situ capacitance probe. Dissolved oxygen concentration and dilution rate were controlled along the process and after 5 days of cell expansion, the hepatic differentiation was integrated in sequential steps for 28 days. The hiPSC were able to grow as 3D aggregates and the expression of hepatic markers and albumin production after differentiation confirmed that hepatocyte differentiation improved when compared to 2D culture. These hiPSC-HLC exhibited functional characteristics of hepatocytes including glycogen storage and drug metabolization capacity. Our results also show a good correlation between the cell permittivity measured online and the aggregate biovolume measured by standard offline methods, demonstrating for the first time the potential of dielectric spectroscopy to monitor hiPSC expansion and differentiation in STB.

Human Extracellular-Matrix Functionalization of 3D hiPSC-Based Cardiac Tissues Improves Cardiomyocyte Maturation.

Human induced pluripotent stem cells (hiPSC) possess significant therapeutic potential due to their high self-renewal capability and potential to differentiate into specialized cells such as cardiomyocytes. However, generated hiPSC-derived cardiomyocytes (hiPSC-CM) are still immature, with phenotypic and functional features resembling the fetal rather than their adult counterparts, which limits their application in cell-based therapies, in vitro cardiac disease modeling, and drug cardiotoxicity screening. Recent discoveries have demonstrated the potential of the extracellular matrix (ECM) as a critical regulator in development, homeostasis, and injury of the cardiac microenvironment. Within this context, this work aimed to assess the impact of human cardiac ECM in the phenotype and maturation features of hiPSC-CM. Human ECM was isolated from myocardium tissue through a physical decellularization approach. The cardiac tissue decellularization process reduced DNA content significantly while maintaining ECM composition in terms of sulfated glycosaminoglycans (s-GAG) and collagen content. These ECM particles were successfully incorporated in three-dimensional (3D) hiPSC-CM aggregates (CM+ECM) with no impact on viability and metabolic activity throughout 20 days in 3D culture conditions. Also, CM+ECM aggregates displayed organized and longer sarcomeres, with improved calcium handling when compared to hiPSC-CM aggregates. This study shows that human cardiac ECM functionalization of hiPSC-based cardiac tissues improves cardiomyocyte maturation. The knowledge generated herein provides essential insights to streamline the application of ECM in the development of hiPSC-based therapies targeting cardiac diseases.

Toward a Microencapsulated 3D hiPSC-Derived in vitro Cardiac Microtissue for Recapitulation of Human Heart Microenvironment Features.

The combination of cardiomyocytes (CM) and non-myocyte cardiac populations, such as endothelial cells (EC), and mesenchymal cells (MC), has been shown to be critical for recapitulation of the human heart tissue for in vitro cell-based modeling. However, most of the current engineered cardiac microtissues still rely on either (i) murine/human limited primary cell sources, (ii) animal-derived and undefined hydrogels/matrices with batch-to-batch variability, or (iii) culture systems with low compliance with pharmacological high-throughput screenings. In this work, we explored a culture platform based on alginate microencapsulation and suspension culture systems to develop three-dimensional (3D) human cardiac microtissues, which entails the co-culture of human induced pluripotent stem cell (hiPSC) cardiac derivatives including aggregates of hiPSC-CM and single cells of hiPSC-derived EC and MC (hiPSC-EC+MC). We demonstrate that the 3D human cardiac microtissues can be cultured for 15 days in dynamic conditions while maintaining the viability and phenotype of all cell populations. Noteworthy, we show that hiPSC-EC+MC survival was promoted by the co-culture with hiPSC-CM as compared to the control single-cell culture. Additionally, the presence of the hiPSC-EC+MC induced changes in the physical properties of the biomaterial, as observed by an increase in the elastic modulus of the cardiac microtissue when compared to the hiPSC-CM control culture. Detailed characterization of the 3D cardiac microtissues revealed that the crosstalk between hiPSC-CM, hiPSC-EC+MC, and extracellular matrix induced the maturation of hiPSC-CM. The cardiac microtissues displayed functional calcium signaling and respond to known cardiotoxins in a dose-dependent manner. This study is a step forward on the development of novel 3D cardiac microtissues that recapitulate features of the human cardiac microenvironment and is compliant with the larger numbers needed in preclinical research for toxicity assessment and disease modeling.

Unveiling the molecular crosstalk in a human induced pluripotent stem cell-derived cardiac model.

In vitro cell-based models that better mimic the human heart tissue are of utmost importance for drug development and cardiotoxicity testing but also as tools to understand mechanisms related with heart disease at cellular and molecular level. Besides, the implementation of analytical tools that allow the depiction and comprehensive understanding of the molecular mechanisms of the crosstalk between the different cell types is also relevant. In this work, we implemented a human cardiac tissue-like in vitro model, derived from human-induced pluripotent stem cell (hiPSC), and evaluated the relevance of the cell-cell communication between the two of the most representative cell populations of the human heart: cardiomyocytes (hiPSC-CM) and endothelial cells (hiPSC-EC). We observed that heterotypic cell communication promotes: (a) structural maturation of hiPSC-CM and (b) deposition of several extracellular matrix components (such as collagens and fibronectin). Overall, the toolbox of analytical techniques used in our study not only enabled us to validate previous reports from the literature on the importance of the presence of hiPSC-EC on hiPSC-CM maturation, but also bring new insights on the molecular mechanisms involved in the communication between these two cell types when cocultured in vitro.

Impact of hydrodynamics on iPSC-derived cardiomyocyte differentiation processes.

Cardiomyocytes (CMs), derived from pluripotent stem cells (PSCs), have the potential to be used in cardiac repair. Addition of physical cues, such as electrical and mechanical stimulations, have proven to significantly effect morphology, density, cardiogenesis, maturity and functionality of differentiated CMs. This work combines rigorous fluid dynamics investigation and flow frequency analysis with iPSC differentiation experiments to identify and quantify the flow characteristics leading to a significant increase of differentiation yield. This is towards a better understanding of the physical relationship between frequency modulation and embryoid bodies suspension, and the development of dimensionless correlations applicable at larger scales. Laser Doppler Anemometry and Fast Fourier Transform analysis were used to identify characteristic flow frequencies under different agitation modes. Intermittent agitation resulted in a pattern of low intensity frequencies at reactor scale that could be controlled by varying three identified time components: rotational speed, interval and dwell times. A proof of concept biological study was undertaken, tuning the hydrodynamic environment through variation of dwell time based on the engineering study findings and a significant improvement in CM yield was obtained. This work introduces the concept of fine-tuning the physical hydrodynamic cues within a three-dimensional flow system to improve cardiomyocyte differentiation of iPSC.

Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches.

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3day-1 dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7×106cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells' pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors.