Cost-Effective Mechanical Aggregation of Cardiac Progenitors and Encapsulation in Matrigel Support Self-Organization in a Dynamic Culture Environment.

Human iPSC-derived self-organized cardiac tissues can be valuable for the development of platforms for disease modeling and drug screening, enhancing test accuracy and reducing pharmaceutical industry financial burden. However, current differentiation systems still rely on static culture conditions and specialized commercial microwells for aggregation, which hinders the full potential of hiPSC-derived cardiac tissues. Herein, we integrate cost-effective and reproducible manual aggregation of hiPSC-derived cardiac progenitors with Matrigel encapsulation and a dynamic culture to support hiPSC cardiac differentiation and self-organization. Manual aggregation at day 7 of cardiac differentiation resulted in 97% of beating aggregates with 78% of cTnT-positive cells. Matrigel encapsulation conjugated with a dynamic culture promoted cell migration and the creation of organized structures, with observed cell polarization and the creation of lumens. In addition, encapsulation increased buoyancy and decreased coalescence of the hiPSC-derived cardiac aggregates. Moreover, VEGF supplementation increased over two-fold the percentage of CD31-positive cells resulting in the emergence of microvessel-like structures. Thus, this study shows that the explored culture parameters support the self-organization of hiPSC-derived cardiac microtissues containing multiple cardiac cell types. Additional stimuli (e.g., BMP) in long-term scalable and fully automatized cultures can further potentiate highly structured and mature hiPSC-derived cardiac models, contributing to the development of reliable platforms for high-throughput drug screening and disease modeling.

Biophysical study of human induced Pluripotent Stem Cell-Derived cardiomyocyte structural maturation during long-term culture.

Human induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs) have an enormous potential for the development of drug screening and modeling cardiac disease platforms. However, early hiPSC-CMs usually exhibit low structural development, precluding the applicability of these cells. Here, we follow during 120 days the progressive structural maturation of hiPSC-CM microtissues obtained using the Wnt signaling modulation protocol. For this purpose, we designed a user friendly custom-written program to quantify cardiac fiber alignment and sarcomere length. Cardiomyocyte shape, cardiac fiber density and multinucleation were also analyzed. Derived cardiomyocytes showed significant progression in cardiomyocyte fiber density and sarcomere length during the long-term culture, with a peak at day 90 of 40% multinucleated cells. In addition, cardiomyocyte microtissues remained functional with progressive maturation leading to a decrease in the percentage of cTnT positive cells from 59% to 22% at day 120, a value similar to the content present in tissues of the adult left ventricle. These data and the framework that we provide to quantify cardiomyocyte structural features can be important to set new metrics to develop applications for drug screening and disease modeling.