Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Model Chronic Cardiac Disorders

Cardiomyopathy is a leading cause of death in patients affected by Alström syndrome (ALMS) and Duchenne muscular dystrophy (DMD). Both disorders have a genetic origin, caused by mutations in the ALMS1 and DMD gene, respectively. Despite intense research, their precise pathologic function remains unclear.

With the discovery of human embryonic (ESCs) and induced pluripotent stem cells (iPSCs) efforts have been made towards directing stem cell differentiation to the cardiovascular lineage. The generation of cardiovascular cells from human pluripotent stem cells (PSCs) provides a renewable source of human cardiomyocytes (CMs) to model heart-associated disorders in vitro. Moreover, PSCs hold potential for other clinical applications, including drug screening and toxicity testing as well as regenerative medicine. However, a major obstacle to more extensive use is their immature phenotype.

Here, we described cardiac differentiation strategies from human PSCs to obtain more mature CMs for disease modeling purposes. Through the addition of Activin A (ActA) during early cardiac induction, we induced an endodermal-like cell subpopulation, which enhanced the early maturation state of PSC-derived CMs (PSC-CMs), probably through CRIPTO-1 signaling. Interestingly, this ActA-induced embryoid body (EB)-based CM differentiation model could reveal a persistent proliferation activity of postnatal CMs, suggesting defects in the CM cell cycle of ALMS patients with certain ALMS1 gene mutations. Moreover, via CRISPR/Cas9 gene editing, we could correct the genetic mutation, restoring ALMS1 protein expression levels and consequently rehabilitate the mitogenic cardiomyopathy in ALMS iPSC-CMs. Another strategy to increase the maturity of iPSC-CMs is to further differentiate them embedded in an extracellular matrix (ECM), mimicking the tissue-like environment. We developed human three-dimensional (3D) engineered heart tissue (EHT) constructs to address cardiomyopathy-related disease mechanisms in the DMD pathology, given that dystrophin (DYS) has a structural and signaling function in the heart. First, we addressed a more immature phenotype within iPSC-CMs that were differentiated in a classical two-dimensional (2D) monolayer protocol compared to iPSC-CMs that were further matured in a 3D differentiation environment. Secondly, we confirmed and identified potential mediators in DMD-induced cardiomyopathy, showing significantly dysregulated RNA transcripts.

In conclusion, we described 3D EB and EHT cardiac differentiation systems from iPSCs isolated from ALMS and DMD patients to obtain more mature CMs. Our stem cell-based models could be of high interest to elucidate disease mechanisms, eventually pointing out novel therapeutic targets to ameliorate or tackle disease onset or progression.