*4.3. Human iPSCs*

The contribution of animal models to our overall understanding of DCM has been indispensable. However, many important differences exist between animal models and humans. Additionally, cardiac tissues from DCM patients are difficult to obtain and exhibit a low survival rate in long-term culture. The emergence of induced pluripotent stem cells (iPSCs) [130], and the rapidly advancing technology associated with them have made it possible to obtain functional cardiac myocytes through the differentiation of human iPSCs derived from DCM patients [113,114]. Stem cell-derived cardiac myocytes from cells isolated directly from patients with cardiomyopathies recapitulate certain aspects of human cardiovascular disease and represent a powerful new model system to study the basic mechanisms of inherited cardiomyopathies. Thus, human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) are an important complement to experimental animal models to study the cellular, molecular, and physiological mechanisms associated with the pathogenesis of DCM, as well as to establish high-throughput platforms for drug screening in human cells.

DCM was first modeled by Sun and coworkers using iPSCs-CMs derived from a member of a family with DCM carrying a heterozygous R173W mutation in cardiac troponin T (TNNT2) [111]. iPSC-derived cardiac myocytes from this patient recapitulated some of the morphological and functional phenotypes of familial DCM with inherited mutations in troponin T. This study describes the first successful modeling of dilated cardiomyopathy in hiPSC-CMs. Another patient-specific DCM iPSC line was generated from a single member of a family with an autosomal dominant nonsense mutation (p.R225X) in exon 4 of the lamin A/C (LMNA) gene. hiPSC-CMs from this patient showed morphologic changes, including a higher prevalence of nuclear bleb formation, micronucleation, as well as nuclear senescence and cellular apoptosis [112]. Additionally, Tse and colleagues generated hiPSC-CMs derived from a DCM patient with a novel heterozygous mutation of p.A285V codon conversion on exon 4 of the desmin (DES) gene [113]. In this study, hiPSC-CMs were able to provide histologic and functional confirmation that the candidate gene variant detected by whole exome sequencing was responsible for the disease.

To study the molecular mechanisms underlying DCM in DMD, Lin and co-workers generated cardiac myocytes (CMs) from DMD patients and healthy control induced pluripotent stem cells (iPSCs). Using DMD patient-derived iPSC-CMs, they have established an *in vitro* model that manifests the major phenotypes of DCM in DMD patients, and uncovered a potential new disease mechanism [114]. In this regard, Lin and co-workers examined a collection of muscular dystrophies (including DMD and Becker Muscular Dystrophy) and healthy hiPSC-derived cardiac myocytes. This included demonstration that loading of the treated DMD hiPSC-derived cardiac myocytes with the calcium sensitive dye, Rhod-2AM, revealed increased cytosolic calcium concentration. The use of calcium assays in hiPSC derived cardiac myocytes is becoming commonplace due to the ease and availability of high speed/resolution optical imaging techniques. Typically, this uses voltage-sensitive dyes or genetically-encoded voltage indicators to measure action potentials and calcium wave propagation. Indeed, Guan and coworkers showed a two-fold increase in T50 (duration of recovery) of calcium transients in hiPSC-derived cardiac myocytes from DMD patients compared to healthy hiPSC derived cardiac myocytes [131]. In addition, Tsurumi et al. reported that the measurement of the fluorescent ratio (410/490 nm) of indo-1 demonstrated that the intracellular calcium concentration was much higher in cardiac myocytes differentiated from DMD-hiPSCs than in those differentiated from control-hiPSCs [132].

HiPSC-CMs represent a unique platform to study basic mechanisms of cardiomyopathies using a human cell-based system. The literature published in the past decade demonstrates the utility of patient-specific iPSCs in disease modeling of cardiomyopathy, and has provided unique insights into disease mechanisms. However, the hiPSC-CMs that are available today still largely represent an immature version of the adult cardiac myocyte, and thus have inherent limitations in the study of cardiovascular disease. Systems to induce greater maturation in hiPSC-CMs are being developed, but considerable work remains to be done to further advance this model system.
