For iPSCs to dominate a wide spectrum of biomedical fields, their effective differentiation into specific cell types is of extreme importance. In vitro CM differentiation from iPSCs is achieved by modulation of signaling pathways known to be involved in cardiac development during normal embryogenesis [
12]. To date, there have been three main strategies developed to obtain functional CMs from iPSCs: (1) co-culture with visceral endoderm-like cells (END-2); (2) embryoid body (EB)-based differentiation, and (3) two-dimensional culture. During embryonic development in vivo, visceral END-2 releases factors that lead to cardiac differentiation of the nearby mesoderm [
13]; this discovery was the basis of the co-culture strategy in which PSCs cultured either in the presence of END-2 cells or in END-2-conditioned medium enter cardiac fate. Although this protocol was successfully applied to both ESCs [
14] and iPSCs [
15], the CM yield is very low (less than 10%) [
16]. EB-based differentiation is a serum-mediated three-dimensional method relying on the capability of PSCs to form floating cell aggregates when cultivated as single cells in low attachment substrate. These aggregates, known as embryoid bodies (EBs), spontaneously produce derivative cells of all the three germ layers [
17]. However, the EB-differentiation procedure, due to the presence of serum, suffers from low reproducibility and inter-line variability [
18]. Serum was later replaced by cytokines and growth factors known to be involved in heart development such as Wnt proteins [
19], bone morphogenetic proteins (BMPs) and activin A [
12,
20], and Notch signals [
21], together with their corresponding inhibitors [
22,
23]. Different small molecules have been tested for their ability to promote in vitro cardiac differentiation; activators (CHIR99021) and inhibitors (IWR, XAV, IWP2) of the Wnt pathway have been proved to increase cardiac differentiation [
24]. However, this strategy requires a high number of starting cells and has a low efficiency. To overcome the limitations of methods based on EB formation, differentiation protocols have been developed based on cell monolayers but with the use of the same molecular factors described for EB differentiation [
25,
26]. A monolayer-based strategy allowed significant improvement of the yield of cardiac differentiation and the phenotype of derived cardiac cells exhibiting typical features of ventricular, atrial, or nodal cardiomyocytes [
27]. The “matrix sandwich” method is a modification of monolayer assay, consisting of covering confluent iPSCs, previously treated with specific growth factors and cytokines to induce cardiac differentiation, with an overlay composed of matrix (i.e., Matrigel) and culture medium. This method relies on the pivotal role that the extracellular matrix plays in the differentiation process leading to high CM purity and yield [
28]. Other differentiation methods require two steps: during the first step iPSCs are induced to differentiate into cardiac progenitor cells (CPCs), which in turn can be further differentiated into different cellular fates including CMs, smooth muscle cells (SMCs), and endothelial cells [
29,
30]. Moreover, it was recently demonstrated that induced CPCs can be directly generated using mouse fibroblasts, skipping the intermediate stage of iPSCs [
31,
32]. Although there are currently available protocols providing a differentiation efficiency of up to 80% or more in terms of CM purity [
27,
30,
33], all the so far reported strategies show major limitations such as heterogeneity and immaturity of the cardiac population [
34]. The low purity and high heterogeneity of the differentiated CM population constitutes an important obstacle for their use in cell-based therapy that requires efficient purification methods to enrich the cardiac population. So far, several studies have developed efficient isolation methods based on the identification of specific cardiac markers such as SIRPA and VCAM1 [
35,
36,
37]. Other studies have instead developed protocols to differentiate human iPSCs into specific subtypes of functional cardiac cells, such as atrial-, ventricular- [
38,
39,
40], nodal-like [
41] and pacemaker cells [
42]. Although the cardiac cells obtained from iPSCs can start beating very early during differentiation, they resemble, morphologically and functionally, fetal cardiomyocytes. iPSC-CMs display a disorganized morphology, reduced contractile capacity, alteration of glycolytic metabolism, abnormal electrophysiological properties, and reduced automaticity [
43]. This immaturity renders adult-onset heart disease modeling very challenging, owing to the uncertainty regarding the ability of relatively immature iPSC-CMs to fully recapitulate adult disease phenotypes or as a function of aging, while the understanding of early-stage pathological events is not affected by low iPSC-CM maturity. Moreover, the incomplete maturity of iPSC-derived CMs could narrow the effectiveness of these cells in mimicking the pathology, e.g., if this is caused by a gene mutated postnatally, with negative impact on their usefulness for studies on drug effect/toxicity. In order to improve the differentiation strategy of iPSCs toward the generation of a high mature and homogeneous cardiomyocyte population, new differentiation methodologies and technical modifications have been proposed. A long culture period (80–120 days) results in multinucleated iPSC-CMs exhibiting mature sarcomeres and increased electrophysiological properties compared to 20–40-day-old CMs [
44]. This higher grade of maturity of long-term culture CMs is strictly related to mitochondrial metabolism regulation, which is necessary for energy production and increased cell contractility [
45]. Other methods aimed to improve CM maturation include addition of T3 hormone [
46] or dexamethasone [
47] in culture medium and stressing CMs with mechanical and electrical stimuli [
48]. Among the methods developed to increase the maturation of iPSC-CMs, in vivo environments provided the most mature phenotype of iPSC-CMs [
49,
50]. Currently, nanotechnology-based approaches offer new perspectives in many fields of biomedical research, including cardiovascular research [
51]. Three-dimensional scaffolds produced starting from natural or synthetic materials and functionalized to reach specific mechanical and chemical features can be used for direct iPSC differentiation into cardiomyocytes. Scaffolds designed for cardiac differentiation should possess specific properties such as good elasticity to allow cardiac cell contraction and properties to allow cardiomyocytes to arrange in a polarized and organized structure typical of native myocardium. These properties are retained for example by poly (vinyl alcohol) [
52], polyethylene oxide [
53], poly(lactic-co-glycolic acid) [
54], and poly(caprolactone) [
55]. CMs cultured on 3D structures show an enhanced calcium signaling respect to monolayer culture [
56]; however, it is mandatory to combine this method with electrical and physiological stimulation to obtain cardiac cells with a complete degree of maturity [
57]. The number of differentiated cells obtained using classical culture methods represents an additional shortcoming for the application of iPSCs in cell therapy, given that up to one billion CMs need to be transplanted within the infarcted myocardium to replace damaged tissue [
58]. Large-scale production of CMs from iPSCs can be achieved using bioreactors that make the process scalable and reproducible via the continuous control and stabilization of culture parameters [
59]. Bioreactors create a dynamic suspension culture in which there is a constant flow of nutrients and homeostasis of pH and oxygen levels [
60]. iPSCs cultured in a spinner flask form aggregates that can be used as starting materials for CM production after treatments with molecules acting on the Wnt pathway. The spinner flask methods allow critical variables to be tightly supervised, such as aggregate size and cytokine release, augmenting differentiation efficiency [
61]. The culture in the suspension of cells that are adhesion-dependent for survival and proliferation can be obtained through the use of supporting matrices known as microcarriers [
62]. Laco et al. (2020) developed a microcarrier culture system in a tank bioreactor that allowed scalable iPSC expansion and CM differentiation and purification, reaching a yield of ~40 CMs per iPSC seeded after 22 days in culture [
63]. A future intension of bioreactor use will be the production of large amounts of high quality CMs in GMP manufacturing, to improve their use in clinical practice.