Cardiac Differentiation

Since the original discovery of iPSCs by Takahashi and Yamanaka, who described the induction of a pluripotent state (first in mice (Takahashi and Yamanaka, 2006) and then in 

3 iPSC-BASED DISEASE MODELING AND DRUG TESTING 

The KCNQl and KCNH2 genes code for two major cardiac repolarizing currents, and mutations in these channels have been shown to cause long QT syndrome (LQTS) type 1 and type 2, respectively (Modell and Lehmann, 2006). iPSC-CMs carrying mutations in these cardiac potassium channels 

In addition, iPSC-CMs have been used to model structural heart diseases, including familial dilated cardiomyopathy (DCM) (Sun et al., 2012) and hypertrophic cardiomyopathy (HCM) (Lan et al., 2013) caused by mutations in cardiac troponin T (cTnT) and myosin heavy chain 7 (MYH7), respectively. In both studies, iPSC-CMs recapitulated the disease phenotype at the cellular level with pharmacological rescue by chronic treatment with p-adrenergic blocker (propranolol or metoprolol) or the L-type Ca channel blocker (verapamil) (Sun et al., 2012 Lan et al., 2013) (Table 22.1). Collectively, these studies reinforce the ability of iPSC-CMs to model channelopathies and cardiomyopathies as well as to test the clinical efficacy of drugs. 

Standard patch clamp, calcium imaging Standard patch clamp 

---

Retinoic acid (RA), forskolin, and HDAC inhibitors have been shown/used to induce neuronal differentiation of hippocampal adult neural progenitor cells. However, these factors are either pleiotropic or of undefined physiologic relevance RA has neuronal subtype patterning activity and also been reported to induce cardiac differentiation (27) as well as pancreatic differentiation (28) HDAC inhibitors are nonspecific and forskolin activates protein kinase A and serves to increase the cellular levels of the general signaling molecule cAMP (29). 

Choi SC, et al. 5-azacytidine induces cardiac differentiation of PI 9 embryonic stem cells. Exp. Mol. Med. 2004 36 515-523. Tsuji-Takayama K, et al. Demethylating agent, 5-azac)4idine, reverses differentiation of embryonic stem cells. Biochem. Biophys. Res. Commun. 2004 323 86-90. 

The method was first introduced by Doetschman et al. using mouse ES (mES) cells [88] and demonstrating that spontaneous cell aggregation leads to formation of random-sized EBs, while Kehat et al. first used this method for cardiac differentiation studies in human ES cells [89],... 

Another approach for cardiac differentiation is based on co-culture of undifferentiated hESCs with mouse visceral endodermal like cell line (END-2), particularly in the absence of serum [98, 99]. hES cells or clumps are co-cultured with END-2 cells instead of MEF culture for 3 weeks in serum-free media, and beating cell patches are scored from day 5 onwards. 

Cardiac differentiation from stem cells is controlled by complex networks, and currently cardiac differentiation is effectively induced by multiple growth factors. 

For efficient cardiac differentiation, the choice of growth factors, combinations, concentrations, and exposure time need to be adjusted for different cell lines. 

In addition to growth factors, small molecules that induce the cardiogenesis pathway can enhance cardiomyocyte differentiation from hPSCs. A number of small molecules have been screened for promotion of cardiogenesis. The BMP signaling inhibitors [102, 103], p38 MAPKsignaling inhibitors [100], Wnt signaling activators [104], and Wnt singling inhibitors [105-107] are reported to promote the cardiac differentiation. 

In this directed cardiac differentiation protocol, combination of activin A and BMP4 induces a primitive streak-like population and mesoderm in hESCs. The addition of Wnt inhibitor DKK1 induces the cardiac mesoderm formation, while VEGF is included to promote the expansion and maturation of KDR+ population. The additional presence of bFGF supports the continuous expansion of the developing cardiovascular lineages. 

Fig. 4 Schematic representation of cardiac differentiation protocol using hESC...

Small molecule-induced cardiac differentiation in hPSC [110] The protocol implies a growth factor-free directed cardiac differentiation method. Dissociate the hPSC into single cells with accutase for 5 min at room temperature, and consequently plate them at the... 

Ao A, Hao JJ, Hopkins CR, Hong CC (2012) DMH1, a novel BMP small molecule inhibitor, increases cardiomyocyte progenitors and promotes cardiac differentiation in mouse embryonic stem cells. PLoS One 7(7) e41627... 

The EST has been developed with the aim to exploit the characteristics and differentiation potential of mouse embryonic stem cells (ES cells), established from the early embryo in 1981 [4, 5], ES cells are cultured in suspension to induce the formation of embryoid bodies, and afterwards they are transferred in 24-well dishes to allow attachment and differentiation in contracting cardiomyocytes. The toxicological endpoint is the inhibition of cardiac differentiation. In parallel a cytotoxicity test is performed on undifferentiated ES cells and a control somatic (fibroblast) cell line (3T3). The concentrations of testing chemicals that induce 50 % of differentiation inhibition (ID50) and 50 % cytotoxicity (IC50) in ES cells and 3T3 cells are inserted in a validated prediction model to classify the test chemical as non-embryotoxic, moderate, or strong embryotoxic [2, 6, 7], The validation of the method has been... 

Zhang J et al (2009) Functional cardiomyo-cytes derived from human induced pluripotent stem cells. Circ Res 104(4) e30-e41 Zwi L et al (2009) Cardiomyocyte differentiation of human induced pluripotent stem cells. Circulation 120(15) 1513-1523 Burridge PW et al (2012) Production of de novo cardiomyocytes human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell 10(l) 16-28 Rattman SJ et al (2011) Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and... 

Zhang J et al (2012) Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells the matrix sandwich method. Circ Res 111 (9) 1125-1136... 

The currently available cardiomyocytes differentiation system of the hES is essentially spontaneous, and is characterized by relatively low efficacy. Understanding the mechanisms that drive early-cardiac differentiation of the hES cells may also have important clinical implications. A major obstacle for the use of these cells in future myocardial regeneration strategies is the insufficient number of cardiomyocytes achieved by the currently available differentiation scheme. Therefore, further efforts aimed at developing a more productive cardiomyocyte differentiation system are essential for the generation of the large quantities of cells needed to achieve the successful application of this strategy. 

There is also evidence to suggest that lessons learned from early cardiac differentiation in the model systems (as described above) may also apply to the hES cells. The cardiogenic inductive role of the primitive visceral endoderm (VE) was also shown to play a role in cardiomyocyte differentiation of the hES fine in an elegant study conducted by Mummery et al. [59[. Go-cultur-ing of a human ES ceU line (hES2) that does not regularly differentiate spontaneously to... 

---