In our long term cultures of human

In our long-term cultures of human iPSC CMs, the cTnI:ssTnI ratio stalled at ∼2:98, making it fetal-like with regard to the TnI isoform profile. This stalled marker of development was observed using different differentiation protocols, in multiple hiPSC lines (five hiPSC-CMs line obtained from different labs) and over long-term culture (9+ months). In an attempt to modify this ratio via extrinsic signaling mechanisms, we employed varied media modifications including thyroid hormone supplementation. In rodent myocytes in culture, T3-supplemented media accelerated the transition from ssTnI to cTnI; however, in human iPSC-CMs, no change in the TnI isoform ratio was obtained. The basis for the lack of effect of T3 on hiPSC-CMs to influence changes in TnI isoforms is not clear. Recent studies have shown T3 to have an effect on αMHC and SERCA2a muscarinic profiles in hiPSC-CMs, indicating that key components of the T3 signaling cascade are present (Ivashchenko et al., 2013; Yang et al., 2014). The only successful method used here to more closely approach the mature adult TnI signature was via direct gene transfer of TNNI3, which was highly efficient in stoichiometric replacement of ssTnI with cTnI, similar in practice to our previous work in adult cardiac myocytes (Westfall et al., 1997; Day et al., 2006). Along with implementing TnI isoform ratio as a maturation marker, it is well known that TnI isoforms have significant physiological implications for cardiac myocyte performance (Siedner et al., 2003; Davis et al., 2008). There is considerable debate regarding identifying the specific properties of stem cell-derived cardiac myocytes most suitable in terms of effecting physiologically meaningful regeneration/repair in disease myocardium in vivo. In terms of the profile of TnI isoform expression, there are positive and negatives attributed to the fetal and adult forms of the TNNI gene. The TNNI1 gene product, ssTnI, has unique molecular switch properties that would be expected to be an advantage in terms of survival in the harsh environment where stem cell-derived myocytes are placed upon transfer to ischemic and diseased hearts. Here, the ssTnI protein confers robust Ca2+ activated contraction in an environment where blood supply is inadequate and in the biochemical milieu of acidosis (Westfall et al., 1997; Day et al., 2006), as would be expected during cellular transplantation. A negative aspect of ssTnI function relates to its detrimental effects on relaxation performance owing to comparatively slow inactivation of the thin filament in diastole (Davis et al., 2008). In comparison, the adult TNNI3 gene product, cTnI, by virtue of its unique 32-amino-acid N-terminal extension containing target PKA-responsive serines, confers enhanced relaxation performance to the myofilaments that is physiologically important to overall normal heart performance (Davis et al., 2008). However, a negative attribute of cTnI is its marked downregulation in function in ischemic/hypoxic conditions that would be expected in the milieu of the transplanted myocytes (Westfall et al., 1997; Day et al., 2006). In this light, the ideal TnI molecule would retain the best properties of both cTnI and ssTnI isoforms. We have used structure-function and evolutionary biology concepts to guide construction of a unique TNNI gene product that incorporates an acidosis resistant histidine button while retaining fast inactivation properties required to cardiac lusitropy during adrenergic stimulation (Day et al., 2006). We would propose this engineered molecular switch protein to be ideal in genetic design of stem cell-derived cardiac myocytes for repair in vivo. While the TnI isoform ratio is proposed here to be necessary for tracking maturation of stem cell-derived cardiac myocyte, it is not sufficient, on its own, to establish the mature adult cardiac myocyte state. Many other well-defined physiological and structural markers must also assemble in concert to fully define a myocyte as adult, including highly refined Ca2+ handling, exquisite cell morphology and subcellular structure, and unique physiological forces and motion. Ultimately, these features, which are critical but limited by their restricted use in isolation (owing to issues of fetal-like reversion in stress and disease states), make the TnI switch profile valuable as an immutable barometer to gauge methods seeking to advance stem cell-derived myocytes toward the mature adult state. Mindful of the complexities listed above, the cTnI:ssTnI protein isoform ratio will enable a differentiation status standard on which cardiac myocytes can be directly compared between research groups as we have tested in this study. This could be useful in cardiac stem cell myocyte applications in the lab and the clinic.