Tracking the maturation status of stem cell derived cardiac

Tracking the maturation status of stem cell-derived cardiac myocytes by ratiometric analysis of the cTnI:ssTnI protein profile provides a tool to permit direct comparisons of myocyte developmental status from different cell lines and with using different culture methods and differentiation protocol across laboratories. Tracking the cTnI/ssTnI isoform e1 activating enzyme profile will be useful in cataloging the large array of other pertinent markers of myocyte development including molecular, structural, electrical, morphological, and functional features of maturing stem cell-derived cardiac myocytes. Ultimately, to be defined as an adult myocyte would require acquisition of the well-known structural and functional profile of the mature myocyte, and we propose the adult designation would require the exclusive expression of the cTnI isoform (Bhavsar et al., 1991; Siedner et al., 2003). Key features of the TnI protein isoform switch as a quantitative standard for cardiac stem cell-derived myocyte maturation are (1) its stoichiometric conservation, (2) its normal developmental irreversibility, and (3) its physiological significance (Siedner et al., 2003; Davis et al., 2008). Conservation of a strict one-to-one stoichiometric expression pattern provides a unique internal standard on which the cTnI:ssTnI isoform ratio informs a direct quantitative marker of development. This differs from tracking, for example, the relative abundance of a single cardiac marker in developing cells, such as cardiac actin, α-actinin, or myosin light chains, because a reference internal “fetal” standard is lacking thus limiting their use. Further, often used cardiac development markers such as cell morphology and striation appearance are inherently subjective in terms of precise tracking and cataloging maturation progression. Irreversibility in the ssTnI to cTnI transition is of value as a maturation marker. At the level of the cardiac sarcomere, as cTnI (TNNI3 gene product) progressively replaces ssTnI (TNNI1) during maturation, there is no reactivation of the fetal TNNI1 gene program, even in disease states including stress, hypertrophy (physiological or maladaptive), ischemia, or in heart failure (Bhavsar et al., 1991; Solaro, 1999). In all mammalian hearts, including human, it is the complete stoichiometric replacement of ssTnI by cTnI that is as an immutable profile of acquiring the mature adult state. Accordingly, to be termed adult, a cardiac myocyte requires a profile of 100% cTnI and 0% (i.e., undetectable) ssTnI. It would be insufficient, for example, to simply detect cTnI expression (as a single cardiac marker by IF or fluorescence-activated cell sorting [FACS]) to track differentiation status, without also documenting in unison the complete stoichiometric loss of ssTnI expression. We propose the following cTnI:ssTnI protein isoform ratio, quantified by western blot analysis (with a pan TnI isoform antibody as shown here), be implemented for the developmental stage classification of populations of stem cell-derived cardiac myocytes: immature = 0:100 to 15:85; neonatal-like = >15: <85 to 90: 10; mature profile = 100:0 (Figure 6). Other techniques such as IF or FACS would not be adequate for quantifying this TnI isoform profile because of limitations in precise ratiometric analysis of the stoichiometrically conserved cTnI:ssTnI isoform ratio. Numerous cardiac markers, including molecular, structural, and physiological, have been used in attempt to track and classify cardiac cells derived from stem cell progenitors or from direct reprogramming (Ieda et al., 2010; Qian et al., 2012). The sarcomere markers cTnT and myosin are in frequent use, along with the detection of myofilament striations, myocyte morphology, Ca2+ handling, rhythmic beating, action potential shape, and others for cardiac lineage assignment and development assessment (Radisic et al., 2004; Lundy et al., 2013). Limitations of these markers include questions of lineage specificity (e.g., cTnT is also expressed in smooth muscle) and developmental progression. For example, markers such as TnT and MyHC show isoform developmental regulation with the complicating feature of reversion to the “fetal” program in disease (Parker et al., 1990; Kinugawa et al., 2001). The failing adult cardiac myocyte reactivates the fetal program at many levels, including structural, hormonal, and metabolic gene profile by downregulating adult gene transcripts (Parker et al., 1990; Kinugawa et al., 2001). For instance, transcript levels of ANF, BNP, skeletal actin, βMHC, and metabolic genes such as GLUT1 are higher in the human fetal and failing hearts than in the normal adult heart (Pasternac and Cantin, 1990; Tanaka et al., 1995; Shioi et al., 2000). In concert, transcript levels of adult genes such as αMHC and SERCA-2a, ion channels, and metabolic genes such as GLUT4 are reduced in failing human heart (Pasternac and Cantin, 1990; Tanaka et al., 1995; Lowes et al., 1997; Shioi et al., 2000). Previous studies have used one or more of these genes to evaluate maturation of in vitro derived cardiac myocytes (Radisic et al., 2004; Chan et al., 2013b; Lundy et al., 2013). Therefore, it cannot be certain by testing, for example, MyHC isoforms or Ca2+ handling function that the myocytes in question are at a fetal stage of development or rather are reverting in profile owing to latent activation of the fetal program. In other words, markers in frequent use today in cardiac stem cell biology are highly plastic in nature, making difficult any specific assignment of myocyte maturation. As mentioned, because cardiac TnT is also expressed in smooth muscle cells, the use of cTnT to quantify CMs must be interpreted with caution. Therefore, it is important to incorporate at least a second cardiac-specific marker for cardiac lineage assignment. Equally important, the use of MLC2a and MLC2v to define maturation is not straightforward. This is because although at early developmental time points most hiPSC-CMs express MLC2a and not MLC2v, at later time points, they express robust MLC2v, with MLC2a still significantly expressed. To be classified as a mature ventricular myocyte, stem cell-derived CMs require 100% cTnI (and 0% ssTnI) that is colocalized with MLC2v. MLC2v alone cannot serve as a mature marker in CMs, as we show MLC2V-positive myocytes express primarily ssTnI (the immature marker) (Figure S4).