In Huber et al reported the

In 2007, Huber et al. reported the generation of stable transgenic human embryonic stem cell lines for identification and selection of differentiating CMs. Although the authors used the human MLC-2v promoter reporter construct, they did not characterize the atrial- or ventricular-like features of their purified CMs. They demonstrated that the sorted MLC-2v-GFP+ cells showed high expression of the cardiac-specific genes MLC-2v, MLC-2a, and α-MHC but did not distinguish between the atrial or ventricular lineage. Here, we showed by RT-PCR and WB that the BJ MLC-2v-GFP+ sorted cells had low MLC-2a and α-MHC expression and robust MLC-2v expression. Huber et al. (2007) found that MLC-2v+ cells displayed embryonic-like electrophysiological properties with maximal diastolic potentials (MDP) of ~−54mV which is slightly more depolarized than our sorted ventricular-like CMs (~−58mV). It is possible that the EB differentiation protocol used by Huber et al. (2007) led to cardiac cultures with lower level of maturity than the matrigel sandwich protocol and a higher proportion of double MLC-2a/MLC-2v positive cells which fail to display characteristics of relatively differentiated ventricular CMs. In addition, in our study, we did not establish a stable transgenic cell line but used transient adenovirus-mediated gene transfer to purify MLC-2v from MLC-2a positive CMs later in the differentiation protocol (between day 30 and day 40) after emergence of the MLC-2v and MLC-2a gene expression in the culture (day 15 and day 5, respectively). The differentiation protocol and the timeline of selection might explain the major differences that we find with Huber et al. (2007), as follows: 1) higher percentage of recovered cells, 2) greater expression of the ventricular myosin isoforms, Cx43 and ventricular transcription factor HEY2 than atrial myosin isoforms, Cx40 and atrial transcription factor TBX5, 3) demonstration of the emergence of an early ventricular (double MLC-2a/MLC-2v positive) population in contrast with the relatively more differentiated ventricular CMs (single MLC-2v positive), 4) typical electrophysiological properties of ventricular-like CMs. We have demonstrated that MLC-2a and MLC-2v promoter activity-based selection can be used to segregate atrial- from ventricular-like differentiating hiPSC-CMs with distinct functional phenotypes. We showed that hiPSC-CMs selected based on MLC-2 viral reporters recapitulate electrophysiological and calcium cycling properties of human atrial- and ventricular-like CMs. Single cell patch-clamp experiments were performed to quantify the AP characteristics of each cell type. Our data showed that the Cy3 hydrazide manufacturer duration, which mostly depends on the balance between a depolarizing calcium current and repolarizing potassium currents, was significantly shorter in the atrial-like CMs compared to ventricular-like CMs. We also demonstrate that the action potential amplitude was smaller in the atrial-like cells (Fig. 7). Stimulated action potential morphologies in the BJ hiPSC-CMs also matched values reported for adult human atrial and ventricular CMs (Grandi et al., 2010, 2011). Interestingly, we found that, ventricular-like CMs had spontaneous calcium transients with larger amplitudes and lower frequencies than the atrial-like CMs which correlates with the calcium wave characteristics (Supplementary Fig. 9). As discussed earlier, the developmental heterogeneity of the MLC-2a+ population seems greater than MLC-2v+ cells. However, action potential parameters such as resting membrane potential (most negative value of membrane potential reached after repolarization determined by an inward potassium current) and phase-0 upstroke velocity (depending on the amount of depolarizing inward sodium current) did not reflect these differences. Cardiac myocytes selected using the MLC-2a and MLC-2v promoters contained a measurable population of MLC-2a/MLC-2v double positive cells, as detected by immunocytochemistry with more single MLC-2v positive cells identified in the MLC-2v selected population. In the MLC-2a+ population, the majority of the cells exhibited strong staining for MLC-2a, organized at the sarcomere with faint and non-organized MLC-2v staining. These cells may represent early atrial CMs. Conversely, cells with strong staining and a clear sarcomeric organization for MLC-2v may be in transition to ventricular-like CMs. In the MLC-2v+ population, we distinguished different level of maturity depending on the degree of MLC-2a expression and its localization pattern. The minor population of cells with a stronger MLC-2a than MLC-2v staining might represent the most immature cells commited to become ventricular. It is tempting to speculate that the relative proportions of MLC-2a/MLC-2v may indicate the transition from a less to more mature ventricular CMs, as is seen in vivo. In that case, cells with less MLC-2a and more MLC-2v expression would represent a more mature population of ventricular CMs. Finally, this population may evolve into the most differentiated population that we detect, MLC-2v positive/MLC-2a negative ventricular CMs. The percentage of single positive cells was much higher in the MLC-2v positive population (26.9%) than in the MLC-2a positive population (4%), suggesting a higher level of maturity of the MLC-2v+ cells. The presence of MLC-2a/MLC-2v double positive cells in hiPS-CM cultures also has been described by other groups (Zhang et al., 2009; Lee et al., 2012). We have shown that MLC-2a gene expression precedes MLC-2v during cardiac-directed differentiation of hiPSCs (Fig. 1D) and MLC-2a is expressed before MLC-2v in the heart tube (Kubalak et al., 1994). Later in development, MLC-2a expression is downregulated in the developing ventricle and becomes restricted to the atria in the adult (Kubalak et al., 1994). MLC-2a is expressed in differentiating cells destined to become either atrial or ventricular CMs while MLC-2v is more selective. Thus, it is not suprising that the use of the MLC-2v adenovirus led to more homogeneous populations than the MLC-2a adenovirus. Immunostaining demonstrated a more organized sarcomere in the MLC-2v+ population but only few MLC-2a+ cells exhibited clear MLC-2a sarcomeric organization probably due to the presence of distinct ranges of maturation in these cultures (Supplementary Fig. 5). Importantly, in the sorted populations the expression of MLC-2a and MLC-2v transcripts is not lost with time in culture but instead the level of these transcripts increases over time. In accordance with previous studies, PSC-CMs are not fully differentiated and exhibit a phenotype more consistent with embryonic heart tube CMs (Fijnvandraat et al., 2003). Taken together, our data show that robust MLC-2v promoter activity is sufficient to define a subset of hiPSC-CM differentiating into a ventricular myocyte lineage that remains stable in culture. In contrast, the MLC-2a promoter is active in multiple hiPSC-CM cell lineages, including cells entering the atrial lineage and cells destined to become ventricular CMs. Improved culture conditions that drive hiPSC-CMs into a more fully differentiated state may be needed to allow clean segregation of both atrial and ventricular like hiPSC-CM populations using these, or other promoters.