To realize the full scientific and clinical potential of human embryonic stem cell (hESC)-cardiomyocytes, strategies to overcome the high degree of heterogeneity of differentiated populations are required. Here we demonstrate the utility of two transgenic approaches in enrichment of cardiomyocytes derived from HUES-7 cells: (i) negative selection of proliferating cells with the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system; and (ii) positive selection of cardiomyocytes expressing a bicistronic reporter [ green fluorescent protein (GFP)-internal ribosome entry site (IRES)-puromycin-N-acetyltransferase (PAC)] from the human amyosin heavy chain promoter. Parental and transgenic HUES-7 cells were similar with regard to morphology, pluripotency marker expression, differentiation, and cardiomyocyte electrophysiology. Whereas immunostaining of dissociated cardiomyocyte preparations expressing HSVtk or PAC contained <7% cardiomyocytes, parallel cultures treated with GCV or puromycin, respectively, contained 33.4 +/- 2.1% or 91.5 +/- 4.3% cardiomyocytes corresponding to an enrichment factor of 6.7- or 14.5-fold. Drug-selected cardiomyocytes responded to chronotropic stimulation and displayed cardiac-specific action potentials, demonstrating that functionality was retained. Both transgenic strategies will be generically applicable and should readily translate to the enrichment of many other differentiated lineages derived from hESCs.

Differentiation of human embryonic stem cells (hESCs) into cardiomyocytes in culture may offer unique opportunities for modeling genetic disorders, screening potentially cardiotoxic pharmaceutical agents or replacing cells of the diseased heart. However, before clinical utility can be realized, numerous hurdles must be overcome. Comprehensive molecular and phenotypic characterization is required but has so far been restricted to cardiomyocytes derived from a limited subset of hESC lines. Thus, we have initiated analysis of cardiomyocyte differentiation and function from a further two independently derived lines, BG01 and HUES-7. The challenge of improving cardiac cell induction, enrichment and maturation must also be addressed to meet the demands of high throughput pharmaceutical screening or to provide sufficient cells to repair an infarcted heart. Transplanted cells must functionally integrate without inducing arrhythmias, while survival and evasion of immune surveillance must be accomplished without tumorigenicity. This review evaluates the opportunities presented by hESC-derived cardiomyocytes and the progress towards surmounting the challenges of clinical translation.

1. It has previously been shown that ATP and UTP stimulate P2Y receptors in vascular smooth muscle cells (VSMCs), but the nature of these receptors, in particular the contribution of P2Y2 and P2Y4 subtypes, has not been firmly established. Here we undertake a further pharmacological analysis of [3H]inositol polyphosphate responses to nucleotides in cultured rat VSMCs. 2. ATP generated a response that was partial compared to UTP, as reported earlier. 3. In the presence of a creatine phosphokinase (CPK) system for regenerating nucleoside triphosphates, the response to ATP was increased, the response to UTP was unchanged, and the difference between UTP and ATP concentration-response curves disappeared. Chromatographic analysis showed that ATP was degraded slightly faster than UTP. 4. The response to UDP was always smaller than that to UTP, but with a shallow slope and a high potency component. In the presence of hexokinase (which prevents the accumulation of ATP/UTP from ADP/UDP), the maximum response to UDP was reduced and the high-potency component of the curve was retained. By contrast, the response to ADP was weaker throughout in the presence of hexokinase. 5. ATP gamma S was an effective agonist with a similar EC50 to UTP, but with a lower maximum. ITP was a weak agonist compared with UTP. 6. Suramin was an effective antagonist of the response to UTP (pA2=4.48), but not when ATP was the agonist. However, suramin was an effective antagonist (pA2=4.45) when stimulation with ATP was in the presence of the CPK regenerating system. 7. Taken together with the results of others, these findings indicate that the response of cultured rat VSMCs to UTP and to ATP is predominantly at the P2Y2 receptor, and that there is also a response to UDP at the P2Y6 receptor.