Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell-derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell-derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.

Abstract-Cell replacement therapy is a promising approach for the treatment of cardiac diseases, but is challenged by a limited supply of appropriate cells. We have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem (hES) cells. Cardiomyocyte differentiation was evaluated using 3 parent (H1, H7, and H9) hES cell lines and 2 clonal (H9.1 and H9.2) hES cell lines. All cell lines examined differentiated into cardiomyocytes, even after long-term culture (50 passages or Ϸ260 population doublings). Upon differentiation, beating cells were observed after one week in differentiation conditions, increased in numbers with time, and could retain contractility for over 70 days. The beating cells expressed markers characteristic of cardiomyocytes, such as cardiac ␣-myosin heavy chain, cardiac troponin I and T, atrial natriuretic factor, and cardiac transcription factors GATA-4, Nkx2.5, and MEF-2. In addition, cardiomyocyte differentiation could be enhanced by treatment of cells with 5-aza-2Ј-deoxycytidine but not DMSO or retinoic acid. Furthermore, the differentiated cultures could be dissociated and enriched by Percoll density centrifugation to give a population containing 70% cardiomyocytes.