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.

The pertussis toxin (PTX) insensitive heterotrimeric G protein G 12 has been implicated in mitogenesis and transformation, but its direct effectors remain unknown. To define potential signaling pathways utilized by G 12 , we expressed an activated mutant of its ␣ subunit, G␣ 12 , which belong to the mitogen activated family of protein kinases (MAPKs), stimulate the transactivation potential of the c-Jun component of the transcription factor AP-1 by phosphorylating it at residues Ser-63 and Ser-73 (1, 2). The JNKs are strongly activated by exposure to UV irradiation and osmotic stress (2-4). The JNKs are also activated by mitogens including epidermal growth factor and the oncogenic v-Src tyrosine kinase and v-Ras, and by proinflammatory cytokines such as tumor necrosis factor ␣ (2, 5, 6). The activation of JNK by growth factors and cytoplasmic oncogenes suggests a role for JNK in cellular proliferation or differentiation (7). Consistent with this hypothesis, blockade of the JNK pathway inhibits transformation of NIH3T3 fibroblasts by v-Src (5).JNK regulation by cytoplasmic oncogenes and growth factors has recently been shown to require activation of the low molecular weight G protein Ras (8) and two Rho subfamily low molecular weight G proteins, Cdc42 and Rac (5, 9). While Ras is also an efficient activator of the extracellular signal regulated kinases (ERKs), Rac and Cdc42 are involved only in activation of JNK and the related p38 MAPK (5,[10][11][12]. It has also been demonstrated that G protein-coupled muscarinic receptors expressed in NIH3T3 and Rat 1a cells can activate JNKs (13,14). A recent report demonstrates that M 1 and M 2 muscarinic acetylcholine receptors (mAChRs) activate JNKs through the G ␤␥ subunit, suggesting this as the mechanism of JNK regulation for other G protein-coupled receptors (15).Although its effectors are unknown, the pertussis toxin (PTX) insensitive heterotrimeric G protein G 12 has been implicated in cellular transformation and proliferation. Expression of an activated form of G␣ 12 , G␣ 12 (Q229L) (referred to here as activated G␣ 12 ) induces transformation in NIH3T3 and Rat1 cells (16,17). We recently demonstrated by microinjection of antibodies to G␣ 12 that the PTX insensitive DNA synthesis induced by thrombin in 1321N1 astrocytoma cells requires G 12 (18). In addition, transient expression of activated G␣ 12 can dramatically stimulate AP-1-dependent gene expression in a Ras-dependent manner (18). These results suggest that G␣ 12 stimulates a Ras-dependent signaling pathway leading to mitogenesis and AP-1 activation. The studies reported here examine the possibility that the ␣ subunit of G 12 activates Rasand Rac-dependent pathways and thereby stimulates JNK and AP-1 activity. MATERIALS AND METHODS TransfectionHEK293 cells were plated at 2 ϫ 10 5 cells per 60-mm dish 3 days before transfection and grown at 5% CO 2 in ␣-minimum essential medium supplemented with 10% fetal calf serum and penicillin/streptomycin. Cells were transfected by calcium phosphate coprecipitation (19) ...

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Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12 has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of G alpha 12 increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of G alpha 12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-G alpha 12 resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of G alpha o did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-G alpha 12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for G alpha 12 in the mitogenic response to thrombin in human astroglial cells.

The electrophoretic mobility of liposomes containing a negatively charged derivative of phosphatidylethanolamine with a large headgroup composed of the hydrophilic polymer polyethylene glycol (PEG-PE) was determined by Doppler electrophoretic light scattering. The results show that this method is improved by the use of measurements at multiple angles to eliminate artifacts and that very small mobilities can be measured. The electrophoretic mobility of liposomes with 5 to 10 mol% PEG-PE is approximately -0.5 mu ms-1/Vcm-1 regardless of PEG-PE content compared with approximately -2 mu ms-1/Vcm-1 for similar liposomes but containing 7.5% phosphatidylglycerol (PG) instead of PEG-PE. Measurements of surface potential by distribution of an anionic fluorescent probe show that the PEG-PE imparts a negative charge identical to that by PG, consistent with the expectation of similar locations of the ionized phosphate responsible for the charge. The reduced mobility imparted by the surface bound PEG is attributed to a mechanism similar to that described for colloidal steric stabilization: hydrodynamic drag moves the hydrodynamic plane of shear, or the hydrodynamic radius, away from the charge-bearing plane, that of the phosphate moities. An extended length of approximately 50 A for the 2,000 molecular weight PEG is estimated from the reduction in electrophoretic mobility.

In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G proteincoupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2', diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxininsensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that Ga12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells.

Cytokines and extracellular matrix proteins initiate signaling cascades that regulate cell migration and proliferation. Evidence is provided that the adaptor protein Shc can differentially regulate these processes. Specifically, under growth factor–limiting conditions, Shc stimulates haptotactic cell migration without affecting anchorage-dependent proliferation. However, when growth factors are present, Shc no longer influences cell migration; rather, Shc is crucial for DNA synthesis. Mutational analysis of Shc demonstrates that, while tyrosine phosphorylation is required for both DNA synthesis and cell migration, the switch in Shc signaling is associated with differential use of Shc's phosphotyrosine interacting domains; the PTB domain regulates haptotaxis, while the SH2 domain is selectively required for proliferation.

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