BackgroundThe use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry.ResultsHere we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture.ConclusionsSimple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.

-During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca 2ϩ overload, and mitochondrial permeability transition pore (mPTP) opening, as the crucial mechanism of cardiomyocyte injury, remains intriguing. Here, we investigated whether an induction of a partial decrease in mitochondrial membrane potential (⌬⌿ m) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca 2ϩ , and mPTP opening. The magnitude of APCinduced decrease in ⌬⌿m was mimicked with the protonophore 2,4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC-and DNP-induced decrease in ⌬⌿ m. In cardiomyocytes, ⌬⌿ m, ROS, mPTP opening, and cytosolic and mitochondrial Ca 2ϩ were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca 2ϩ uptake were determined spectrofluorometrically. In cells exposed to oxidative stress, APC and DNP increased cell survival, delayed mPTP opening, and attenuated ROS production, which was reversed by mitochondrial repolarization with pyruvate. In isolated mitochondria, depolarization by APC and DNP attenuated ROS production, but not Ca 2ϩ uptake. However, in stressed cardiomyocytes, a similar decrease in ⌬⌿m attenuated both cytosolic and mitochondrial Ca 2ϩ accumulation. In conclusion, a partial decrease in ⌬⌿m underlies cardioprotective effects of APC by attenuating excess ROS production, resulting in a delay in mPTP opening and an increase in cell survival. Such decrease in ⌬⌿m primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca 2ϩ uptake.cardioprotection; oxidative stress; mitochondria; reactive oxygen species DAMAGE DURING ISCHEMIA and reperfusion (I/R) of the heart involves complex processes where the cellular machinery itself becomes a source of deleterious mediators of injury. Such processes include excessive production of mitochondrial reactive oxygen species (ROS) and cellular Ca 2ϩ overload (6, 37), which trigger opening of the mitochondrial permeability transition pore (mPTP) (16, 19), a critical event in the transition towards cell death (18). The mPTP opening instantly dissipates mitochondrial membrane potential (⌬⌿ m ), ceases mitochondrial ATP production, and initiates cell death pathways (6).Studies suggest that most of the cell death occurs during reperfusion (51, 53), which can be attenuated with antioxidants (52), indicating an important role of ROS. In cardiomyocytes, ROS are primarily generated at complexes I and III of the mitochondrial electron transport chain (50). During I/R, accumulation of cytosolic Ca 2ϩ , which drives accumulation of mitochondrial Ca 2ϩ (45), is primarily attributed to insufficient ATP production and derangement of intracellular ion homeostasis (37). It is suggested that excess ROS production and mitochondrial Ca 2ϩ overload are mutually de...

Background Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients, relatively inaccessible for research purposes. We tested whether anesthetic-induced preconditioning (APC) with isoflurane elicits competent protective mechanisms in hESC-derived cardiomyocytes against oxidative stress to be used as a model of human cardiomyocytes for studying preconditioning. Methods H1 hESC cell line was differentiated into cardiomyocytes using growth factors activin A and bone morphogenetic protein-4. Living ventricular hESC-derived cardiomyocytes were identified using lentiviral vector expressing a reporter gene (enhanced green fluorescent protein) driven by a cardiac-specific human myosin light chain 2v promoter. Mitochondrial membrane potential, reactive oxygen species production, opening of mitochondrial permeability transition pore, and survival of hESC-derived cardiomyocytes were assessed using confocal microscopy. Oxygen consumption was measured in contracting cell clusters. Results Differentiation yielded a high percentage (∼85%) of cardiomyocytes in beating clusters that were positive for cardiac-specific markers and exhibited action potentials resembling mature cardiomyocytes. Isoflurane depolarized mitochondria, attenuated oxygen consumption, and stimulated generation of reactive oxygen species. APC protected these cells from oxidative stress-induced death and delayed mitochondrial permeability transition pore opening. Conclusions APC elicits competent protective mechanisms against oxidative stress in hESC-derived cardiomyocytes, suggesting the feasibility to use these cells as a model of human cardiomyocytes for studying APC and potentially other treatments/diseases. Our differentiation protocol is very efficient and yields a high percentage of cardiomyocytes. These results also suggest a promising ability of APC to protect and improve engraftment of hESC-derived cardiomyocytes into the ischemic heart.

Introduction Anesthetic preconditioning protects cardiomyocytes from oxidative stress-induced injury, but it is ineffective in patients with diabetes mellitus. To address the role of hyperglycemia in the inability of diabetic individuals to be preconditioned, we used human cardiomyocytes differentiated from induced pluripotent stem cells generated from patients with or without type 2 diabetes mellitus (DM-iPSC- and N-iPSC-CMs, respectively) to investigate the efficacy of preconditioning in varying glucose conditions (5, 11, and 25 mM). Methods Induced pluripotent stem cells were induced to generate cardiomyocytes by directed differentiation. For subsequent studies, cardiomyocytes were identified by genetic labeling with enhanced green fluorescent protein driven by a cardiac-specific promoter. Cell viability was analyzed by lactate dehydrogenase assay. Confocal microscopy was utilized to measure opening of the mitochondrial permeability transition pore and the mitochondrial adenosine-5′-triphosphate-sensitive potassium channels. Results Isoflurane (0.5 mM) preconditioning protected N-iPSC- and DM-iPSC-CMs from oxidative stress-induced lactate dehydrogenase release and mitochondrial permeability transition pore opening in 5 mM and 11 mM glucose. Isoflurane triggered mitochondrial adenosine-5′-triphosphate-sensitive potassium channel opening in N-iPSC-CMs in 5 mM and 11 mM glucose and in DM-iPSC-CMs in 5 mM glucose. 25 mM glucose disrupts anesthetic preconditioning-mediated protection in DM-iPSC- and N-iPSC-CMs. Conclusions The opening of mitochondrial adenosine-5′-triphosphate-sensitive potassium channels are disrupted in DM-iPSC-CMs in 11 mM and 25 mM glucose and in N-iPSC-CMs in 25 mM glucose. Cardiomyocytes derived from healthy donors and patients with a specific disease, such as diabetes in this study, open possibilities in studying genotype and phenotype related pathologies in a human relevant model.