SUMMARY Induced pluripotent stem cells (iPSCs) outwardly appear to be indistinguishable from embryonic stem cells (ESCs). A study of gene expression profiles of mouse and human ESCs and iPSCs suggests that, while iPSCs are quite similar to their embryonic counterparts, a recurrent gene expression signature appears in iPSCs regardless of their origin or the method by which they were generated. Upon extended culture, hiPSCs adopt a gene expression profile more similar to hESCs; however, they still retain a gene expression signature unique from hESCs that extends to miRNA expression. Genome-wide data suggested that the iPSC signature gene expression differences are due to differential promoter binding by the reprogramming factors. High-resolution array profiling demonstrated that there is no common specific subkaryotypic alteration that is required for reprogramming and that reprogramming does not lead to genomic instability. Together, these data suggest that iPSCs should be considered a unique subtype of pluripotent cell.

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O 2 at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F 1 F 0 ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.

The mitochondrial theory of aging proposes that reactive oxygen species (ROS) generated inside the cell will lead, with time, to increasing amounts of oxidative damage to various cell components. The main site for ROS production is the respiratory chain inside the mitochondria and accumulation of mtDNA mutations, and impaired respiratory chain function have been associated with degenerative diseases and aging. The theory predicts that impaired respiratory chain function will augment ROS production and thereby increase the rate of mtDNA mutation accumulation, which, in turn, will further compromise respiratory chain function. Previously, we reported that mice expressing an error-prone version of the catalytic subunit of mtDNA polymerase accumulate a substantial burden of somatic mtDNA mutations, associated with premature aging phenotypes and reduced lifespan. Here we show that these mtDNA mutator mice accumulate mtDNA mutations in an approximately linear manner. The amount of ROS produced was normal, and no increased sensitivity to oxidative stress-induced cell death was observed in mouse embryonic fibroblasts from mtDNA mutator mice, despite the presence of a severe respiratory chain dysfunction. Expression levels of antioxidant defense enzymes, protein carbonylation levels, and aconitase enzyme activity measurements indicated no or only minor oxidative stress in tissues from mtDNA mutator mice. The premature aging phenotypes in mtDNA mutator mice are thus not generated by a vicious cycle of massively increased oxidative stress accompanied by exponential accumulation of mtDNA mutations. We propose instead that respiratory chain dysfunction per se is the primary inducer of premature aging in mtDNA mutator mice.mitochondria ͉ mtDNA mutator mice T he free radical theory of aging, formulated 50 years ago by Harman (1), proposes that aging and associated degenerative diseases can be attributed to deleterious effects of reactive oxygen species (ROS). Intracellular ROS are primarily generated by the mitochondrial electron transport chain, making the mitochondrial network a prime target of oxidative damage. Building on this, the mitochondrial theory of aging predicts that a vicious cycle contributes to the aging process. (i) Normal metabolism causes ROS production by the electron transport chain. (ii) ROS production induces damage to lipids, proteins, and nucleic acids in mitochondria. (iii) ROS-induced mtDNA mutations lead to the synthesis of functionally impaired respiratory chain subunits, causing respiratory chain dysfunction and augmented ROS production (2). (iv) This vicious cycle is proposed to cause an exponential increase of mtDNA mutations over time, resulting in aging and associated degenerative diseases. A substantial amount of correlative data from morphological, bioenergetic, biochemical, and genetic studies of mammalian tissues supports this theory (3). Mitochondria are larger and fewer in older individuals, and mitochondrial abnormalities such as vacuoles, abnormal cristae, and paracrystalline inclusions...

A large number of mutations in the gene encoding the catalytic subunit of mitochondrial DNA polymerase γ (POLγA) cause human disease. The Y955C mutation is common and leads to a dominant disease with progressive external ophthalmoplegia and other symptoms. The biochemical effect of the Y955C mutation has been extensively studied and it has been reported to lower enzyme processivity due to decreased capacity to utilize dNTPs. However, it is unclear why this biochemical defect leads to a dominant disease. Consistent with previous reports, we show here that the POLγA:Y955C enzyme only synthesizes short DNA products at dNTP concentrations that are sufficient for proper function of wild-type POLγA. In addition, we find that this phenotype is overcome by increasing the dNTP concentration, e.g. dATP. At low dATP concentrations, the POLγA:Y955C enzyme stalls at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The POLγA:Y955C enzyme will compete with wild-type POLγA for primer utilization, and this will result in a heterogeneous population of short and long DNA replication products. In addition, there is a possibility that POLγA:Y955C is recruited to nicks of mtDNA and there enters an idling mode preventing ligation. Our results provide a novel explanation for the dominant mtDNA replication phenotypes seen in patients harboring the Y955C mutation, including the existence of site-specific stalling. Our data may also explain why mutations that disturb dATP pools can be especially deleterious for mtDNA synthesis.

We wish to clarify that Fig 4A and B intentionally displayed duplicate controls and that the first panel in Figs 1A and S1A was intentionally duplicated.The low (control) and high (FCCP) traces were purposefully duplicated in panels from Fig 4A and B to allow for direct, experimentally unbiased comparisons between the effects of antimycin and sodium oxamate on pluripotent stem and differentiated cells. Panels shown in Fig 4A and B are representative of one single experiment for N = 2 equivalently performed experiments. The panels were separated into parts 4A (antimycin) and 4B (sodium oxamate) for clarity of presentation since the traces and the colors of these traces have overlaps, which makes clear visualization difficult. We apologize for not explicitly stating that these traces were derived from a single, representative experiment.In addition, the first panel in Fig 1A and the first panel of Supplementary Fig S1A were purposefully reproduced to highlight different points. Figure 1A was provided to show how the steady-state pluripotent mitochondrial network appears in relation to the networks shown from three additional pluripotent stem cell lines and one differentiated cell type in the other panels of Fig 1A. Fig S1A was provided to clearly show the progressive effect of a differentiation time course induced by removal of basic fibroblast growth factor over 5 days on the mitochondrial network using a side-by-side comparison with the network in the pluripotent state. We apologize for not explicitly stating that these images were duplicated to highlight these specific, different features.All authors concur with this statement, and we regret not being more explicit in the purposeful use of these duplicated materials in the original publication.

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Protocol Splitting human embryonic stem cells (hESCs) plated on mouse embryonic fibroblasts (MEFs)Usually a confluent hESC plate can be split 1:6 to 1:10, depending on the particular hESC line. The split plate will become confluent again 5-7 days after splitting. DiscussionThis video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. In the last step before plating, when the hESCs are resuspended, they should be of roughly uniform colony size and shape. Carefully pipette the hESC suspension up and down a few times to make the colonies smaller and more uniform, but not so much that single cells or very small colonies are generated.Immunofluorescence staining and microscopy or flow cytometry for hESC pluripotency markers, such as Oct-4 and SSEA-4, are needed to confirm maintenance of hESCs in an undifferentiated state during culture.

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This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.Protocol hESC culture daily maintenance Splitting hESCs from MEFs onto MatrigelUsually a confluent 6-well plate of hESCs on MEFs can be split 1:2 to 1:3 onto Matrigel 6-well plates, with the wells becoming confluent again 4-5 days after splitting. DiscussionThis video demonstrates how to passage hESCs from MEF plates to feeder cell-free Matrigel plates. Note that the colony density on Matrigel is higher than the density of colonies by usual splitting on MEFs. Immunofluorescence staining and microscopy or flow cytometry for hESC pluripotency markers, such as Oct-4 and SSEA-4, are needed to confirm maintenance of hESCs in an undifferentiated state in feeder-free culture conditions. , place in a 50ml falcon tube, and warm to 37°C in a water bath. Once the media is warmed, add bFGF stored at 4°C to a final concentration of 10ng/ml. Put the bFGF stock back at 4°C immediately after use! 2. Remove from the 6-well plates all but~500µl of media. Make sure to swirl the culture dish to suspend debris for removal. Make sure the bFGF in the hESC culture media is thoroughly mixed and add 3ml of fresh media back to each well of a 6-well plate. Put the plate back in the incubator.1. When splitting hESCs onto Matrigel, MEF-conditioned media (CM) is used to maintain pluripotency. MEF-conditioned media is made in advance. On the first day, 1.5×10 7 γ-irradiated MEFs are seeded into a T75 flask. On the second day, MEFs are washed once with room temperature 1×PBS, pH 7.4, and then 15ml of ES media supplemented with 5ng/ml bFGF is added to the flask. (NOTE: MEFs can be plated into smaller or bigger flasks but proportion between cell density and volume of ES media should be constant.) The flask is incubated at 37°C for another 24 hrs. On the third day, the CM is collected and replaced with 15ml of fresh ES media supplemented with 5ng/ml bFGF. The collected CM is stored at 4°C. MEF-conditioned media from one plating of MEFs are harvested over seven consecutive days to generate 15 × 7 = 105ml. After seven days, all CM fractions are combined, sterile filtered, aliquoted and stored at -20°C. CM media prepared as described can be stored and used for 1 month. 2. One day before hESC splitting, put Matrigel aliquots in eppendorf tubes at 4°C to thaw overnight (one aliquot contains 76.2mg of Matrigel, and this is the amount needed for one 6-well plate). On the day of splitting, take one vial of the thawed Matrigel for one 6-well plate, put the vial and 6ml of cold DMEM/F12 media on ice. Transfer the Matrigel into the DMEM/F12 media and mix well. Add 1ml of the solution to each well of a 6-well plate. Swirl the plate to distribute Matrigel on surface and incubate the Matrigel-covered plate at room temperature for at least 1 hr before splitting the hESCs. 3. hESCs on MEFs are washed once with 1×PBS, pH 7.4. Subsequently 1ml of warm collagenase IV ...