A study of methods to achieve somatic cell reprogramming

While the potential of pluripotent cells and their role in the future of regenerative medicine has rapidly evolved since the derivation of human embryonic stem cells in 1998, ethical issues surrounding the derivation and clinical use of pluripotent cells still remain. Somatic cell reprogramming offers an ethically preferred, potentially patient specific method for deriving pluripotent cells, but this technology is based on integration of genes that have been linked to oncogenesis and therefore limit clinical usefulness. This project, started in late 2005, has explored new methods towards achieving somatic cell reprogramming with the specific goal of reprogramming human somatic cells without altering genomic DNA. Through the use of cytoplasm from pluripotent cells, total RNA from pluripotent cells, and specific mRNAs coding for known reprogramming factors, attempts to reprogram somatic cells were made, along with the goal to better understand the process of reprogramming and the associated gene expression changes that catalyse it. To gauge reactivation of the embryonic genome, an Oct4-GFP fibroblast reporter line was successfully established. A protocol for the isolation of membrane encapsulated, nuclear DNA free pluripotent cell cytoplasm, or cytoplasts, was developed. Following fusion of cytoplasts to somatic reporter cells resulted in temporary OCT4 activation, but no pluripotent cells were isolated. Subsequently, an electroporation protocol was developed and optimised to transfect total RNA and total mRNA from pluripotent cells into somatic cells, in place of cytoplasts. This method successfully showed temporary upregulation of key pluripotency genes, but not full reversion to a pluripotent state. In 2006, it was shown that only four factors (OCT4, SOX2, cMYC, and KLF4) are required for somatic cell reprogramming, but the issue of DNA manipulation remained. Synthetically produced mRNA coding for the key reprogramming factors was then made and transfected into human fibroblast cells. It was found that transfected mRNA can successfully upregulate specific genes of interest, including pluripotency factors, in a more controlled and predictable manner than DNA. Although mRNA only causes upregulation for 3-4 days, in some cases lasting changes on endogenous expression of pluripotency genes were detected, including OCT4. This work shows that mRNA transfection can be a useful tool for temporary upregulation of specific gene expression and, with further optimisation, may provide a method for catalysing somatic cell reprogramming without genetic alteration. Additionally, mRNA has potential as an important tool for differentiation, transdifferentiation, and pluripotency studies

Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach

Background: Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS) cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine. Methodology/Principal Findings: In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT) and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES) cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3 beta, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine) and cultured in human embryonic stem cell (ES) medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days. Conclusion/Significance: Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells

Effect of thermal state and thermal comfort on cycling performance in the heat

Purpose: To determine the effect of thermal state and thermal comfort on cycling performance in the heat. Methods: Seven well-trained male triathletes completed 3 performance trials consisting of 60 min cycling at a fixed rating of perceived exertion (14) followed immediately by a 20-km time trial in hot (30°C) and humid (80% relative humidity) conditions. In a randomized order, cyclists either drank ambient-temperature (30°C) fluid ad libitum during exercise (CON), drank ice slurry (-1°C) ad libitum during exercise (ICE), or precooled with iced towels and ice slurry ingestion (15g/kg) before drinking ice slurry ad libitum during exercise (PC+ICE). Power output, rectal temperature, and ratings of thermal comfort were measured. Results: Overall mean power output was possibly higher in ICE (+1.4%1.8% [90% confidence limit]; 0.4 > smallest worthwhile change [SWC]) and likely higher PC+ICE (+2.5%1.9%; 1.5 > SWC) than in CON; however, no substantial differences were shown between PC+ICE and ICE (unclear). Time-trial performance was likely enhanced in ICE compared with CON (+2.4%2.7%; 1.4 > SWC) and PC+ICE (+2.9%3.2%; 1.9 > SWC). Differences in mean rectal temperature during exercise were unclear between trials. Ratings of thermal comfort were likely and very likely lower during exercise in ICE and PC+ICE, respectively, than in CON. Conclusions: While PC+ICE had a stronger effect on mean power output compared with CON than ICE did, the ICE strategy enhanced late-stage time-trial performance the most. Findings suggest that thermal comfort may be as important as thermal state for maximizing performance in the heat

Chemically defined generation of human cardiomyocytes

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification

Measurement of psi (2S) production cross-sections in proton-proton collisions at v s=7 and 13 TeV

The cross-sections of \u3c8(2 S) meson production in proton-proton collisions at s=13TeV are measured with a data sample collected by the LHCb detector corresponding to an integrated luminosity of 275pb-1. The production cross-sections for prompt \u3c8(2 S) mesons and those for \u3c8(2 S) mesons from b-hadron decays (\u3c8(2S)-from-b) are determined as functions of the transverse momentum, pT, and the rapidity, y, of the \u3c8(2 S) meson in the kinematic range 2<20GeV/c and 2.0 < y< 4.5. The production cross-sections integrated over this kinematic region are \u3c3(prompt\u3c8(2S),13TeV)=1.430\ub10.005(stat)\ub10.099(syst)\u3bcb,\u3c3(\u3c8(2S)-from-b,13TeV)=0.426\ub10.002(stat)\ub10.030(syst)\u3bcb.A new measurement of \u3c8(2 S) production cross-sections in pp collisions at s=7TeV is also performed using data collected in 2011, corresponding to an integrated luminosity of 614pb-1. The integrated production cross-sections in the kinematic range 3.5<14GeV/c and 2.0 < y< 4.5 are \u3c3(prompt\u3c8(2S),7TeV)=0.471\ub10.001(stat)\ub10.025(syst)\u3bcb,\u3c3(\u3c8(2S)-from-b,7TeV)=0.126\ub10.001(stat)\ub10.008(syst)\u3bcb.All results show reasonable agreement with theoretical calculations

Measurement of the eta(c)(1S) production cross-section in p p collisions at root s=13TeV

Using a data sample corresponding to an integrated luminosity of 2.0 fb-1, collected by the LHCb experiment, the production of the \u3b7c(1 S) state in proton\u2013proton collisions at a centre-of-mass energy of s=13TeV is studied in the rapidity range 2.0 < y< 4.5 and in the transverse momentum range 6.5<14.0GeV. The cross-section for prompt production of \u3b7c(1 S) mesons relative to that of the J/ \u3c8 meson is measured using the pp\uaf decay mode and is found to be \u3c3\u3b7c(1S)/\u3c3J/\u3c8=1.69\ub10.15\ub10.10\ub10.18. The quoted uncertainties are, in order, statistical, systematic and due to uncertainties on the branching fractions of the J/\u3c8\u2192pp\uaf and \u3b7c\u2192pp\uaf decays. The prompt \u3b7c(1 S) production cross-section is determined to be \u3c3\u3b7c(1S)=1.26\ub10.11\ub10.08\ub10.14\u3bcb, where the last uncertainty includes that on the J/ \u3c8 meson cross-section. The ratio of the branching fractions of b-hadron decays to the \u3b7c(1 S) and J/ \u3c8 states is measured to be Bb\u2192\u3b7cX/Bb\u2192J/\u3c8X=0.48\ub10.03\ub10.03\ub10.05, where the last uncertainty is due to those on the branching fractions of the J/\u3c8\u2192pp\uaf and \u3b7c\u2192pp\uaf decays. The difference between the J/ \u3c8 and \u3b7c(1 S) masses is also determined to be 113.0\ub10.7\ub10.1MeV, which is the most precise single measurement of this quantity to date