Automated,scalable culture of human embryonic stem cells in feeder‐free conditions

Large‐scale manufacture of human embryonic stem cells (hESCs) is prerequisite to their widespread use in biomedical applications. However, current hESC culture strategies are labor‐intensive and employ highly variable processes, presenting challenges for scaled production and commercial development. Here we demonstrate that passaging of the hESC lines, HUES7, and NOTT1, with trypsin in feeder‐free conditions, is compatible with complete automation on the CompacT SelecT, a commercially available and industrially relevant robotic platform. Pluripotency was successfully retained, as evidenced by consistent proliferation during serial passage, expression of stem cell markers (OCT4, NANOG, TRA1‐81, and SSEA‐4), stable karyotype, and multi‐germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Automation of hESC culture will expedite cell‐use in clinical, scientific, and industrial applications. Biotechnol. Bioeng. 2009;102: 1636–1644. © 2008 Wiley Periodicals, Inc.     

Cooperative nature of gating transitions in K+ channels as seen from dynamic importance sampling calculations

The growing dataset of K⁺ channel x‐ray structures provides an excellent opportunity to begin a detailed molecular understanding of voltage‐dependent gating. These structures, while differing in sequence, represent either a stable open or closed state. However, an understanding of the molecular details of gating will require models for the transitions and experimentally testable predictions for the gating transition. To explore these ideas, we apply dynamic importance sampling to a set of homology models for the molecular conformations of K⁺ channels for four different sets of sequences and eight different states. In our results, we highlight the importance of particular residues upstream from the Pro‐Val‐Pro (PVP) region to the gating transition. This supports growing evidence that the PVP region is important for influencing the flexibility of the S6 helix and thus the opening of the gating domain. The results further suggest how gating on the molecular level depends on intra‐subunit motions to influence the cooperative behavior of all four subunits of the K⁺ channel. We hypothesize that the gating process occurs in steps: first sidechain movement, then inter‐S5‐S6 subunit motions, and lastly the large‐scale domain rearrangements. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

The Protein Kinase C�� Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint

Protein kinase Cδ (PKCδ) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCδ is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCδ-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKCδ-cat caused a pronounced G₂/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G₂/M arrest, PKCδ-cat induced phosphorylation of Cdk1 (Tyr¹⁵), a critical event in the G₂/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCδ-cat-induced G₂/M arrest, suggesting that PKCδ-cat is functioning downstream of ATM/ATR in the G₂/M checkpoint. To better understand the role of PKCδ and PKCδ-cat in the cell cycle response to DNA damage, we exposed wild-type and PKCδ null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G₂/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKCδ null MEFs were resistant to these effects. Expression of PKCδ-green fluorescent protein, but not caspase-resistant or kinase-inactive PKCδ, was able to restore G₂/M checkpoint integrity in PKCδ null MEFs. The function of PKCδ in the DNA damage-induced G₂/M cell cycle checkpoint may be a critical component of its tumor suppressor function.     

Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.     

Osteoarthritis and nutrition. From nutraceuticals to functional foods: a systematic review of the scientific evidence

The scientific and medical community remains skeptical regarding the efficacy of nutrition for osteoarthritis despite their broad acceptation by patients. In this context, this paper systematically reviews human clinical trials evaluating the effects of nutritional compounds on osteoarthritis. We searched the Medline, Embase, and Biosis databases from their inception to September 2005 using the terms random, double-blind method, trial, study, placebo, and osteoarthritis. We selected all peer-reviewed articles reporting the results of randomised human clinical trials (RCTs) in osteoarthritis that investigated the effects of oral interventions based on natural molecules. Studies on glucosamine and chondroitin sulfate were excluded. The quality of the RCTs was assessed with an osteoarthritic-specific standardised set of 12 criteria and a validated instrument. A best-evidence synthesis was used to categorise the scientific evidence behind each nutritional compound as good, moderate, or limited. A summary of the most relevant in vitro and animal studies is used to shed light on the potential mechanisms of action. Inclusion criteria were met by 53 RCTs out of the 2,026 identified studies. Good evidence was found for avocado soybean unsaponifiables. Moderate evidence was found for methylsulfonylmethane and SKI306X, a cocktail of plant extracts. Limited evidence was found for the Chinese plant extract Duhuo Jisheng Wan, cetyl myristoleate, lipids from green-lipped mussels, and plant extracts from Harpagophytum procumbens. Overall, scientific evidence exists for some specific nutritional interventions to provide symptom relief to osteoarthritic patients. It remains to be investigated whether nutritional compounds can have structure-modifying effects.     

Cryptococcal meningitis in the immunocompromised host: intracranial hypertension and other complications

Cryptococcosis as a complication of the immunocompromised host has dramatically increased in frequency since the start of the AIDS epidemic. This trend has heightened awareness of the complications of cryptococcal meningitis; of these, intracranial hypertension is common, severe, and life-threatening, as exemplified by three cases in our institutions presented here in detail. An aggressive approach to management of this complication has not been the standard of care, but neurosurgical interventional studies combined with physiologic observations suggest early intervention may reduce the devastating morbidity and mortality. This revised version was published online in June 2006 with corrections to the Cover Date.     

Infrared body temperature measurement of mice as an early predictor of death in experimental fungal infections

Temperatures of mice were measured using an infrared high performance non-contact thermometer, after the device had been calibrated using implantable microchips containing temperature transponders. Mice were infected with three species of Candida (isolates) and the resultant disseminated infections monitored. Mouse temperatures could be reliably measured using the infrared device and this measurement caused little distress to the mice. We were further able to demonstrate that mice rarely recovered if their body temperature dropped below 33.3 degrees C (sensitivity 68%, specificity 97%). Adoption of a 33.3 degrees C endpoint in fungal sepsis experiments measured by infrared non-contact thermometer would significantly reduce the suffering in the terminal stages of this type of infection model.     

Oxidation of pyocyanin, a cytotoxic product from Pseudomonas aeruginosa, by microperoxidase 11 and hydrogen peroxide

Pyocyanin (1-hydroxy-N-methylphenazine) is a cytotoxic pigment secreted by the bacterial species Pseudomonas aeruginosa, which frequently infects the lungs of immunosuppressed patients as well as those with cystic fibrosis. Pyocyanin toxicity results presumably from the ability of the compound to undergo reduction by NAD(P)H and subsequent generation of superoxide and H2O2 directly in the lungs. We report that in the presence of peroxidase mimics, microperoxidase 11, or hemin, pyocyanin undergoes oxidation by H2O2, as evidenced by loss of the pigment's characteristic absorption spectrum and by EPR detection of a free radical metabolite. The oxidation of pyocyanin is irreversible, suggesting an extensive modification of the pigment's phenazine chromophore. Oxidation of pyocyanin was observed also when exogenous H2O2 was replaced by a H2O2-generating system consisting of NADH and the pigment itself. That the oxidation involves the phenolate group of pyocyanin was verified by the observation that a related pigment, phenazine methosulfate, which is devoid of this group, does not undergo oxidation by microperoxidase 11/H2O2. In contrast to intact pyocyanin, oxidized pyocyanin was less efficient in NADH oxidation and stimulation of interleukin-8 release by human alveolar epithelial A549 cells in vitro, suggesting that oxidation of pyocyanin leads to its inactivation. This study demonstrates that pyocyanin may play a dual role in biological systems, first as an oxidant and ROS generator, and second as a substrate for peroxidases, contributing to H2O2 removal. This latter property may cause pyocyanin degradation and inactivation, which may be of considerable biomedical interest.     

Activation of caspase-9 is required for UV-induced apoptosis of human keratinocytes

UV radiation from the sun activates both the membrane death receptor and the intrinsic or mitochondrial apoptotic signaling pathways in epidermal keratinocytes, triggering apoptosis and affording protection against skin cancer formation. We have investigated the involvement of caspase-9 in the UV death effector pathway in human keratinocytes, since this is the initiating caspase in the mitochondrial pathway required for UV-induced apoptosis in some, but not all, cell types. UV radiation triggered activation of caspase-3, caspase-9, and caspase-8 with similar kinetics, although the rank order of activation was caspase-3 > caspase-9 > caspase-8. Inhibition of caspase-9 with either the peptide inhibitor benzyloxycarbonyl-Leu-Glu(OCH(3))-His-Asp(OCH(3))-fluoromethyl ketone, or expression of a catalytically inactive caspase-9 by retroviral transduction, protected normal keratinocytes from UV-induced apoptosis. HaCaT keratinocytes harboring mutant p53 alleles were also protected from UV-induced apoptosis by the dominant negative caspase-9. The dominant negative caspase-9 blocked UV-induced activation of caspase-3, caspase-9, and caspase-8, and also protected cells from the loss of mitochondrial membrane potential. In contrast, the dominant negative caspase-9 did not protect from anti-Fas-induced apoptosis or caspase activation. These results identify caspase-9 as the critical upstream caspase initiating apoptosis by UV radiation in human keratinocytes, the relevant cell type for this important environmental carcinogen.     

Modification of the fatty acid composition of cultured human fibroblasts.

The fatty acid composition of human skin fibroblasts grown in 10% dialyzed fetal calf serum can be modified considerably by adding supplemental fatty acids to the culture medium. The degree of modification was dependent on the concentration of added fatty acid over the range tested, 2.5 X 10(-5) to 1 X 10(-4) M. At the higher concentration, the extent of the modifications was as those which can be produced in nonhuman or malignant cell lines. Although the greatest changes were produced in the neutral lipid fraction, the cellular phospholipids also exhibited appreciable modifications. The phospholipids isolated from a microsomal fraction prepared from the cell homogenate exhibited similar changes in fatty acyl composition. These findings indicate that the human fibroblast can tolerate considerable variability in fatty acid composition, even in membrane phospholipids. The triglyceride content of the cells increased when they were grown in the presence of added fatty acids, but the phospholipid and cholesterol content remained unchanged. Growth was not affected by either oleic or linoleic acids, but it was reduced up to 50% when palmitic linolenic, or arachidonic acid was added in concentrations of 5 X 10(-5) M or above. Extensive modifications in phospholipid fatty acid composition also were produced in confluent monolayers of these fibroblasts. This suggest that some membrane lipid turnover occurs even when the cultures are not rapidly growing. Fatty acid modifications also were produced in the commercially available IMR-90 strain of human lung fibroblasts, suggesting that the ability to tolerate considerable differences in fatty acid composition is not a special property of the skin fibroblast line that was isolated locally.