The ROCK Inhibitor Y-27632 Improves Recovery of Human Embryonic Stem Cells after Fluorescence-Activated Cell Sorting with Multiple Cell Surface Markers

Background

Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions.

Methodology/Principal Findings

HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions.

Conclusions/Significance

The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research.     

Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes. Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.     

Reconstruction of firing rate changes across neuronal populations by temporally deconvolved Ca2+ imaging

Methods to record action potential (AP) firing in many individual neurons are essential to unravel the function of complex neuronal circuits in the brain. A promising approach is bolus loading of Ca(2+) indicators combined with multiphoton microscopy. Currently, however, this technique lacks cell-type specificity, has low temporal resolution and cannot resolve complex temporal firing patterns. Here we present simple solutions to these problems. We identified neuron types by colocalizing Ca(2+) signals of a red-fluorescing indicator with genetically encoded markers. We reconstructed firing rate changes from Ca(2+) signals by temporal deconvolution. This technique is efficient, dramatically enhances temporal resolution, facilitates data interpretation and permits analysis of odor-response patterns across thousands of neurons in the zebrafish olfactory bulb. Hence, temporally deconvolved Ca(2+) imaging (TDCa imaging) resolves limitations of current optical recording techniques and is likely to be widely applicable because of its simplicity, robustness and generic principle.     

Analysis of MVP and VPARP promoters indicates a role for chromatin remodeling in the regulation of MVP

Multi-drug-resistant cancer cells frequently express elevated levels of ribonucleoprotein complexes termed vaults. The increased expression of vault proteins and their mRNAs has led to the suggestion that vaults may play a direct role in preventing drug toxicity. To further understand vault component up-regulation, the three proteins that comprise the vault, the major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), and telomerase-associated protein-1 (TEP1), were examined with respect to gene amplification and drug-induced chromatin remodeling. Gene amplification was not responsible for increased vault component levels in multi-drug-resistant cancer cell lines. The TATA-less murine MVP and human VPARP promoters were identified and functionally characterized. There was no significant activation of either the MVP or VPARP promoters in drug-resistant cell lines in comparison to their parental, drug-sensitive counterparts. Treatment of various cell lines with sodium butyrate, an inhibitor of histone deacetylase (HDAC), led to an increase in vault component protein levels. Furthermore, treatment with trichostatin A (TSA), a more specific inhibitor of HDAC, caused an increase in MVP protein, mRNA, and promoter activity. These results suggest that up-regulation of MVP in multi-drug resistance (MDR) may involve chromatin remodeling.     

Class-selective drug detection: fluorescently-labeled calmodulin as the biorecognition element for phenothiazines and tricyclic antidepressants

A small-scale, homogeneous, rapid sensing system for phenothiazines and tricyclic antidepressants (TCAs) has been developed by employing fluorescently labeled mutant calmodulin (CaM) as the recognition element. A calmodulin mutant containing a unique cysteine residue at position 109 on the protein was expressed in Escherichia coli. Following purification, the environment-sensitive, thiol-specific fluorophores N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), and 4-[N-(2-(iodoacetoxy)ethyl)-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester) were coupled to the C109 site of the mutant protein. The response of labeled CaM in the presence of calcium to increasing concentrations of chlorpromazine hydrochloride (CPZ), as well as other phenothiazines and structurally related antipsychotics and antidepressants, was investigated. Fluorescence measurements were performed on benchtop and microtiter plate fluorometers. The responses were characterized as a change in the signal intensity of the labeled protein upon ligand binding, and the stability of the system was monitored over a nine-month period. The assay showed specificity for the phenothiazine and TCA classes of drugs, with limits of detection in the micromolar range. Selectivity studies indicated negligible response of the biosensing system to structurally unrelated compounds. This work represents a proof-of-concept assay for rapid, homogeneous detection of drugs employing binding proteins as the biorecognition element.     

Different responses to temperature in three closely-related sympatric cereal aphids

Cotesia glomerata L. (Hymenoptera: Braconidae) is a parasitoid of early instar larvae of Pieris brassicae L. (Lepidoptera: Pieridae). Late instars of P. brassicae can more often overcome parasitization by hemocytic encapsulation of C. glomerata eggs. Short-term hemocyte responses to parasitization were examined in third and fourth instar larvae of P. brassicae. Total and differential hemocyte counts did not differ between parasitized and unparasitized host larvae. A rapid, but temporary decrease of total hemocyte as well as plasmatocyte numbers was observed immediately after oviposition. Numbers of hemocytes adhering to tissues were shown to be the same in untreated, wounded and parasitized P. brassicae larvae by tracing hemocytes with monoclonal antibodies as markers. The in vitro spreading ability of hemocytes from unparasitized third and fourth instar larvae was lower than that of the last instar's; parasitization, however, had no influence on hemocyte spreading. We therefore suggest that the higher parasitization success of C. glomerata in earlier instars of P. brassicae is mainly due to the low spreading ability of the hemocytes. Abbreviations: ACS – anticoagulant saline; BSA – bovine serum albumin; DABCO – 1,4-diazabicyclo-[2,2,2]-octane; DHC – differential hemocyte count; FITC – fluorescein isothiocyanate; GR – granular cells; LPS – lipopolysaccharide; mAb – monoclonal antibody; OE – oenocytoids; PL – plasmatocytes; PRO – prohemocytes; PS – Pieris saline; PVP – polyvinylpyrrolidone; TBS – tris-buffered saline; THC – total hemocyte count.     

Thermoregulation: What Role for UCPs in Mammals and Birds?

Mammals and birds are endotherms and respond to cold exposure by the means of regulatory thermogenesis, either shivering or non-shivering. In this latter case, waste of cell energy as heat can be achieved by uncoupling of mitochondrial respiration. Uncoupling proteins, which belong to the mitochondrial carrier family, are able to transport protons and thus may assume a thermogenic function. The mammalian UCP1 physiological function is now well understood and gives to the brown adipose tissue the capacity for heat generation. But is it really the case for its more recently discovered isoforms UCP2 and UCP3? Additionally, whereas more and more evidence suggests that non-shivering also exists in birds, is the avian UCP also involved in response to cold exposure? In this review, we consider the latest advances in the field of UCP biology and present putative functions for UCP1 homologues.