A scalable label-free approach to separate human pluripotent cells from differentiated derivatives.

The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)-10(7) cells per min and up to 50% removal of specific cell types per single pass. The level of enrichment and depletion of soft, pluripotent hESC in the respective channels was found to rise with increasing stiffness of the differentiating cells, suggesting CEM can serve as a major discriminator. Our results demonstrate the principle of a scalable, label-free, solution for separation of heterogeneous cell populations deriving from human pluripotent stem cells.

Nitric oxide and thiol reagent modulation of Ca2+-activated K+ (BKCa) channels in myocytes of the guinea-pig taenia caeci.

The modulation of large conductance Ca2+-activated K+ (BKCa) channels by the nitric oxide (NO) donors S-nitroso-L-cysteine (NOCys) and sodium nitroprusside (SNP) and agents which oxidize or reduce reactive thiol groups were compared in excised inside-out membrane patches of the guinea-pig taenia caeci. When the cytosolic side of excised patches was bathed in a physiological salt solution (PSS) containing 130 mM K+ and 15 nM Ca2+, few BKCa channel openings were recorded at potentials negative to 0 mV. However, the current amplitude and open probability (NPo) of these BKCa channels increased with patch depolarization. A plot of ln(NPo) against the membrane potential (V) fitted with a straight line revealed a voltage at half-maximal activation (V0.5) of 9.4 mV and a slope (K) indicating an e-fold increase in NPo with 12.9 mV depolarization. As the cytosolic Ca2+ was raised to 150 nM, V0.5 shifted 11.5 mV in the negative direction, with little change in K (13.1 mV). NOCys (10 microM) and SNP (100 microM) transiently increased NPo 16- and 3. 7-fold, respectively, after a delay of 2-5 min. This increase in NPo was associated with an increase in the number of BKCa channel openings evoked at positive potentials by ramped depolarizations (between -60 and +60 mV). Moreover, this NOCys-induced increase in NPo was still evident in the presence of 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 10 microM), the specific blocker of soluble guanylyl cyclase. The sulfhydryl reducing agents dithiothreitol (DTT; 10 and 100 microM) and reduced glutathione (GSH; 1 mM) also significantly increased NPo (at 0 mV) 7- to 9-fold, as well as increasing the number of BKCa channel openings evoked during ramped depolarizations. Sulfhydryl oxidizing agents thimerosal (10 microM) and 4,4'-dithiodipyridine (4,4DTDP; 10 microM) and the thiol-specific alkylating agent N-ethylmaleimide (NEM; 1 mM) significantly decreased NPo (at 0 mV) to 40-50% of control values after 5-10 min. Ramped depolarizations to +100 mV evoked relatively few BKCa channel openings. The effects of thimerosal on NPo were readily reversed by DTT, while the effects of NOCys were prevented by NEM. It was concluded that both redox modulation and nitrothiosylation of cysteine groups on the cytosolic surface of the alpha subunit of the BKCa channel protein can alter channel gating.

Acute study on the efficacy and safety of an auditory brainstem prosthesis.

Patients with profound binaural sensorineural hearing loss can be treated with cochlear implantation. In recent years, patients who have lost the integrity of the auditory nerve between the spiral ganglion and the cochlear nucleus in the brainstem, and cannot benefit from a cochlear implant, have reported auditory sensations following direct stimulation of the cochlear nucleus with an auditory brainstem prosthesis. To examine the safety and efficacy of such a prosthesis, the cochlear nuclei of guinea-pigs were acutely implanted and stimulated unilaterally with bipolar surface electrodes using the parameters of human implants. The activation of the central auditory pathway by the prosthesis was demonstrated using the 2-deoxyglucose technique. There was broad 2-deoxyglucose labelling in the ipsilateral cochlear nucleus and bilaterally in the inferior colliculi, indicating unusual stimulation of the ipsilateral ascending pathway. Histological examination was performed on all cochlear nuclei. The volumes of cochlear nuclei and the neuron sizes and density in the cochlear nuclei were analysed with three-dimensional reconstruction techniques, and comparisons were made between the stimulated and unstimulated sides. No histological difference, either by direct visual observation or by statistical comparisons, was observed between the stimulated cochlear nuclei and the control sides. These results suggest that in the acute case the auditory brainstem prostheses can safely and effectively activate the auditory pathway in guinea-pigs.

Acute study on the neuronal excitability of the cochlear nuclei of the guinea pig following electrical stimulation.

To help deaf patients who cannot benefit from the cochlear implant due to interruption of the auditory nerve, a central auditory prosthesis has been developed to directly stimulate the cochlear nucleus in the brainstem. The electrode array lies on the surface of the cochlear nucleus and is designed to stimulate at 250 pulses/sec. To examine the safety of this prosthesis, guinea pig cochlear nuclei were stimulated acutely with bipolar surface electrodes using charge-balanced biphasic current pulses at rates of 250, 500 or 1,000 pulses/s and charge intensities of 1.8, 2.8, 3.5 or 7.1 microC/phase cm(-2). The electrically evoked auditory brainstem response (EABR) was used to monitor neuronal excitability of the cochlear nuclei following this acute electrical stimulation. Respiration rate and body temperature were also monitored during the experiment. The amplitudes and latencies of the EABR waves were measured and compared among the before, during and after stimulation periods. The results showed that respiration rate and body temperature remained within normal limits for the duration of the acute stimulation. During and after electrical stimulation, no change was found in the EABR waveform, dynamic ranges and threshold with up to 6 h direct continuous stimulation of the cochlear nucleus. There was no significant change in the amplitudes and latencies of the EABR waves after stimulation. However, a slight temporary reduction in the amplitude of the EABR waves was observed at 30-60 min during the course of acute stimulation using the highest charge density (7.1 microC/phase cm(-2)). This reduction showed a stronger correlation with the stimulus current, charge/phase and charge density than threshold. The present findings suggest that acute bipolar electrical stimulation with surface electrodes at rates up to 1,000 pulses/s and charge density up to 7.1 microC/phase cm(-2) is safe for neuronal excitability of the cochlear nucleus in guinea pig.

Histological and physiological effects of the central auditory prosthesis: surface versus penetrating electrodes.

To rehabilitate profoundly deaf patients who are not suitable for cochlear implants, central auditory prostheses have been implanted. To compare two possible electrode configurations - penetrating and surface ones - electrical stimulation of the cochlear nucleus with both types of arrays was tested on guinea pigs and cats. Electrophysiological, autoradiographic and histological measures were used to study effects of the central auditory prostheses on the auditory pathway. The results showed that a successful electrically evoked auditory brainstem response could be recorded with both surface and penetrating electrodes in cats and guinea pigs. In guinea pigs the penetrating electrodes had advantages over surface arrays in the sense of lower thresholds and wider dynamic ranges. In cats penetrating electrodes showed lower thresholds than surface ones. In cats and guinea pigs stimulated with either surface or penetrating electrodes, evoked 2-deoxyglucose (2-DG) label was found in the auditory pathway from the cochlear nucleus to the inferior colliculus. No non-auditory tissues were found with evoked 2-DG label. Histological results showed that in subdivisions of the guinea pig cochlear nucleus stimulated with penetrating electrodes the neurone density was decreased, and the mean soma area was increased compared with the control side. In the cat, penetrating electrodes were associated only with increased mean soma area in parts of the stimulated cochlear nucleus. These results suggest that the physiological advantages of penetrating electrodes over surface ones were achieved with some trade-off in safety, especially in the guinea pig.

Phase I and pharmacokinetics studies of prochlorperazine 2-h i.v. infusion as a doxorubicin-efflux blocker.

In an earlier phase I study, we reported that the maximal tolerated dose (MTD) of prochlorperazine (PCZ) given as a 15-min i.v. infusion was 75 mg/m2. The highest peak plasma PCZ concentration achieved was 1100 ng/ml. The present study was conducted to determine if PCZ levels high enough to block doxorubicin (DOX) efflux in vitro could be achieved and sustained in vivo by increasing the duration of i.v. infusion from 15 min to 2 h. The treatment schedule consisted of i.v. prehydration with at least 500 ml normal saline (NS) and administration of a fixed standard dose of 60 mg/m2 DOX as an i.v. bolus over 15 min followed by i.v. doses of 75, 105, 135, or 180 mg/m2 PCZ in 250 ml NS over 2 h. The hematologic toxicities attributable to DOX were as expected and independent of the PCZ dose. Toxicities attributable to PCZ were sedation, dryness of mouth, anxiety, akathisia, hypotension, cramps, and confusion. The MTD of PCZ was 180 mg/m2. Large interpatient variation in peak PCZ plasma levels (91-3215 ng/ml) was seen, with the plasma half-life (t1/2 alpha) being approximately 57 min in patients given 135-180 mg/m2 PCZ. The volume of distribution (Vd), total clearance (ClT), and area under the curve (AUC) were 350.1 +/- 183.8 1/m2, 260.7 +/- 142.7 l m2 h-1 and 1539 +/- 922 ng ml h-1, respectively, in patients given 180 mg/m2 PCZ and the respective values for patients receiving 135 mg/m2 were 48.9 +/- 23.76 l/m2, 33.2 +/- 2.62 l m2 h-1, and 4117 +/- 302 ng ml h-1. High PCZ plasma levels (> 600 ng/ml) were sustained in all patients treated with 135 mg/m2 PCZ for up to 24 h. DOX plasma elimination was biphasic at 135 and 180 mg/m2 PCZ, and a > 10-ng/ml DOX plasma level was maintained for 24 h. Partial responses were seen in three of six patients with malignant mesothelioma, in two of ten patients with non-small-cell lung carcinoma, and in the single patient with hepatoma. Our data show that PCZ can be safely given as a 2-h infusion at 135 mg/m2 with clinically manageable toxicities. The antitumor activity of the combination of DOX and PCZ needs to be confirmed in phase II trials.

Prochlorperazine as a doxorubicin-efflux blocker: phase I clinical and pharmacokinetics studies.

Doxorubicin (DOX) efflux in drug-resistant cells is blocked by phenothiazines such as trifluoperazine (TFP) and prochlorperazine (PCZ) in vitro. The present phase I study was conducted in 13 patients with advanced, incurable, nonhematologic tumors to determine whether PCZ plasma levels high enough to block DOX efflux could be achieved in vivo. The treatment schedule consisted of prehydration and i.v. administration of 15, 30, 50, and 75 mg/m2 PCZ followed by a standard dose of 60 mg/m2 DOX. The hematologic toxicities attributable to DOX were as expected and independent of the PCZ dose used. Toxicities attributable to PCZ were sedation, dryness of the mouth, cramps, chills, and restlessness. The maximal tolerated dose (MTD) of PCZ in this schedule was 75 mg/m2. Pharmacokinetic analysis indicated a large interpatient variation in peak plasma PCZ levels that ranged from 95 to 1100 ng/ml. The three plasma half-lives of PCZ were: t1/2 alpha (+/- SE), 20.9 +/- 5.3 min; t1/2 beta, 1.8 +/- 0.3 h; and t1/2 gamma, 21.9 +/- 5.3 h. The volume of distribution (Vd), total clearance (ClT), and area under the curve (AUC) for PCZ were 2254 +/- 886 l/m2, 60.2 +/- 13.5 l m-2 h-1, and 1624 +/- 686 ng ml-1 h, respectively. DOX retention in tumor cells retrieved from patients during the course of therapy indicated the appearance of cells with enhanced DOX retention. The combination of DOX and high-dose i.v. PCZ appeared to be safe, well tolerated, and active in non-small-cell lung carcinoma.

Levels of three hemostatic factors in relation to serum lipids. Monocyte procoagulant activity, tissue plasminogen activator, and type-1 plasminogen activator inhibitor.

To explore the relationship between blood lipid levels and a predisposition to thrombosis, levels of three hemostatic factors were measured in 41 human subjects and correlated with serum lipids. Procoagulant activity associated with peripheral blood monocytes isolated and purified after a 2-hour incubation in whole blood was not significantly related to lipid levels. However, activity in monocytes incubated with 100 ng/ml of bacterial endotoxin was significantly correlated with high density lipoprotein (HDL) cholesterol (r = 0.55, p less than 0.005), while net procoagulant activity (endotoxin-challenged minus basal) was significantly correlated with both HDL cholesterol (r = 0.61, p less than 0.005) and total cholesterol (r = 0.50, p less than 0.01). Plasma levels of the fibrinolytic factor, tissue plasminogen activator, were significantly correlated with total cholesterol (r = 0.41, p less than 0.01), while those of the type-1 plasminogen activator inhibitor were significantly correlated with both total cholesterol (r = 0.46, p less than 0.01) and total triglycerides (r = 0.31, p less than 0.05). The balance between the fibrinolytic factors was not significantly related to serum lipids. These results suggest that the expression of procoagulant activity by peripheral blood monocytes exposed to endotoxin may be enhanced in cases where HDL cholesterol levels are high. In addition, these results suggest that hypertriglyceridemia may be associated with a decreased fibrinolytic capacity due to elevated secretion of plasminogen activator inhibitor.