Synchronous firing patterns of induced pluripotent stem cell-derived cortical neurons depend on the network structure consisting of excitatory and inhibitory neurons.

The balance between glutamate-mediated excitation and GABA-mediated inhibition is critical to cortical functioning. However, the contribution of network structure consisting of the both neurons to cortical functioning has not been elucidated. We aimed to evaluate the relationship between the network structure and functional activity patterns in vitro. We used mouse induced pluripotent stem cells (iPSCs) to construct three types of neuronal populations; excitatory-rich (Exc), inhibitory-rich (Inh), and control (Cont). Then, we analyzed the activity patterns of these neuronal populations using microelectrode arrays (MEAs). Inhibitory synaptic densities differed between the three types of iPSC-derived neuronal populations, and the neurons showed spontaneously synchronized bursting activity with functional maturation for one month. Moreover, different firing patterns were observed between the three populations; Exc demonstrated the highest firing rates, including frequent, long, and dominant bursts. In contrast, Inh demonstrated the lowest firing rates and the least dominant bursts. Synchronized bursts were enhanced by disinhibition via GABAA receptor blockade. The present study, using iPSC-derived neurons and MEAs, for the first time show that synchronized bursting of cortical networks in vitro depends on the network structure consisting of excitatory and inhibitory neurons.

Silicone sheet containing all-trans retinoic acid and hydroquinone for the treatment of epidermal melanosis.

BACKGROUND: Although bleaching treatment using all-trans retinoic acid (RA) and hydroquinone (HQ) improves epidermal melanosis, the application of two medications and the irritant dermatitis induced by RA inconvenience patients. To overcome these problems, we developed a silicone sheet containing RA and HQ. OBJECTIVE: To compare the efficacy of a silicone sheet containing RA and HQ with that of conventional bleaching treatment. METHOD: Silicone sheets containing 1% RA and 5% HQ were applied at night during the bleaching phase of 4 weeks, followed by application of sheets containing 5% HQ during the healing phase of 4 weeks. Hemifacial epidermal melanosis, for which the sheets were applied, was compared with a contralateral face which was treated conventionally using RA and HQ. Twenty-four Japanese women who were enrolled in this study and followed up for more than 6 months were analyzed. RESULTS: RA/HQ sheets improved epidermal melanosis, as did the conventional bleaching method, but irritant dermatitis occurred less in patients treated using silicone sheets. CONCLUSION: RA/HQ sheets, which are easily applied to face skin, can improve epidermal melanosis to the same extent as conventional bleaching.

Long-Term Developmental Process of the Human Cortex Revealed In Vitro by Axon-Targeted Recording Using a Microtunnel-Augmented Microelectrode Array.

OBJECTIVE: We aimed to develop a method for evaluating developmental changes in the synchronized activity of human induced pluripotent stem cell (hiPSC)-derived neurons without extrinsic signals from feeder astrocytes. METHODS: Microelectrode arrays (MEAs) and microtunnels were fabricated with photolithography and soft lithography. hiPSCs were induced to differentiate into cortical neurons, and seeded to conventional and microtunnel MEAs. Spontaneous activity was recorded every ten days, and spiking and bursting activities were elucidated. RESULTS: First, hiPSC-derived neurons were cultured on conventional MEAs. They formed aggregates and subsequently detached from the culture substrate. Hence, no MEAs showed spontaneous synchronized activity beyond 300 days post-induction. Next, we applied a microtunnel structure designed to keep the axons on the array. Synchronized activity was then recorded from all microtunnel MEAs by 450 days post-induction. The proportion of electrodes showing neural activity was greater than that in conventional MEAs. The activity pattern reached a steady state after approximately 330 days, which may be the maturation time of the human neuronal network. CONCLUSION: The use of a microtunnel MEA enables the monitoring of the long-term development of human neuronal networks of cell populations that are relatively natural given their lack of astrocyte feeders. SIGNIFICANCE: We report a more accurate method for culturing cortical neurons differentiated from hiPSCs, validating their use in elucidating cortical development and pathogenic mechanisms in humans.

Cell-cycle-dependent Ca2+ transients in human induced pluripotent stem cells revealed by a simultaneous imaging of cell nuclei and intracellular Ca2+ level.

Cell cycle phase and [Ca2+]i are key determinants of self-renewal and differentiation in pluripotent stem cells. However, little is known about their relationship in human pluripotent stem cells owing to the lack of an effective method. Here, we applied an imaging-based approach for evaluating the relationship between the cell cycle and Ca2+ transients in human induced pluripotent stem (iPS) cells. Ca imaging and DNA staining was simultaneously performed at the same site. Then, individual cells were recognized and the cell cycle phase was estimated from the image of nuclei. We found that 18 ± 4% of human iPS cells exhibited spontaneous Ca2+ transients and their inter-transient interval was 119 ± 19 s. Ca wave events were observed in 64% of the sample and the [Ca2+]i elevation propagated among 47 ± 30 cells with a duration of 57 ± 22 s. With the imaging-based approach, we demonstrated that the ratio of cells exhibiting Ca2+ transients significantly decreased during cell cycle progression, suggesting that the relationship previously described in mouse cells holds true in the human context as well. These results suggest that our method is suitable for evaluating Ca2+ transients, the cell cycle phase, and their relationship with densely cultured cells.

Salvage of infected tissue expanders using a new continuous irrigation method with intermittent aspiration.

When tissue expander sites are infected, it often results in removal of the expander. To salvage the infected expander and achieve full expansion, we devised a new continuous irrigation method with intermittent aspiration. In this method, the continuous irrigation of the tissue expander pocket was performed without removal of the expander. Saline was continuously infused at 50 ml h(-1) via the IVH catheter and intermittent aspiration was done at 10 cm H(2)O negative pressure via the suction drainage tube for 3 min per hour until the infection was under control. We performed this method on two cases of infection of tissue expander sites and salvaged both expanders. After controlling the infection, reconstructions were successfully performed with enough skin expansion. In this method, the expander left in the pocket acts not only in maintaining the expanded pocket but also helps in irrigating the inner surface of the skin pocket. This method can perform effective irrigation with a relatively small amount of saline (1200 ml per day) and salvage the tissue expander.

Blood gas analysis of the jejunum in the supercharge technique: to what degree does circulation improve?

BACKGROUND: The supercharge technique has become widely prevalent in the field of esophageal reconstruction. Despite the logical advantages with this technique, the actual degree of its effect on the blood circulation is not clear. There may be cases in which the supercharge technique is not necessary for survival of the jejunum. To decide whether or not the supercharge technique is indicated, it is crucial to know how effective it is in improving blood flow to the jejunum. METHODS: The effect of the additional vessel anastomosis in the pedicled jejunal transfer was evaluated by blood gas analysis of the venous blood in the mesenteric vein. In 27 patients undergoing pedicled jejunal transfer with additional vessel anastomosis using the internal mammary vessels for reconstruction of the thoracic esophagus, intraoperative blood sampling was performed three times: before anastomosis, after venous anastomosis, and after venous and arterial anastomosis. RESULTS: The venous partial pressure of oxygen showed little increase after the venous anastomosis (mean, 115.7 percent; p = 0.0022). In contrast, venous partial pressure of oxygen increased markedly after the arterial and venous anastomosis in most of the patients (mean, 177.8 percent; p < 0.0001). Similarly, venous partial pressure of carbon dioxide, after both anastomoses, decreased to a lower level than before the additional anastomosis in most patients (mean, 93.1 percent; p = 0.035). CONCLUSION: The authors conclude that the additional anastomosis of both the artery and the vein is recommended if it is possible.