Induced pluripotent stem cell-based assays recapture multiple properties of human astrocytes.

The majority of the population of glial cells in the central nervous system consists of astrocytes, and impairment of astrocytes causes various disorders. It is useful to assess the multiple astrocytic properties in order to understand their complex roles in the pathophysiology. Although we can differentiate human astrocytes from induced pluripotent stem cells (iPSCs), it remains unknown how we can analyse and reveal the multiple properties of astrocytes in complexed human disease conditions. For this purpose, we tested astrocytic differentiation protocols from feeder-free iPSCs based on the previous method with some modifications. Then, we set up extra- and intracellular assessments of iPSC-derived astrocytes by testing cytokine release, calcium influx, autophagy induction and migration. The results led us to analytic methods with conditions in which iPSC-derived astrocytes behave as in vivo. Finally, we applied these methods for modelling an astrocyte-related disease, Alexander disease. An analytic system using iPSC-derived astrocytes could be used to recapture complexities in human astrocyte diseases.

Involvement of Histamine H3 Receptor Agonism in Premature Ejaculation Found by Studies in Rats.

Several of the drugs currently available for the treatment of premature ejaculation (PE) (e.g., local anesthetics or antidepressants) are associated with numerous safety concerns and exhibit weak efficacy. To date, no therapeutics for PE have been approved in the United States, highlighting the need to develop novel agents with sufficient efficacy and fewer side effects. In this study, we focused on the histamine H3 receptor (H3R) as a potential target for the treatment of PE and evaluated the effects of imetit (an H3R/H4R agonist), ciproxifan (an H3R antagonist), and JNJ-7777120 (an H4R antagonist) in vivo. Our in vivo electrophysiological experiments revealed that imetit reduced mechanical stimuli-evoked neuronal firing in anesthetized rats. This effect was inhibited by ciproxifan but not by JNJ-7777120. Subsequently, we evaluated the effect of imetit using a copulatory behavior test to assess ejaculation latency (EL) in rats. Imetit prolonged EL, although this effect was inhibited by ciproxifan. These findings indicate that H3R stimulation suppresses mechanical stimuli-evoked neuronal firing in the spinal-penile neurotransmission system, thereby resulting in prolonged EL. To our knowledge, this is the first report to describe the relationship between H3R and PE. Thus, H3R agonists may represent a novel treatment option for PE.

Basic fibroblast growth factor promotes the generation of microtubule-associated protein 2-positive cells from microglia.

We recently demonstrated that microglia as multipotential stem cells give rise to microtubule-associated protein 2 (MAP2)-positive and glial fibrillary acidic protein (GFAP)-positive cells and that microglia-derived MAP2-positive cells possess properties of functional neurons. In this study, we investigated the role of fibroblast growth factor (FGF) signaling in the molecular mechanism underlying the generation of microglia-derived MAP2-positive and GFAP-positive cells. Real-time quantitative PCR analyses demonstrated that mRNA levels of a family of three FGF receptors, Fgfr1-3, were upregulated in microglia treated with 70% fetal bovine serum (FBS). Immunocytochemical analyses demonstrated that basic FGF (bFGF) promoted the generation of microglia-derived MAP2-positive and GFAP-positive cells, and the FGF receptor tyrosine kinase inhibitor SU5402 and the MEK inhibitor PD98059 both inhibited this process. Western blot analyses demonstrated that bFGF increased phosphorylated ERK1/2 levels without altering total ERK1/2 levels. These results suggest that bFGF promotes the generation of microglia-derived MAP2-positive and GFAP-positive cells via FGF receptors and the ERK-MAP kinase pathway.

A role for SOX2 in the generation of microtubule-associated protein 2-positive cells from microglia.

We recently demonstrated that, as a type of multipotential stem cells, microglia give rise to microtubule-associated protein 2 (MAP2)-positive and glial fibrillary acidic protein (GFAP)-positive cells. In this study, we investigated the role of SOX2, a high-mobility group DNA binding domain transcription factor, in the generation of microglia-derived MAP2-positive and GFAP-positive cells. Western blot analysis demonstrated that expression of SOX2 was upregulated by treatment with 70% fetal bovine serum treatment. Immunocytochemical analyses demonstrated that SOX2 expression was evident in the nuclei of microglia-derived MAP2-positive and GFAP-positive cells, whereas it was not present in the nuclei of microglia. These assays also showed that Sox2 siRNA inhibited the generation of MAP2-positive and GFAP-positive cells from microglia. Interestingly, this activity was also inhibited by Smad4 siRNA, which reduces SOX2 expression. These results indicate that SOX2 upregulation is involved in the generation of microglia-derived MAP2-positive and GFAP-positive cells through SMAD4.

A molecular pathway involved in the generation of microtubule-associated protein 2-positive cells from microglia.

We have recently demonstrated that microglia as multipotential stem cells give rise to microtubule-associated protein 2 (MAP2)-positive and glial fibrillary acidic protein (GFAP)-positive cells and that microglia-derived MAP2-positive cells possess properties of functional neurons. In this study, we investigated the molecular pathways involved in the generation of microglia-derived MAP2-positive and GFAP-positive cells. Western blot analyses demonstrated that expression levels of Id2 protein, an inhibitory basic helix-loop-helix transcription factor of the inhibitor of differentiation and DNA binding family, and Smad proteins were upregulated under differentiation conditions. Immunocytochemical analyses demonstrated that the generation of MAP2-positive and GFAP-positive cells from microglia was promoted by bone morphogenetic proteins (BMPs) and was inhibited by noggin which is a BMP antagonist, Smad4 siRNA and Id2 siRNA. These results indicate that activation of BMP signaling through Smad and Id2 proteins is one of the molecular pathways involved in the generation of microglia-derived MAP2-positive and GFAP-positive cells.

Microtubule-associated protein 2-positive cells derived from microglia possess properties of functional neurons.

Microglia are believed to play an important role in the regulation of phagocytosis, neuronal survival, neuronal cell death, and inflammation. Recent studies have demonstrated that microglia are multipotential stem cells that give rise to neurons, astrocytes, and oligodendrocytes. However, the functional properties of neurons derived from microglia are poorly understood. In this study, we investigated the possibility that microglia differentiate into functional neurons. Immunocytochemical study demonstrated that microtubule-associated protein 2 (MAP2)-positive cells were derived from microglia under differentiation conditions. Intracellular Ca(2+) imaging study demonstrated that KCl caused no significant changes in [Ca(2+)](i) in microglia, whereas it caused a remarkable increase in [Ca(2+)](i) in microglia-derived cells. Furthermore, electrophysiological study demonstrated that the spike waveform, firing rate, and tetrodotoxin sensitivity of extracellular action potentials evoked by 4-aminopyridine from microglia-derived MAP2-positive cells were nearly identical to those from cultured cortical neurons. These results suggest that microglia-derived MAP2-positive cells possess properties of functional neurons.

The first private-hospital based proton therapy center in Korea; status of the Proton Therapy Center at Samsung Medical Center.

PURPOSE: The purpose of this report is to describe the proton therapy system at Samsung Medical Center (SMC-PTS) including the proton beam generator, irradiation system, patient positioning system, patient position verification system, respiratory gating system, and operating and safety control system, and review the current status of the SMC-PTS. MATERIALS AND METHODS: The SMC-PTS has a cyclotron (230 MeV) and two treatment rooms: one treatment room is equipped with a multi-purpose nozzle and the other treatment room is equipped with a dedicated pencil beam scanning nozzle. The proton beam generator including the cyclotron and the energy selection system can lower the energy of protons down to 70 MeV from the maximum 230 MeV. RESULTS: The multi-purpose nozzle can deliver both wobbling proton beam and active scanning proton beam, and a multi-leaf collimator has been installed in the downstream of the nozzle. The dedicated scanning nozzle can deliver active scanning proton beam with a helium gas filled pipe minimizing unnecessary interactions with the air in the beam path. The equipment was provided by Sumitomo Heavy Industries Ltd., RayStation from RaySearch Laboratories AB is the selected treatment planning system, and data management will be handled by the MOSAIQ system from Elekta AB. CONCLUSION: The SMC-PTS located in Seoul, Korea, is scheduled to begin treating cancer patients in 2015.

Development of highly functional long-term culture method of liver slice embedded in agarose gel for bioartificial liver.

It is difficult to a produce highly functional bioartificial liver (BAL) using only hepatocytes, because it is believed that liver-specific three-dimensional structure is necessary to maintain high function for BAL. But it is difficult to construct a culture system with liver-specific three-dimensional structure in vitro. To realize a highly functional culture system with liver-specific three-dimensional structure, we developed a culture system using liver slices that keep liver-specific architecture, such as liver lobule and hepatic microvascular system. Liver slices were embedded in agarose gel to maintain them under a moist and three-dimensional environment. We examined the viability and function of liver slices by using various shapes of agarose gel. Liver slices were cultured 1) under stationary condition (control), 2) directly embedded in gel, and 3) embedded in cylindrical gel for good drainage of medium and ventilation of air. The viability and function of the incubated liver slices were evaluated by LDH leakage, histomorphology, and immunohistochemistry. At 10 days, the morphological condition and function of liver slices embedded in cylindrical gel were maintained better than liver slices directly embedded in gel or in the stationary condition. We suggest that high functionality and morphological condition of liver slices could be maintained by embedding in cylindrical gel. In the future, it is possible that this method could be used to develop a highly functional bioartificial liver.