Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

[Background:] Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson’s disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. [Objective:] To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. [Methods:] We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. [Results:] The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. [Conclusion:] These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses

Optineurin with amyotrophic lateral sclerosis-related mutations abrogates inhibition of interferon regulatory factor-3 activation

Optineurin has been shown to be involved in primary open-angle glaucoma. We recently found that optineurin is involved in familial amyotrophic lateral sclerosis (ALS). On the other hand, optineurin has been shown to inhibit transcription factors related to innate immunity such as NF-kappa B and interferon regulatory factor-3 (IRF3). In the present study, the effect of ALS-associated optineurin mutations on IRF3 activation was investigated. Optineurin inhibited IRF3 activation induced by melanoma differentiation-associated gene 5 or Toll-IL-1 receptor domain-containing adaptor-inducing interferon-beta. The inhibition was abrogated by mutations related to ALS but not by a mutation related to glaucoma. Reporter assay indicated that the JAK-STAT signaling pathway was not affected by optineurin. These results show that ALS-related optineurin is involved in the IRF3 activation pathway. Pathogenesis of ALS may be associated with some kind of innate immunity, especially that against virus infection, through IRF3 activation

雌ラットにおいて生殖腺の状態とエストロゲン環境が視床下部オキシトシン遺伝子発現および血清オキシトシンレベルに及ぼす影響

Oxytocin (OT) and its receptor (OTR) play various roles in the central and peripheral regulation of appetite and body weight. Previously, we have shown that the administration of OT markedly decreased appetite and body weight gain in ovariectomized (OVX) obese rats. In addition, recent studies have shown that the endogenous OT system is also affected by endogenous or exogenous estrogen. In this study, we showed that ovariectomy decreased rats' hypothalamic OT/OTR mRNA and serum OT levels, but did not affect their visceral fat OTR mRNA levels. The chronic administration of estradiol (E2) abrogated these ovariectomy-induced changes; i.e., it increased the rats' hypothalamic OT/OTR mRNA and serum OT levels, and may be associated with reductions in food intake and body weight gain. In addition, acute E2 administration increased the rats' hypothalamic OTR mRNA and serum OT levels, but did not affect their hypothalamic OT mRNA levels. Taken together, these results suggest that endogenous OT and/or OTR expression might be positively regulated by E2 and that the suppressive effects of E2 on appetite and body weight gain might be mediated, at least in part, by the OT system. Thus, we consider that OT might be a target hormone to pursue subsequent interventions of menopause for menopause-induced metabolic disorders

Alpha7 nicotinic ACh receptor mediated neuroprotective action by nicotine and GTS-21: An approach by the hippocampal organotypic slice cultures.

Nicotine, main constituent of tabaco, is known as a nicotinic acetylcholine receptor (nAChR) agonist and increases cognitive performance. 3-(2,4-dimethoxybenzylidene)-anabaseine (GTS-21) is derived from the marine worm toxin, anabaseine, and is alpha7-selective nAChR (D7-nAChR) agonist. Both nicotine and GTS-21 were expected as therapeutic agents of Alzheimer’s disease. Several studies showed that nicotine and GTS-21 protected neuron by activating nAChR, especially D7-nAChR. It has been reported that D7-nAChR has been shown to be an essential regulator of inflammation. The purpose of this study is to examine the neuroprotective and anti-inflammatory effects of nicotine and GTS-21 using organotypic hippocampal slice cultures. Kainic acid (KA, 5-50µM) induced concentration- and time-dependent neuronal cell death in the hippocampal organotypic slice cultures. The pretreatment with nicotine and GTS-21 tended to decrease in KA toxicity. In a CA3 area-specific analysis, pretreatment with nicotine resulted in significant inhibition of KA-induced neurotoxicity. The results suggest that nicotine may protect KA-induced neuronal cell death via D7-nAChR. We also examined anti-inflammatory effects of nicotine and GTS-21. Hippocampal slices were pretreated with nicotine or GTS-21, and then treated with lipopolysaccharide (LPS). LPS treatment induced concentration-dependent increases in TNFD and IL-1E gene expressions. LPS-induced TNFD gene expression, but not IL-1E was suppressed by GTS-21 pretreatment. These results suggest that D7-nAChR might be involved in the microglia activation towards a neuroprotective role by suppressing inflammatory cytokine

A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells

Nakagawa, M.et al. A novel efficient feeder-free culture system forthe derivation of human induced pluripotent stem cells.Sci. Rep.4, 3594; DOI:10.1038/srep03594 (2014)

Strukturierung des Unterrichts anhand der Lehrer:innenfragen. Perspektiven der Jugyo Kenkyu

Analog zum Leipziger Ansatz der qualitativen Unterrichtsforschung stellt das Team der Hiroshima Universität sein spezifisches Vorgehen der Lesson Study vor. Auf der Basis der normativen Lesson Study mit Fokus auf die Gruppenbildung im Klassenzimmer zeigt die Analyse ihre Praxisnähe und moniert ein gewisses Defizit der Untersuchungsmaterialien, die aus dem Kontext der qualitativen Unterrichtsforschung bereitgestellt wurden. Die forschungsmethodologische Differenz zeigt sich darin, dass der Fokus auf die Lehrer:innenfrage im qualitativen Zugang Prozesse der Offenheit und Lenkung und in der Jugyo Kenkyu Strukturen der Gruppenbildung in den Blick rückt. (DIPF/Orig.

A novel PCOS rat model and an evaluation of its reproductive, metabolic, and behavioral phenotypes

Background: Although animal models of PCOS have been used in many studies, none of them can reproduce both the reproductive and metabolic phenotypes of PCOS. In addition, behavioral parameters have not been evaluated in PCOS animal models. Purpose: We tried to produce an improved rat model of PCOS, and the reproductive, metabolic, and behavioral phenotypes of the model rats were evaluated. Methods: Female rats were implanted with silicon tubes containing oil-dissolved dihydrotestosterone (Oil-DHT) as a new PCOS model. Their phenotypes were compared with those of conventional PCOS model rats (DHT), into which tubes containing crystalline DHT were implanted, and non-DHT-treated rats (control). Results: Both the Oil-DHT and DHT rats showed greater body weight gain, food intake, and fat depot weight than the control rats. Furthermore, these groups showed fewer estrous stages and increased numbers of cystic follicles. The DHT rats exhibited lower ovarian and uterine weights than the control rats, whereas no such changes were observed in the Oil-DHT rats. The Oil-DHT and DHT rats showed less locomotor activity in the light phase than the control rats. Conclusions: Our proposed PCOS model reproduced both the reproductive and metabolic phenotypes of PCOS and may have potential for PCOS research