幹細胞・細胞分化1 Stem cells and differentiation 1
O1-6-1-1 マウス人工多能性幹細胞の増殖および神経前駆細胞への分化に対するセロトニン受容体刺激の影響 Involvement of 5-hydroxytryptamine receptors on proliferation and differentiation into neural progenitor cells of mouse induced pluripotent stem cells ○石塚俊晶1, 小澤亜也子1, 五嶋葉月1, 渡辺康裕1 ○Toshiaki Ishizuka1, Ayako Ozawa1, Hazuki Goshima1, Yasuhiro Watanabe1 防衛医科大学校薬理学1 Dept Pharmacol, National Defense Medical College, Tokorozawa, Japan1 [Introduction] Our previous study reported that treatment with 5-hydroxytryptamine (5-HT) may affect proliferation and differntiation into neural progenitor cells (NPCs) of mouse induced pluripotent stem (iPS) cells. However, it remains unclear whether the effect of 5-HT is dependent on activation of 5-HT receptors. In the present study, we examined whether pretreatment with selective 5-HT receptor antagonists regulates proliferation or differentiation into NPCs of mouse iPS cells. [Materials and Methods] Mouse iPS cells were cultured under feeder-free conditions in the presence of leukemia inhibitory factor (LIF). The cells were pretreated by WAY100635 (a 5-HT1 receptor antagonist; 0.1 -1 μM), Ketanserin (a 5-HT2 receptor antagonist; 0.1 -1 μM), or GR113808 (a 5-HT4 receptor antagonist; 1 -10 μM) and treated by 5-HT (0.3 μM) for 48 h. Proliferation of mouse iPS cells was examined by MTT assay. Mouse iPS cell differentiation was initiated by embryoid body (EB) formation under LIF-free condition. The EBs were pretreated by WAY100635 (0.1 -1 μM), Ketanserin (0.1 -1 μM), or GR113808 (1 -10 μM) and stimulated with all trans retinoic acid (ATRA; 1 μM) and/or 5-HT (0.03 μM) for 4 days and then transferred to fibronectin-coated dishes. The differentiation potential into NPCs was evaluated by Nestin expression using western blot analysis. [Results] Pretreatment with either WAY100635 or Ketanserin significantly inhibited 5-HT-enhanced proliferation of mouse iPS cells. Although pretreatment with neither WAY100635 nor Ketanserin affected 5-HT-enhanced Nestin expression in the differentiated cells, the pretreatment with GR113808 significantly inhibited 5-HT-enhanced Nestin expression. [Conclusion] These results suggest that stimulation with either 5-HT1 or 5-HT2 receptor enhances the proliferation of mouse iPS cells and then stimulation with 5-HT4 receptor enhances the differentiation into NPCs.	O1-6-1-2 胎生期脊髄におけるケラタン硫酸によるオリゴデンドロサイト発生の制御 Keratan sulfate regulates oligodendrocyte development in the embryonic spinal cord ○橋本弘和1,2, 石野雄吾2, 吉村武1,2, 内村佳子3, 内村健治3, 門松健治3, 池中一裕1,2 ○Hirokazu Hashimoto1,2, Yugo Ishino2, Takeshi Yoshimura1,2, Yoshiko Uchimura3, Kenji Uchimura3, Kenji Kadomatsu3, Kazuhiro Ikenaka1,2 総合研究大学院大学　生命科学研究科　生理科学専攻1, 自然科学研究機構　生理学研究所　分子神経生理部門2, 名古屋大学大学院　医学研究科　分子生物学3 Department of Physiological Sciences, The Graduate University for Advanced Studies, Japan1, Division of Molecular Physiology, National Institute for Physiological Sciences, Japan2, Department of Biochemistry, Nagoya University Graduate School of Medicine, Japan3 In the embryonic spinal cord, several secretory factors, such as Wnt, BMP and Shh, act as morphogen. Wnt and BMP are secreted from the roof plate and Shh are secreted from the floor plate and the notochord. They are involved in the patterning of the spinal cord. Wnts and Shh bind to acidic sugar chains, for example keratan sulfate (KS). We hypothesized that the interaction between morphogens and acidic sugar chains play essential roles in the pattern formation of embryonic spinal cord. In this study, we analyzed involvement of KS.First, we analyzed localization of KS and sulfotransferases that are involved in KS synthesis in the embryonic spinal cord. Highly sulfated KS was expressed in the floor plate and the notochord. This expression pattern colocalized with Shh expression. In the E10.5 spinal cord, KSGal6ST, C6ST1 and GlcNAc6ST1 were broadly expressed. At E12.5, C6ST1 and GlcNAc6ST1 were observed in the ventricular zone (VZ) and differentiated cells, respectively. Next, to estimate roles of KS, we analyzed the KS null mouse spinal cord. This mouse is deficient of GlcNAc6ST1. GlcNAc6ST1 is very important for KS chain elongation. We could not find any change at E10.5. At E12.5, however, the domain structure shifted ventrally in the KS null spinal cord. We also obtained a similar result with IHC. Especially, pMN domain shifted ventrally and the length of p3 domain was decreased. Then we focused on cell types generated from the pMN domain. The pMN domain generates motor neurons and subsequently oligodendrocyte. PDGFRΑ, a marker for oligodendrocyte progenitor cell, was hardly expressed in the E12.5 spinal cord. Moreover, motor neuron production detected by Islet1/2 expression was increased and the VZ became wider. It is possible that the switch from motor neuron production to oligodendrocyte production development is delayed in KS null mouse.Since KS depletion affected oligodendrocyte development, KS may regulate Shh signaling and distribution of Shh.
O1-6-1-3 MIFにより制御され神経幹細胞の生存および維持に貢献する因子の同定 Identification of a novel macrophage migration inhibitory factor (MIF)-regulated factor that promotes the survival and maintenance of neural stem/progenitor cells ○大多茂樹1, 三沢彩1, 岡野栄之2, 河上裕1, 戸田正博3 ○Shigeki Ohta1, Aya Misawa1, Hideyuki Okano2, Yutaka Kawakami1, Masahiro Toda3 慶應義塾大学・医・ 先端研1, 慶應義塾大学・医・生理学2, 慶應義塾大学・医・脳神経外科3 Inst. for Adv. Med. Res., Keio Univ Sch of Medicine, Tokyo1, Dept of Physiology, Keio Univ Sch of Medicine, Tokyo2, Dept of Neurosurgery, Keio Univ Sch of Medicine, Tokyo3 Macrophage migration inhibitory factor (MIF) was identified as a factor that can support the proliferation and/or survival of murine neural stem/progenitor cells (NSPCs) in our previous study. We newly discovered that MIF increases the gene expression of Sox6, but not Sox1 or Sox2 in NSPCs in vitro. During neural development, Sox6 was expressed in the ventricular zone of ganglionic eminence (GE) of mouse brains at embryonic day 14.5 (E14.5), cultured NSPCs from E14.5 GE, and NSPCs and neural progenitor cells in the subventricular zone (SVZ) around the lateral ventricle (LV) of the adult mouse forebrain. Retrovirally-expressed Sox6 in NSPCs increased cell viability and the number of primary and secondary neurospheres, thereby supporting cell survival and/or self-renewal ability. Sox6 increased Hes1 and Bcl-2 expression and the phosphorylation of Akt in NSPCs. MIF was shown to activate Stat3 in NSPCs in our previous study. Constitutively-activated Stat3 up-regulated Sox6 gene expression and ChIP analysis showed that MIF increased Stat3 binding to the Sox6 promoter in NSPCs, indicating that Stat3 is a downstream molecule regulated by MIF which promotes Sox6 gene expression. The ability of MIF, which can increase the number of primary and secondary neurospheres was inhibited by Sox6 gene silencing, suggesting that Sox6 may play a key role as a downstream molecule of MIF to support NSPC stemness.	O1-6-1-4 ゼブラフィッシュの錐体視細胞は錐体タイプ特異的前駆細胞の対称分裂により産生される Functionally distinct cone photoreceptor types are generated by symmetric terminal divisions of dedicated progenitors in zebrafish ○鈴木祥宏1, 武智正樹2, 河村正二2 ○Sachihiro C. Suzuki1, Adam Bleckert1, Philip R. Williams1, Masaki Takechi2, Shoji Kawamura2, Rachel O. Wong1 , 東京大院・新領域・先端生命科学2 Department of Biological Structure, University of Washington1, Dept. of Integrated Biosciences, Graduate School of Frontier Sciences, Univ. of Tokyo, Chiba, Japan2 Color vision requires integration of information from functionally distinct cone photoreceptor types, each of which has a maximum sensitivity to a different wavelength of light. In some vertebrates, there are stereotypic proportions of cone photoreceptor types across the retina. Thus, it is important to understand how the appropriate numbers and proportions of different cone types are attained during development. However, what type of progenitors and what modes of cell division generate each type of cone photoreceptor in vivo are not well understood. To answer these questions, we performed in vivo time-lapse imaging analysis of cone photoreceptor genesis in the zebrafish retina. We discovered a novel mechanism by which zebrafish cone photoreceptors sensitive to ultraviolet (UV), long (L), medium (M) or short (S) wavelengths are produced by symmetric divisions of their own dedicated progenitors. Loss and gain-of-function experiments show that L-opsin expression requires thyroid hormone receptor β2 (trβ2) activity prior to cone differentiation, likely at the dedicated progenitor stage. L-opsin expression can be triggered by trβ2 after cones differentiate but this results in some cones expressing more than one opsin type. Differences in the onset of trβ2 activity may explain why some species have pure cone types and others have cones with mixed opsins. We also propose that symmetric division of the dedicated progenitors is an efficient strategy for rapidly attaining the final numbers and proportions of cone types (L>M, UV>S) in the larval zebrafish retina, without the need to coordinate opsin choice of each progeny across all cell divisions. Based on these findings, we are currently determining whether dedicated progenitors exist in development but not during cone regeneration, to produce distinct cone photoreceptor types.

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