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mTOR蛋白複合體之訊息傳遞在小鼠胚胎幹細胞分化出之神經細胞中的角色

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(1)國立臺灣師範大學生命科學系博士論文. mTOR 蛋白複合體之訊息傳遞在小鼠胚 胎幹細胞分化出之神經細胞中的角色 The role of mTOR complexes signaling pathway in neurons differentiated from mouse embryonic stem cells. 研 究 生:莊. 仁. 華. Jen-Hua Chuang. 指導教授:林. 炎. 壽 博士. Yenshou Lin, Ph.D.. 中華民國一百零二年六月. i.

(2) 致謝 終於到了寫致謝的這一刻,這也是我與師大生物系/生命科學系/生命科學所 十三年來密切的關係畫下句點的歷史性的一刻。回想當年爸爸極力推薦我到師大 念書,從剛開始的不適應、不認同自己是一個生物系的學生,一直到今天在這裡 把博士學位完成,變成學弟妹口中的師大之魂,這一切漸漸的變化,令我自己驚 訝,也是這裡充滿人情味的師長、學弟妹、系辦人員讓我有了這些變化。 這些年收過很多本學弟妹畢業的論文,每次我都會想著何時我可以寫致謝,屆 時應該要寫好長一串的名字吧!的確,好多好多我想感謝的人,很想把大家的名 字紛紛列出來,但這並不是一件容易的事情,所以我決定把朋友們的名字謹記在 心而不一一列出。 首先,一定要感謝的是口委老師們,李桂楨老師、謝秀梅老師、廖永豐老師、 黃怡萱老師和林炎壽老師,謝謝您們百忙中還幫我看論文,指導我並讓我順利通 過口試,尤其是林老師,謝謝您讓我可以參加口試;童麗珠老師和林炎壽老師都 是我在這個學校的最大貴人,遇到您們令我覺得自己是何其幸運,大學和碩班時 的我很糊塗,幸好是遇到無敵親切的童老師,才可以安然地在研究所念下去,實 驗上的許多基本概念是童老師教導我建立起來的。進入林老師實驗室的這五年光 陰,他帶領我進入分生的領域,教導我謹慎的實驗設計、科學倫理的概念,和他 亦師亦友的相處方式,讓我即使實驗挫折時也不會很無助,也很感謝他讓我得以 有 paper 和專利。此外,也要感謝黃慧滇老師、林金盾老師、王玉麒老師、王慈 蔚老師也在這幾年間某些重要階段給予我許多啟發和想法以及鼓勵。 從童老師時期一直到現在實驗室的成員,還有一路扶持的最棒的好同學,璇瑩, 以及常當藏鏡人的育嘉,你們都是我在這幾年最好的工作與朝夕相處的好夥伴, 因為有大家互相打氣和幫忙,無論實驗或生活上才可以順順地過。而其他實驗室 一路上資助我,無論在實驗用品、器材、還是實驗失敗時的打氣加油站,還有八 卦聊天的好友們,也是我在這十幾年來可以一直保持精神和衝勁的來源。另外,. ii.

(3) 即使大學畢業後同學們回到自己國家或者已經在各國高中教書,好友們仍然經常 留訊息關心我或者電話聯絡,只要實驗上有問題,就立刻留訊息互相幫忙,到了 師大一定來找我吃飯,這些都讓我好感動,真的很謝謝朋友們,你們讓我覺得自 己很幸福,這幾年一人在台北的生活可以過得像在第二個家一樣地自在。 最最重要的是我要感謝一路上從沒有阻止我念下去的家人,尤其是媽媽,是我 精神的支柱也是我經濟的來源,每次回家都得聽我幾哩呱啦講不停,不時地又要 聽我抱怨身體哪裡痛,哥哥姐姐們則是每次要想辦法幫我解決身體有毛病的疑慮, 安排活動讓我回家玩樂,而他們的孩子們是我回家放鬆娛樂的最大的功臣,即使 這幾年媽媽的注意力已經轉到小朋友們的身上,我滿滿的愛仍然可以感受得到, 謝謝您們的支持,我最愛的家人。 最後,我也要將這本論文獻給我最親愛的爸爸,是您以前一直鼓勵我念書,我 才會有動力想要往前進,謝謝您,即使整個研究所期間已經不能再聽到您鼓勵我 的話語,但我一直謹記您最後的鼓舞,我會一直努力下去,期許自己能對科學有 一點貢獻!. iii.

(4) CONTENTS Title……………………………………………………………………….i Acknowledgement………………………………………………………..ii Chinese abstract..………………………………………………………viii English abstract…………………………………………………………..x. Chapter I. General Introduction………………………………………1 1.Growth and importance of neurons…………………………………….2 2. Morphological change of neurons……………………………………..4 3. mTOR (mammalian target of rapamycin) signaling pathways………..7 4. Embryonic stem cells and neuronal differentiation………………….11 Experimental rationale…………………………………………………..15. Chapter II. General Materials and Methods.………………………..16 I). Materials………………………………………………………….17. II). Methods…………………………………………………………..18 1. Feeder-independent mESCs culture…………………………...18 2. Neurons differentiated from mESCs-EB formation…………...18 3. EB dissociation and neuronal differentiation………………….19 4. Primary cortical neurons cultured from mice………………….20 5. Immunocytochemistry of neuronal cells and neurite density Analysis………………………………………………………….21 6. Cell lysates and immunoblot assay…………………………….22 7. Immunoprecipitation…………………………………………...23 8. Treatment of drugs/inhibitors on neurons……………………...24 iv.

(5) 9. RNAi viral particles preparation……………………………….24 10. Plasmids construction………………………………………….25 III). Statistical analysis………………………………………………..26. Chapter III. An Approach for Differentiating Uniform Glutamatergic Neurons from mESCs………………………………...27 Introduction……………………………………………………………..28 Material and methods…………………………………………………...30 Results…………………………………………………………………..30 Discussion……………………………………………………………….35 Figures…………………………………………………………………..39 Fig. 1. Flow chart of experimental procedures to differentiate mESCs into neurons…………………………………………….40 Fig. 2. Effects of different timing of EBs trypsinization on neuronal differentiation…………………………………………42 Fig. 3. Heterogeneous neuronal differentiation after trypsinization of whole EBs……………………………………………………44 Fig. 4. Uniform neurons differentiated from mESCs…………...46 Fig. 5. Morphological and biochemical evidence showing that uniform neurons had differentiated from mESCs………………48 Fig. 6. Glutamatergic neurons were differentiated from mESCs.50 Fig. 7. Almost uniform neurons differentiated from mESCs show glutamatergic, pre-, and post-synapse markers…………………52. v.

(6) Chapter IV. mTOR Complex I is Essential for Growth and Differentiation of Neurons Derived from mESCs ...............................53 Introduction……………………………………………………………..54 Material and methods…………………………………………………...56 Results…………………………………………………………………..56 Discussion……………………………………………………………….61 Figures…………………………………………………………………..64 Fig. 8. Raptor knockdown in mESCs retards EB formation and causes failure to differentiate into neurons……………………..66 Fig. 9. mTORC1/rapamycin regulates neuronal differentiation and neurite growth in neurons derived from mESCs………………..68 Fig. 10. Rapamycin-induced neurites loss accompanied with caspase-3 activation……………………………………………..70 Fig. 11. Knockdown of raptor in neurons differentiated from normal mESCs impedes morphology of neurites……………….73 Fig. 12. HEK293T cells transfected with pLMG-S6KT389E could be resistant to rapamycin treatment……………………………..76. Chapter V. The Role of RICAP/mTORC2 in Neurons ……….……77 Introduction……………………………………………………………..78 Material and methods…………………………………………………...81 Results…………………………………………………………………..81 Discussion……………………………………………………………….83 Figures…………………………………………………………………..85. vi.

(7) Fig 13. RICAP exhibited as an associated partner of rictor obviously in overexpressed model……………………………...86 Fig. 14. Neurites gradually loss in primary cortex neuronal cells infected with RICAP RNAi…………………………………….88. Chapter VI. General Discussion………………………………………89. Chapter VII. References………………………………………………94. Appendix……………………………………………….......................113. vii.

(8) 中文摘要 神經細胞的生長與分化需要許多因子傳遞訊息及相關蛋白快速且 大量新合成,以因應如發育過程或環境的刺激。目前有一些訊息傳遞 路徑已被報導參與在神經的生長,如 CAMKII 路徑,又如另一與蛋 白轉譯息息相關的一重要因子- mTOR。以細胞腦組織初級培養的實 驗證實 mTOR 的活化有助於長期記憶的形成及神經纖維的突觸可塑 性等。然而對於 mTOR 是否影響由胚胎幹細胞所分化出的神經細胞 之生長等則所知甚少。另外,對於近年來所發現 mTOR 藉由和不同 蛋白而組合成不同之蛋白複合體,其中 mTOR 蛋白複合體 II 對於神 經之生長或型態的作用也是鮮少被探討,因此我們旨在探討 mTOR 蛋白複合體 I 與 mTOR 蛋白複合體 II 對於由胚胎幹細胞所分化出的 神經細胞生長之影響。首先,我們建立小鼠胚胎幹細胞分化成神經的 模式,發現新鮮的胰蛋白 酶在特定的時間內作用,可以有效地將形成 的胚胎體分解成較小的球體,並且得到平均約 87%均一性的神經細胞, 且這些神經細胞屬於可分泌谷氨酸的興奮性神經元。利用這些神經細 胞進而來探討 mTOR 蛋白複合體 I 對其生長之影響。先以藥物處理方 式給予分化後的神經細胞 mTOR 蛋白複合體 I 的抑制劑,0.2μM 或 1μM 雷帕拉霉素(rapamycin),均在處理後第三天明顯導致神經纖 維的斷裂與神經細胞的死亡;在分子層級方面,以病毒包裹 RNAi 感. viii.

(9) 染胚胎幹細胞、以抑制其 mTORC1 複合體 I 中 raptor 基因表達的實 驗中發現雖然這些幹細胞仍可形成胚胎體,但體積明顯地比對照組的 胚胎體小很多,甚至導致分化失敗。至於 mTORC2 對於神經細胞所 扮演角色之探討,我們以胚胎幹細胞分化成之神經細胞以及小鼠大腦 皮質神經元之初級培養細胞二者為模式加以研究。除了研究 mTOR 蛋白複合體 II 中專一蛋白 rictor 之外,之前在 rictor 基因剃除的胚胎 纖維細胞,本實驗室又發現一與 rictor 有關的蛋白,暫時稱之為 RICAP。 以免疫沉澱方式已驗證 HA-rictor 與 FLAG-RICAP 得以結合,目前亦 發現 rictor/RICAP 對神經的生長極具影響。此等研究了解了 mTOR 蛋白複合體 I 及 II 在小鼠胚胎幹細胞所分化出的神經細胞之重要性, 並且於訊息傳遞領域開創了可能之新穎範疇。. 關鍵字: mTOR 蛋白複合體 I 、mTOR 蛋白複合體 II、小鼠胚胎幹細 胞、胚胎體、雷帕拉霉素. ix.

(10) Abstract Neuronal growth and differentiation need many signal cues and de novo protein synthesis to convey information in order to respond to various environmental stimulations. Some signal pathways have been demonstrated to participate in the neuronal growth, such as Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cell division cyclin 42 (Cdc 42) pathway. Previous studies suggested that mTOR, mammalian target of rapamycin, is important in the formation of long-term potential (LTP)/long-term depression (LTD) by using animal model or primary neuronal cells. However, much less is known regarding the role of mTOR and its complexes in the neurons differentiated from mouse embryonic stem cells (mESCs). In addition, the upstream regulators, downstream molecules, and roles of mTORC2, a newly identified mTOR complex, are also largely unknown. Hence, we aim to investigate the roles of mTORC1 and mTORC2 in the progression of neuronal growth/morphological change by using neurons differentiated from mESCs. First of all, we established a cellular model in which glutamatergic neurons can be uniformly differentiated from mESCs. We found that applying fresh trypsin/EDTA solution to dissociate embryonic bodies (EBs) in critical timing determines that >87% of cells differentiated into glutamatergic neurons. By employing these neurons, we found that neurites loss as well as soma shrinkage after 0.2μM or 1μ M rapamycin treatment for 48 to 72 hr. Likewise, the EBs formation from mESCs infected with raptor shRNAs showed a smaller size, even fail to differentiate into neurons. Interestingly, phosphorylation of ribosomal x.

(11) protein S6 kinase (S6K), but not 4E-binding protein 1(4E-BP1), was decreased in rapamycin- or shRNA- treated neurons. On the other hand, a novel rictor associated protein, named RICAP, is recently revealed in our laboratory through immunoprecipitation (IP) and mass spectrometry analysis. FLAG-RICAP and HA-rictor were demonstrated to be able to associate with each other by using IP. The regulation/ function/ morphology of this complex remains further investigation. Taken together, this study provides a new insight to reveal Mtorc1 dependent mechanism which is involved in neuronal growth. The observation of a difference between S6K and 4E-BP1 in neurons suggests that additional regulation might be involved. Equally important, a groundbreaking research regarding Mtorc2 and its novel partner in neuroscience might shed a light on signal transduction as well.. Key words: mTORC1, mTORC2, mouse embryonic stem cells, embryonic bodies, rapamycin. xi.

(12) Chapter I. General Introduction. 1.

(13) Relying on nervous system, animals can monitor and respond to external environment, even imitate through learning etc. Physiologically, individual organism depends on nervous system to maintain the homeostasis of its intra environment. It also conducts to sense the extra environmental change and integrate the information to deliver appropriate output. Depending on nervous system, organisms could have the abilities to survive. In general, nervous system provides three important function, sensory output, integration, and motor output. Nervous system can be roughly divided to two parts, central nervous system (CNS) and peripheral nervous system (PNS). CNS consists of the brain and the spinal cord. The PNS is the part of the nervous system consisting of the nerves and ganglia outside of the brain and spinal cord. The CNS is the major integrated center that can convey the identified information to muscle or glands in order to respond to various stimuli.. 1. GROWTH AND IMPORTANCE OF NEURONS. Structure and function of neurons No matter CNS or PNS, the basic components are signal conducting cells, neurons, and supporting cells, glia (Vernadakis, 1996). A neuron contains one cell body with the nucleus, most organelles, and numerous processes to manage signal transduction. Dendrites are short but highly branched neurites by which they can increase the contacting area with other cells to receive more information. In contrast to dendrites, axons are longer but fewer by which they deliver information from cell bodies to 2.

(14) terminals of neurites. The special connecting structure on the tips of axons was denoted as synaptic terminals. In the synapse cleft, neurons can convey their signals by various chemical molecules, named neurotransmitters, which may trigger ionic currents to modify membrane potential (Debanne et al., 2011).. Synaptic polarity and plasticity During the nervous system maturation, many cues direct neural fates and even crosstalk to modulate neural dynamic stabilization. These include extracellular signals from other cell types or matrix (Lin and Holt, 2007). Axons were guided to their targets by cues from their tip motile structure, growth cones. Underlying repulsive and attractive guidance such as attractive factor or brain-derived neurotrophic factor (BDNF), cytoskeletons in growth cones are capable to be built up and broke down asymmetrically to steer the axonal growth orientation. The process also needs coordination with many organelles such as polysomes, Golgi and smooth endoplasmic reticulum (ER) to accumulate in growth cones (Pierce et al., 2001). In addition, factors related to translation such as 4E-BP1 also present abundantly around this localization (Doyle and Kiebler, 2011). Interestingly, it was found that mRNA can be transported to growth cones far away from cell bodies by RNA binding proteins (RNPs) and released to polysomes so as to exert local translation (Shan et al., 2003). Thereafter, the synaptic polarity would be constructed gradually. Similarly, the post-synaptic dendritic spines at the other end of. 3.

(15) neurons also rely on local protein synthesis to modulate synaptic efficacy (Lin and Holt, 2007).. 2. MORPHOLOGICAL CHANGE OF NEURONS. In addition to growth, neurons progress morphological change in coordinated with their growth so that physiological function can be executed. This is particularly relevant to the development of neurons.. Morphological change of neurons in vitro During the stages of neuronal development, it requires multiple tasks to regulate neural precursor cells switching to post-mitotic cells and even highly polarized neurons in vitro (Cid-Arregui et al., 1995). The processes of generating neuronal specification in different brain areas are complicated and not easy to be verified and illustrated. To date, the ideal tool suggested to respectively monitor the development process and investigate influent factors of neural cells is the neural cells cultivated in vitro. Taken cultured embryonic hippocampal cells as an example, the development can be divided into five stages, as shown in follows (Dotti et al., 1988; Arimura and Kaibuchi, 2007) : Stage 1. The fast, veil-like structure of lamellipodia is developed and extended around the entire circumference of the cells after dissociation for about 4 h. Stage 2. The lamellipodia was loss and begun to transform into definitive, resemble and short processes, which also defined as “minor processes”. 4.

(16) Minor processes are cylindrical in cross section, not flattened. They may increase or decrease in length but are quite stable to persist for several days. Stage 3. At about 24 h after plating, one of the minor processes develops into an axon, while it shows no difference in the initial rate of elongation compared to other minor processes. The other minor processes keep sprouting out briefly and retracting dynamically. Rare cases exhibit that two or more minor processes arising from one cell. Stage 4. At the following 27 to 36 h, axons grew rapidly with prominent unique growth cones. On the other hand, the significant dendritic growth would be observed just after the axonal extension in a lower rate. In contrast to axons, several dendrites can grow at the same time without contacting with surrounding axons. Stage 5. Afterwards, dendrites began to produce the connection to receive more information from afferent innervations though spines formation. Therefore, these dendrites tend to be more mature, leading the neuronal net works stabilization and complicated.. 5.

(17) Mechanisms involved in these stages Neuritogenesis and axonal guidance are key cellular processes necessary for proper development of the nervous system. As abovementioned, survive and morphological change of neurons are essential for neuritogenesis in situ and polarity formation in the early stages. Various and complicated intrinsic mechanisms in a neuron coordinate to specify dynamic plasticity. Several molecules are engaged in these signaling cascades. For example, two main driving forces during stage 2, phosphatase-Rho GAP and Rho GTPase-guanine nucleotide exchange factor (GEF)-phosphatidylinositide 3-kinases (PI3K), are cooperated to conduct membrane elimination and recruitment. In addition, axon formation and maturation require two separated pathways in which one involved partitioning defect complex 3 (PAR3), protein kinase C (PKC), and Cdc42 and the other involved collapsing response mediator protein 2 (CRMP2) and microtubulin heterodimer (Andersen and Bi, 2000; Bradke and Dotti, 2000; Inagaki et al., 2001; Hogan et al., 2004; Nishimura et al., 2005). Furthermore, CaMKII is also involved in the stages of neuronal growth. Cytoplasmic polyadeneylation element-binding protein (CPEB) , as a translational regulator of poly A tail, can regulate the translation of Ca2+/ CaMKII in dendrites (Wu et al., 1998; Si et al., 2010). Attraction-induced CaMKII activity was balanced by repulsion-stimulated calcineurin (CaN)-phosphotase-1 by which the Ca2+releasing level was switched from elevated to moderate (Atkins et al., 2004). Neurotrophins, such as netrin-1and BDNF, control the influx of Ca2+ to mediate the abovementioned mechanisms. CaMKII can further 6.

(18) activate Src to phosphorylate the tyrosine on zipcode-binding protein 1 (ZBP1) which will lead β-actin mRNA to be carried to synapse for eventual translation rather than direct transport of proteins. CaMKII is essential not only as the synaptic tag to capture mRNA or plasticity-related protein from cell bodies in early developmental stages, but also in yielding synapse remodeling in long-lasting synaptic changes immediately after training for 4-8h, particularly in protein synthesis dependent LTP and long-term memory (LTM) (Bramham and Wells, 2007; Frey and Frey, 2008). Several molecules related to protein synthesis in growth cones and synaptic areas will eventually integrate to mediate the growth cone polarity, spine morphology, and dendritic plasticity (Doyle and Kiebler, 2011).. 3. MTOR (MAMMALIAN TARGET OF RAPAMYCIN) SIGNALING PATHWAYS. In order to increase the complexity of neuronal connection, the processes of neurites arborization and spine formation were constantly occurred during growth/development. This requires various signal pathways to interact. For example, members of the Rho family of proteins, Rho A or Cdc 42, direct actin cytoskeleton development, while Ras-Raf-MAPK pathway involves in accepting extracellular signals into nucleus to modulate dendritic filopodia formation (Wu et al., 2001; Luo, 2002; Alonso et al., 2004). Recently, it was found that mTOR pathway is. 7.

(19) also involved in many aspects to regulate the morphological and functional stability of neurons (Garelick and Kennedy, 2011).. mTOR complexes and signaling mTOR, a serine-threonine protein kinase with multiple domains, belongs to the phosphatidylinositol 3-kinase-related kinases (PIKKs) family (Bosotti et al., 2000). It is responsible for merging extracellular information to govern many cellular processes such as growth, survival, and metabolism (Wullschleger et al., 2006). In mammalian cells, mTOR forms different complexes by associating with different proteins, named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The components of mTORC1 are mTOR, mLST8 and raptor, while mTORC2 is comprised of mTOR, mLST8, mSin1 and rictor (Sarbassov et al., 2006). Two complexes display distinct functions. One of the characterized properties is that mTORC1 is rapamycin-sensitive whereas mTORC2 is rapamycin-insensitive (Jacinto et al., 2004). Rapamycin, produced from Streptomyces hygroscopicus, can bind with FKBP12 to target on mTOR kinase and to selectively inhibit mTORC1 activity (Hara et al., 2002; Kim et al., 2002; Hay and Sonenberg, 2004; Sarbassov et al., 2004). Thus, rapamycin has been used as a strategic approach to elucidate the involvement of mTORC1. The current model/hypothesis involved in mTORC1 pathway is depicted as follows (Wang and Proud, 2011). By exogenous growth factors or nutrient binds on their specific receptors, the receptors trigger a series of signal cascades such as insulin receptor, insulin receptor 8.

(20) substrate 1, and PI3K by mediating though protein/protein interaction (Hay and Sonenberg, 2004).Phosphatidylinositol-3,4,5-triphosphate (PIP3) generated by activated PI3K will recruit both PDK1 and Akt onto membrane where PDK1 will phosphorylate Akt on Thr 308. mTORC2 was demonstrated to be able to phosphorylate substrate Akt on Ser 473 site (Sarbassov et al., 2005). Normally, tuberous sclerosis protein 1 (TSC1) and tuberous sclerosis protein 2 (TSC2) form a heterodimeric complex to suppress mTORC1 activity. TSC2 acts as a GTPase-activating protein (GAP) to enhance GTPase activity which will convert the Rheb into GDP-bound form, an inactive state (Vander Haar et al., 2007). Upon growth factor/nutrient activates the upstream molecules, the TSC1/2 complex will dissociate due to the phosphorylation of TSC2 by activated Akt. This will result in the release of inhibition by which no more GAP activity toward Rheb. This will lead Rheb-GTP to activate mTORC1 (Long et al., 2005). The downstream of mTORC1 action is well characterized on its ability to regulate translation initiation. It employed phosphorylating S6 kinase (S6K) and eukaryotic initiation factor 4E-binding proteins (4E-BPs) to control translation and cell cycle (Fingar and Blenis, 2004; Ekim et al., 2011; Thoreen et al., 2012). Two isoforms of S6K, S6K1 and S6K2, are both present in mammalian cells. S6K1 has two isoforms, cytosolic p70 and nuclear p85, while p54 and p56, the two isoforms of S6K2, are in nucleus (Lagasse and Clerc, 1988; Jacinto and Lorberg, 2008).Both of them are belonging to AGC (PKA, PKG and PKC) kinase family, shared with the similar 9.

(21) structure of a small N-terminal lobe and a larger C-terminal lobe to co-ordinate the binding of ATP. Kinase domains of them are 84% identity but quite differ in N-and C-terminus (Jacinto and Lorberg, 2008). S6K1 has been well known as the target of mTORC1, whereas related researches on S6K2 are limited. N-terminus of S6K1 executes as receiving active information to suppress the autoinhibitory C-terminus and its TOR signaling (TOS) motif can carry S6K to be closed with Raptor, one component of mTORC1 so that the active sites of Thr 389/Thr 229 acquire the opportunities to be phosphorylated by mTOR/PDK1 (Pullen and Thomas, 1997; Magnuson et al., 2012). Recent studies utilized S6K1-/- mice to study the importance of mTORC1 in regulating embryogenesis, which resulted in smaller size than that of wild type, but not cell numbers. Nevertheless, the size of S6K2-/-mice is almost similar to that of wild type (Shima et al., 1998; Pende et al., 2000; Pende et al., 2004; Russell et al., 2011; Magnuson et al., 2012). Phosphorylation of S6K1 on Thr389 directly promote activate ribosomal protein S6 to phosphorylate eIF4E to promote translation initiation as well. In addition, mORC1 regulates elongation phase via phosphorylated eEF2 (Wang et al., 2001). The other mTORC1 target, 4E-BP, also has three isoforms and contains one TOS motif in the N terminus to modulate the binding of 4E-BP to raptor for multi-sites phosphorylation. Insulin or serum elevates the phosphorylation of 4E-BP1 on Thr37/46 and thus promotes the activation on Thr70/Ser65 (Schalm et al., 2003; Eguchi et al., 2006). Phosphorylated 4E-BP1 would release itself from eIF4E by which eIF4E 10.

(22) is capable to associate with eIF4G and to attract ribosomes loading onto mRNA, initiating the so called cap-dependent translation (Ruvinsky and Meyuhas, 2006) Similarly, 4E-BP1 also functions in controlling cell size without affecting the cell number (Murakami et al., 2004).. mTOR pathway and neurons mTOR signaling pathway revealed in most cellular types seems to be also observed in neurons. 4E-BP and S6K involved in mTOR-dependent protein translation were found to present in dendrites close to synaptic sites (Richter and Klann, 2009). Rapamycin, was able to prevent the establishment of L-LTP which requires synapse protein-dependent enhancement (Hoeffer and Klann, 2010). In addition, mTORC1 signaling also implies key information for hypothalamus to control food intake. S6K1 knockout mice are resistant to high fat diet induced obesity (Um et al., 2004; Cota et al., 2006). Furthermore, Akt had been proved that its deficiency caused significant mental disorder, due to the terminated dopaminergic signaling (Beaulieu et al., 2009). A recent report further confirmed this mTORC2-related mechanism on brain by using rictor knockout mice (Siuta et al., 2010). However, more detail molecular mechanisms of mTOR complexes involved in those situations still remain unclear.. 4. EMBRYONIC STEM CELLS AND NEURONAL DIFFERENTIATION. 11.

(23) The inner cell mass of blastocysts contains embryonic stem cells which are pluripotent to give rise to three germ layers followed by developed into multiple cellular types. Hence, ex vivo cultured embryonic stem cells (ESCs) can recapitulate the developmental stages and develop into plenty organisms by several unique and specific factors (Martin, 1981). To sustain the pluripotency and stemness of ESCs, three major transcriptional factors, Oct4 (Octamer binding transcription factor-4)/Nanog (Nanog homeobox)/Sox2 (Sex determining region Y box-2) are essential in preventing ESCs differentiation via binding with several specific factors and regulating themselves or each other in terms of autoregulation or negative feedback (Nichols et al., 1998; Chan et al., 2011). In the case of Oct4, as one important marker of stemness, it was reported to interplay with Cdk1 to repress cdx2 expression, a marker of trophectoderm (Strumpf et al., 2005). Around 352 genes were speculated to be activated or repressed by the three factors, such as chromatin remodeling relative genes, self-renew associated protein-STATs and so on (Boyer et al., 2005). Besides, it is also necessary to supply some specific factors in culture medium to maintain ESCs from differentiation, depends on the source of ESCs. LIF (Leukemia inhibitory factor), a member of IL-6 family of lymphokines, is believed to be secreted from the feeder layer of embryonic fibroblasts (Rathjen et al., 1990). Surfaces of mESCs contain two receptors, LIFR β and gp 130. After ligated, phosphorylated tyrosine residues of the heterodimer of the former two receptors can attract downstream STATs and SHP2 to lead SH2 domains activated, which 12.

(24) evoke transcription progression and further regulate cell cycle (Niwa et al., 1998). Albeit the detail mechanisms involved in STATs-mediating cell cycle are still yet clear, it is certain that STATs direct the actions of D-type cyclins and inhibit p27 (Sun et al., 1999; Burdon et al., 2002). Thereby, G1 phase of ESCs is short as only 1.5 hrs and RB-independent so that it is not arrested in ESCs, but differentiated cells require RB as G1 checkpoint regulator. This may elucidate how ESCs maintain their character of self-renewal but not being differentiated (Savatier et al., 1994; Savatier et al., 1996; Robanus-Maandag et al., 1998; Harbour and Dean, 2000). To date, many studies have ruled out various conditions and denoted several developmental stages in differentiating mESCs in vitro. For example, neurosensory progenitors need the interaction of fibroblast growth factors (FGF), sonic hedgehog and bone morphogenetic proteins (BMPs) with facilitated Neurogenin-1 to pattern the otic placode, seemly in propagated mESCs which required retinoic acid and BMP4 for differentiating into sensory-like neurons in vitro (Ohyama et al., 2007; Fritzsch et al., 2010). In the former issues, during the period of differentiation from cultured mESCs, endoderm appeared to generate floating embryonic bodies in bacterial petri dishes at first, and then ectoderm was induced, while only the final developmental stage of mesoderm occurrence could secrete signals to supply neural maturation (Du et al., 2011). Complicated genes modulation and expression are included in neural cells deriving from mESCs (Abranches et al., 2009). Although multiple methods have established to differentiate mESCs into 13.

(25) neuronal cells, only few investigations focus on exploring the critical molecular signaling involved in the developmental process, the formation of EBs and the differentiation of neuronal cells.. 14.

(26) EXPERIMENTAL RATIONALE. Currently, most of studies utilize primary neuronal cells to investigate mTOR related experiments. In order to further study the role of mTOR complexes on neuronal growth and conveniently performed molecule manipulation, additional cellular model in neuroscience might need to be developed. Here, we utilized mESCs to investigate effects of mTOR complexes, especially mTORC1, on the process of neuronal differentiation and followed neurite outgrowth. On the other hand, recent studies indicated that mTORC2 could be stimulated by insulin-PI3K pathway to associate with ribosome to regulate the post-translation of phosphorylating Akt on Ser 473 (Oh et al., 2010; Zinzalla et al., 2011). This provides a new insight to understand mTORC2 in more detail. Because much less is known regarding regulation and function of mTORC2 in contrast to mTORC1 studies, the detail mechanism of mTORC2 function including its role in neurons becomes eagerly warranted. We developed a homogenous population of neuronal cells differentiated from mESCs. By adopting these cells along with primary neuronal culture, we aim to elucidate the detail molecular mechanisms in the regard of mTORC1 in growth/morphology of neurons/neurites differentiated from mESCs. Equally important, we also aim to explore the possible novel partner and function of mTORC2 in neurons.. 15.

(27) Chapter II. General Materials and Methods. 16.

(28) I). Materials. The following materials and reagents were purchased from various companies as indicated. Fetal bovine serum (FBS; Thermo Scientific/Hyclone, Logan, UT, USA); L-glutamine, non-essential amino acids, sodium pyruvate, N2 medium, neural-basal-B27 medium, Neurobasal A medium (Invitrogen/Gibco, Grand Island, NY, USA); Leukemia inhibitory factor (LIF; Millipore ESGRO, Billerica, MA, USA); Bacterial dishes (Greiner, Kremsmünster, Austria); Nylon cell strainer (BD Falcon, Franklin Lakes, NJ, USA); Laminin (Roche, Mannheim, Germany); Fluoromount G (Southern Biotech, Birmingham, AL, USA); Monoclonal anti-type III beta tubulin antibodies (Abcam, Cambridge, UK); Monoclonal anti-GFAP antibodies (Millipore/Chemicon, Billerica, MA, USA); AMPA receptor 1 (AMPAR 1) (Abcam, Cambridge, UK), PSD-95, Oct4 (BD Falcon, Franklin Lakes, NJ, USA) Polyclonal anti-vGLUT1 antibodies (SYSY, Gottingen, Germany); Alexa 488-conjugated anti-mouse and anti-rabbit antibodies (Invitrogen); Rhodamine-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA, USA); Horseradish peroxide (HP)-conjugated secondary antibody (Thermo Scientific/Pierce, Rockford, IL, USA); Monoclonal antibodies, anti-phospho p70 S6 Kinase (pT389), anti-phospho Akt1 (pS473), anti-phospho Akt1 (pT308) anti-pan Akt1, and anti-4E-BP1 (Epitomics, Burlingame, CA, USA); Polyclonal anti-phospho 4E-BP1 (pT37/46) and anti-cleaved caspase-3 (Asp175) antibodies (Cell Signaling Technology, Danvers, MA, USA); Polyclonal 17.

(29) anti-p70 S6 kinase and anti-pro caspase-3 antibodies (Santa Cruz, CA, USA). All other reagents/drugs were purchased from Sigma (St. Louis, MO, USA).. II). Methods. 1. Feeder-independent mESCs culture. The mouse embryonic stem (mES) cell line (ES-E14TG2a) which was derived from ATCC CRL-1821 was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Cells were cultivated in ES medium containing GMEM (Sigma) supplemented with 0.05 mM β-mercaptoethanol (Sigma), 1 mM sodium pyruvate, 2 mM L-glutamine, 1x non-essential amino acids, 10% FBS and 500~1000 U/ml LIF. The culture plates were coated with 0.1% gelatin (Sigma) for at least 30 min before use. When cell growth reached 90%~95% confluency, cells were split using trypsin/EDTA (Sigma) and continuously cultivated in a 37°C incubator with a 5% CO2 atmosphere for mESCs maintenance.. 2. Neurons differentiated from mESCs-EB formation. As mESCs grew to 85%~90% confluency, they were trypsinized and collected by centrifugation at 1000 rpm for 5 min. The pellets were resuspended in embryonic bodies (EB) medium which contained ES medium but no LIF. Cells at 1 x 106 were cultivated in 10-cm bacterial 18.

(30) dishes. mESCs gradually proliferated and aggregated to form spherical shapes called EBs. After 2 days of cultivation in bacterial dishes, EBs were collected by passing cells though a 40-μm nylon cell strainer (BD Falcon). Collected cells were placed in fresh EB medium and cultivated in a new bacterial dish. According to this procedure, the EB medium was changed every 2 days.. 3. EB dissociation and neuronal differentiation. To undergo neuronal differentiation, the day mESCs were cultivated in EB medium in bacteria dishes was counted as day 0. On day 4, a final concentration of 5 µM all-trans retinoic acid (Sigma) was directly added to the EB medium. This medium was replaced with fresh medium on day 6. Two days later (i.e., day 8), the EBs were trypsinized and placed on poly-DL-ornithine/laminin-coated plates. Culture plates coated with poly-DL-ornithine/laminin were prepared by immersion in 10 μg/ml poly-DL-ornithine (Sigma) in a 37°C incubator overnight followed by three washes with sterile distilled water. Laminin 5 μg/ml was then added in a 37°C incubator for at least another 2 h before use. When using poly-DL-ornithine, the stock was prepared as 10 μg/μl in borate buffer (150 mM, pH 8.3, stored at 4°C; Sigma) while the working concentration was 0.1 μg/μl diluted in sterile water. To dissociate EBs, the trypsinization buffer was freshly prepared from 0.05% powdered trypsin and 0.02% EDTA (Sigma) dissolved in a phosphate-buffered saline (PBS) solution. When cells were processed, EBs were dissociated and seeded 19.

(31) onto poly-DL-ornithine/laminin-coated plates, and this moment was designated “time after plating”. First, EBs were collected on a cell strainer and washed once with PBS. After washing, residual PBS was removed to avoid diluting the trypsinization efficiency. Freshly made 0.05% trypsin and 0.02% EDTA were added to fully cover the EBs, and the mixture was incubated at 37°C for various times as indicated in the figure. At the indicated times, the EBs were immediately resuspended in EB medium to neutralize trypsinization and further transferred to a 15-ml Falcon tube (BD Falcon). The mixture was gently pipetted up and down to resuspend the EBs which now contained single cells as well as smaller EB aggregations. After centrifugation for 5 min at 1000 rpm at room temperature and aspirating out the supernatant, the pellet in the tube was resuspended in N2 medium and filtered though a 40-μm cell strainer. An optimal cell density of 1.35 x 105/cm2 was determined and seeded onto poly-DL-ornithine/laminin-coated plates. The medium was changed to fresh N2 medium at 2 and 24 h after plating. At 48 h after plating, one-half of the N2 medium was preserved, and the other half was changed to neural-basal-B27 medium. For the long-term culture of neuronal cells, it was necessary to change the neuro-basal-B27 medium every other day. Instead of withdrawing the entire culture medium, the medium was partially replaced with fresh neuro-basal-B27 solution when changing medium thereafter.. 4. Primary cortical neurons cultured from mice. 20.

(32) In the experiments using primary neurons as a positive control, cerebral cortexes were dissociated from BALB/c mice during postnatal day 2 (P2). The protocol was adopted from Brewer et al. (Brewer and Torricelli, 2007) with modifications. Briefly, about 85 mg of dissociated cortex tissue was maintained temporarily in cold Neurobasal A medium. We transferred cell pieces to a tube for papain digestion by shaking for 20 min. The residues after gravity precipitation were incubated in fresh Neurobasal A medium followed by gentle pipetting for 45 sec, then set still for 2 min precipitation. This step of dissociating residues was repeated three times, and each time the supernatants were collected into a new tube. The cells collected were carefully laid onto a prepared OptiPrep (Sigma) gradient tube according to Brewer et al.’s method. The upper layers were aspired out after centrifuging at 2000 rpm for 15 min and the lower layers containing neuronal cells were removed to a new tube containing 5 ml fresh Neurobasal A medium. We cleaned up these neuronal cells two more times to get rid of gradient medium by adding fresh Neurobasal A medium and centrifuging at 1250 rpm for 5 min. They were plated onto poly-D-lysine (Sigma) coated culture dishes at a density of 1.04x105/cm2. Afterward, the medium was changed every three days using Neurobasal A medium.. 5. Immunocytochemistry of neuronal cells and neurite density analysis. Differentiated neurons were seeded on glass coverslips coated with poly-DL-ornithine/laminin. At a given time after differentiation, cells were washed with PBS, fixed in 4% paraformaldehyde (Sigma) prepared 21.

(33) in PBS for 30 min, and permeabilized in 0.3% Triton X100 (Sigma) in PBS for 10 min. The sample was further blocked in 1% bovine serum albumin made in PBS for 1 h at room temperature. A corresponding primary antibody was added for 2 h, followed by washing with PBS. A secondary antibody such as Alexa 488-conjugated anti-mouse, Alexa 488-conjugated anti-rabbit, or rhodamine-conjugated anti-mouse was incubated for 45 min followed by a PBS wash. Nuclei of cells were stained with 4’, 6’-diamidino-2-phenyindole (DAPI) (Sigma) for an additional 15 min, followed by another PBS wash. The coverslip was then mounted on a glass slide using Fluoromount G. Images were collected using a Leica SP2 confocal microscope connected to a CCD camera (Leica, Wetzlar, Germany) or using a Zeiss Axio Observer D1 microscope (Carl Zeiss, Jena, Germany). We defined density of neurites as neurite length divided by neuron number measured by Metamorph software or Image-Pro Plus software. The images were usually taken as three different fields in each experiment, which was repeated three to four times. Therefore, a total of around 9 to 10 images were captured. Each image typically contained more than 50 counted neurons, so a total of around 500 neurons were evaluated. Figures were prepared using Adobe software.. 6. Cell lysates and immunoblot assay. Cells used for the immunoblot assays were processed as previously described (Lin et al., 2002). Briefly, one 3.5-cm dish of frozen cells was 22.

(34) scraped into 0.1 ml of lysis buffer (50 mM Tris base, pH 7.9, 50 mM NaCl, 0.1 mM EDTA, 20 mM β-glycerophosphate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 nM calyculin A, 0.5% Triton X-100, 1 tablet/50 ml of protease inhibitor (Roche)). Lysates were centrifuged at 13,500 rpm for 10 min. Aliquots of the supernatants containing equal amounts of protein, measured by Bradford assay (Bio-Rad, Hercules, CA, USA). For low-molecular-weight proteins such as 4E-BP1, cell lysates were prepared as described above and subjected to tricine sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Schagger’s method (Schagger, 2006). Briefly, the separation gel contained an additional 0.1% glycerol in a regular format. The cathode buffer (0.1 M Tris, 0.1 M Tricine, and 0.1% SDS; pH 8.25) and anode buffer (0.1 M Tris and 0.0225 M HCl; pH 8.9) were loaded onto a gel apparatus, separated, and transferred. Blots were visualized using a horseradish peroxide (HRP)-conjugated secondary antibody followed by chemiluminescence, as recommended by the manufacturer (Thermo Scientific/Pierce, Rockford, IL, USA).. 7. Immunoprecipitation. Cell lysates were prepared as abovementioned procedures. For immunoprecipataion, primary antibodies were conjugated to protein A/G agarose beads at 4°C. After 1 h of incubation, the conjugated antibodies with beads were cleaning up in lysis buffer for 3 times. Cell lysates were put into antibody pre-conjugated beads and shacked for 2 to 3 h at 4°C. 23.

(35) The immunoprecipitate complexes were washed by using lysis buffer for 3 times and by lysis buffer containing 0.5 M LiCl for 2 more times. Before loading into gels, the samples were added sample buffer and heated at 95°C for 10 min.. 8. Treatment of drugs/inhibitors on neurons. As using pharmaceutical approaches to study, the treatment of drugs is described as follows. After EBs dissociation for 2 h, isolated neuronal cells were begun to expose to various inhibitors. These inhibitors included 1) 1 μM rapamycin, a mTORC1 inhibitor 2) 1 μM Wortmannin, a PI3K inhibitor 3) 10 μM z-VAD, a caspase inhibitor. All the drugs were dissolved in DMSO which was also presented in control group. When the medium was changed within 72 h or thereafter, medium always contains these drugs/inhibitors so as to keep the efficacy of reagents.. 9. RNAi viral particles preparation Bacterial clones or plasmids containing short hairpin RNA oligonucleotides of the target genes were obtained from the National RNAi Core Facility (Genomic Research Center, Academia Sinica, Taipei, Taiwan). Functional clones and corresponding effective target sequences of control, raptor, and rictor were verified and are listed as follows: luciferase (control), 5’-CGCTGAGTACTTCGAAATGTC, raptor sequence, 5’- CCTCATCGTCAAGTCCTTCAA, and rictor sequence, 5’-. 24.

(36) GCCAGTAAGATGGGAATCATT. RNAi plasmids were co-transfected with two other viral packaging plasmids, pCMV8.91 and pMD.G, into HEK 293T cells. The virus-containing medium was collected after 40 h of transfection and concentrated by ultracentrifugation at 25,000 rpm for 2 h. Pellets were concentrated and stored at -80 °C until use. We followed a protocol provided by the RNAi Core Facility in Academia Sinica, Taipei, Taiwan to measure the RNAi titer (http://rnai.genmed.sinica.edu.tw/file/protocol/4_1_EstimationLentivirus TiterRIUV1.pdf). To test the virus titer, we also modified this protocol using primary culture of mice cortex neurons—a cellular model that is much more sensitive to virus infection—instead of using A549 cells.. 10. Plasmids construction To develop plasmids carried target genes for overexpression in neuronal cells, we modified the lentivirus packaged plasmid, pLKO.1-puro. Briefly, we aligned the fragments of human cytomegalovirus (CMV) promoter fused with GFP genes with ClaI on 5’-terminal and AgeI on 3’-terminal. This fragment was further fused with polyA sequences by using PCR. The whole fragment was replaced with the U6 promoter and hairpin region in pLKO.1-puro plasmid. We temporally named it pLMG. Based on this vector, we further constructed S6K1 WT/S6K T389E/4E-BP1 WT/4E-BP1 T3746E into this plasmid after GFP gene to get expression of GFP tagged fusion proteins.. 25.

(37) III). Statistical analysis Data are presented as mean ± S.E. Treatment effects were. evaluated using a two-tailed Student’s t test. A p value<0.05 was considered to be statistically significant.. 26.

(38) Chapter III. An Approach for Differentiating Uniform Glutamatergic Neurons from mESCs.. 27.

(39) Introduction. Differentiation and growth of neurons involve many aspects including 1) sprouting, extension, and branching of dendrites and axons during development of the nervous system, 2) re-wiring of neurons in hippocampal tissues during learning and memory processes, and 3) regeneration of severed axons. Because of the importance of the biological aspects of the differentiation and growth of neurons/neurites, a protocol to achieve an appropriate homogenous population of neurons has emerged as key to pursuing many crucial questions. While many neural studies at the cellular level utilized primary neuron culture or neuronal cell lines (e.g., P19 cells), neurons differentiated from ESCs have emerged as another meaningful cellular model to investigate neuronal differentiation and growth (Evans and Kaufman, 1981). The main advantage is that neurons derived from ectoderm ES cell development are developmentally and physiologically significant. To date, various protocols for neuronal differentiation of ESCs involve the formation of embryoid bodies (EBs) with or without retinoic acid treatment (Bain et al., 1995; Fraichard et al., 1995; Lee et al., 2000; Wichterle et al., 2002). The hurdle encountered by many studies using ESCs as a model system is that the eventual cellular population differentiated from ESCs is not uniquely neurons (Stavridis and Smith, 2003). A protocol described by Bardie's group clearly demonstrates that feeder-dependent mESCs can be differentiated into uniform neurons 28.

(40) (Bibel et al., 2004; Bibel et al., 2007). They characterized many aspects of these neurons, such as neuronal markers and electrophysiological properties. They pointed out that many critical steps must be strictly followed in order to acquire optimal results. After all of the crucial factors were deliberately repeated including a step in which cell aggregations of EBs were completed trypsinized, we found that a homogeneous population was still not always obtained. This indicated that some crucial factors and/or details needed to be explored. Therefore, we set out to systemically investigate many possibilities which might have caused such a discrepancy, including the quality of various reagents and precision of parameters we used. We gradually pinpointed this issue and developed a protocol to consistently successfully differentiate neurons from mESCs. The approach presented herein has the potential to establish a cellular model for neuronal research.. 29.

(41) Material and methods. Refer to “General Materials and Methods” in chapter 2.. Results. Feeder-independent mESCs were routinely cultivated in ES medium supplemented with LIF. As depicted in Fig. 1, the protocol was schematically drawn as a time line to indicate the key processes of differentiation. When mESCs were initially cultivated in ES medium without LIF on the bacterial dishes, these cells began to form EBs, and this time was designated day 0. From day 4, these EBs were cultivated in the same medium but supplied with a final concentration of 5 μM all-trans retinoic acid for 4 more days. On day 8 when the differentiated progenitor cells within EBs were about to be separated and placed on poly-DL-ornithine/laminin-coated plates, these EBs were subjected to trypsinization so as to dissociate the aggregations. The timing was hereafter designated time after plating due to the many manipulation steps in the following procedure. The culture medium was eventually switched to neurobasal medium, and neurons were maintained for at least 21 days.. In an effort to establish a method to make ESCs uniformly differentiate into neurons, many crucial steps were strictly followed according to Bibel et al. (Bibel et al., 2007). These steps and/or factors included the quality of materials, the number of cells required for seeding, 30.

(42) the complete dissociation of EBs, etc. However, we ultimately pinpointed that the critical step was not to completely dissociate EBs, but was the optimal timing of EBs treated with the freshly made trypsinization solution. As shown in Fig. 2A, neurites were originally grown out in all groups in the initial 24 h, as the initial seeding numbers were all the same. When time progressed to 48 or 72 h, only EBs treated with an appropriate trypsinization period (i.e., 8 min in this case) continued to grow, maintain neurites, and eventually create neurite networks. The effect of inappropriate timing on EB trypsinization resulted in cell loss as seen in Fig. 2B.. Meanwhile, over-trypsinization of EBs usually caused the cell population to consist of different cell types. A heterozygous population of neurons was observed at 24~48 h after plating and become more significant at 72 h. As seen in Fig. 3 which was taken at 48~72 h, it obviously contained neurons, neurites, and more than 50% of non-neuronal cell types. One of the cell types was neuronal-like with complicated neurites extending from it which were prone to form aggregations. Another one was non-neuronal demonstrating a flatter morphology, and it was distributed all over the field. Determining exactly what these cells are requires further investigation.. These mESCs were well maintained and the separately formed EBs are shown in Fig. 4A and 4B. Having adopted the above-described modifications, we were consistently able to differentiate homogenous 31.

(43) neurons derived from mESCs. Judging from the appearance of the uniform neuronal morphology in Fig. 4C, this could be distinguished as successful differentiation. We further probed these cells with neuron-specific class III β-tubulin using immunocytochemistry. In Fig. 5A, the majority of cells showed nuclear staining (blue) and was stained with the neuronal marker (green). The double-staining rate was calculated to be > 93% in any given field. In order to clearly demonstrate the homogeneity and identify what kinds of cell types these few cells were, differentiated cells from the same experiment were immunostained with both GFAP and DAPI. The results showed that they were non-glial differentiated cells (Fig. 5B). The same GFAP antibody was capable of staining CTX TNA2 cells, an astrocyte cell line (Fig. 5D). Similarly, this glial cell line was barely stained with neuron-specific class III β-tubulin (Fig. 5C). Furthermore, different cell types were blotted with Oct-4 and type III β-tubulin which are respective markers for pluripotent ES cells and differentiated neurons (Fig. 5E). The results demonstrated that only mESCs contained the Oct4 marker, while differentiated neurons did not. This indicates that mESCs were properly maintained in the culture system. On the other hand, differentiated mESCs using the current protocol developed herein contained substantial type III β-tubulin. In the same experiment, HEK 293T cells were used as a negative control. In addition, NeuN and MAP2, which are other neuronal markers, were also used on the same blots. The results showed the same pattern as that seen for type III β-tubulin (data not shown). These neurons and neurite networks could be maintained for at least 3 weeks without morphological changes. 32.

(44) In an effort to further characterize these neurons, these differentiated neurons were immunostained with a vesicular glutamate transporter 1/2 (vGLUT1/2) antibody, a marker for glutamatergic neurons. As shown in Fig. 6A of staining with vGLUT1/2 and Fig. 6B of staining with DAPI, > 87% of cells were stained with both as shown in Fig. 6C. Although few cells were stained with DAPI but not vGLUT1/2, they demonstrated the specificity of this vGLUT1/2 antibody. This phenomenon was seen as early as 3 days after cell plating. We further examined synapse formation with this differentiation method. As seen in Fig. 6D, synaptotagmin, a marker of synaptic vesicles, was uniquely expressed in neurons differentiated from mESCs, but not other cell lines such as mESCs or HEK293T cells. These results further demonstrated that mESCs differentiated using the defined protocol we established uniformly developed into glutamatergic neuron. We further characterized the neurons derived from mESCs. Neurons/neurites were confirmed by staining with class III β-tubulin (red), a neuronal marker, as shown in Fig.7. Within the same field, synaptotagmin (green), which functions as a calcium sensor in the regulation of neurotransmitter release, was also stained on these neurons three days and nine days after differentiation. We further verified these neurons by western blot analysis. As shown in Fig. 7, AMPA receptor 1 (AMPAR 1), an ionotropic transmembrane receptor for glutamate in the central nervous system, began to express on Day 3 after differentiation, and more on Day 9. PSD-95, a protein that is almost exclusively located. 33.

(45) in the post synaptic density of neurons and is involved in anchoring synaptic proteins, also showed a similar pattern to AMPAR 1 or synaptotagmin. The lysates from HEK 293T cells and primary neuronal cultures of mice cerebral cortex functioned as negative and positive controls, respectively, for all antibodies used. Within the same blot, Oct-4 and type III β-tubulin, which are respective markers of mESCs and differentiated neurons, demonstrated that mESCs were properly maintained and neurons were well developed. This also indicated that synapses could potentially be constructed within these neurons.. 34.

(46) Discussion. We adopted an approach which contains many crucial steps to differentiate mESCs into neurons (Bibel et al., 2004; Bibel et al., 2007). Many factors were indeed very crucial in our test as well. However, in almost all the early efforts when we followed the protocol, we obtained neurons and other cellular types. The morphology of which was larger than neurons with no neuronal characteristics such as neurites as seen in Fig. 3. Therefore, many variables were systemically compared in parallel, and trial-and-error experiments were performed. We pinpointed that the critical factor which determined the success of homogenous neuronal differentiation was the dissociation timing of EBs. It was reported that the processes and timing of germ-layer formation derived from EBs in vitro greatly repeated stages of development during the embryonic phase of life in vivo (Leahy et al., 1999; Rohwedel et al., 1999). As to the order and spatial relation existing in these EBs, they developed as endoderm-like cells in the core first, then as ectoderm cells in the rim, followed by mesoderm cells within the entire EB. As we trypsinized the EBs at different times, the right layers of progenitor cells could be isolated at the appropriate time (as seen in Fig. 2). Conversely, over-trypsinization of the EBs caused a mixed population which resulted from more than one germ-layer of progenitor cells.. Based on the formation and development of the embryonic germ layers, they were theoretically more uniform when only the outer layers 35.

(47) of the EBs were trypsinized then seeded onto plates. Nevertheless, we found that successful differentiation greatly depended on an optimal duration of trypsinization, not just on the complete or less-than-complete dissociation of EBs. As long as the trypsinization timing was optimal as seen in those treated for 8 min in Fig. 2, the neurons survived and grew normally. As shown in Fig. 2, neurons/neurites only survived for 24 h in groups of EBs trypsinized for 2, 4, or 6 min. This phenomenon occurred within groups that were inappropriately trypsinized, which could have been due to either apoptosis after EB dissociation (Koyanagi et al., 2008) or cell death because of a lack of sufficient growth cytokines (Cook et al., 2010). Since the initial cell numbers which were dissociated from EBs for plating were the same, the latter factor seems less likely in this case. Recently, it was shown that control over molecular transport of EBs plays a strategic role in stem cell differentiation (Sachlos and Auguste, 2008). Whether this is relevant to the phenomenon seen here and how the appropriate timing of trypsinization creates such a difference remain to be further investigated.. Neurons differentiated from mESCs using this protocol survived for more than 3 weeks with only slight changes in their morphology. Also, the network formed by the neurites gradually grew more complicated and was maintained for the same time. In agreement with Bardie et al.’s results (Bibel et al., 2004; Bibel et al., 2007), the majority of neurons differentiated by the current method were glutamatergic neurons based on the positive staining of vGLUT1/2. Since vGLUT1/2 is a membrane 36.

(48) protein expressed by most pyramidal neurons in the cerebral cortex and hippocampus (Fremeau et al., 2004), the establishment of this method further confirms that this is an excellent cellular model for investigating properties related to the central nervous system (CNS).. Alternatively, there are some other approaches such as gene manipulation to obtain uniform neuron populations. Utilizing insertion of the sox promoter followed by a green fluorescent protein (GFP) and/or other reporter genes, GFP-positive cells can be sorted into homogenous neuronal populations (Li et al., 1998; Ellis et al., 2004; Plachta et al., 2007). Although such an approach will definitely result in uniform neuronal cells, it could interfere with potential applications in medicine because of the consideration of genomic interference and instability. It might be more ideal and physiologically relevant in terms of obtaining uniform neurons by using the current approach developed in this context. Glutamatergic, multipolar neurons, and complicated neurite networks began forming at three to nine days post culture of neurons differentiated from mESCs. On the basis of vGLUT1/2, AMPAR, and PSD-95 blots, they indicate synapses could also be constructed within these neurons. Initially, they were immature, with more bipolar neurons at three days after differentiation, as shown in Fig. 7, although already around 5% had begun to show multipolar neurons. As time passed, by the seventh day, the majority showed mature characteristics, such as multipolar neurons and complicated neuritis. Several papers employing a similar protocol produced glutamatergic neurons like ours. Bibel et al.. 37.

(49) developed a method and reported that essentially all neurons were positive for glutamate vesicular transporter 1/2. By contrast, less than 1% of the cells were positive when stained with antibodies to other neurotransmitter systems after one week of culture. Most neurons that they investigated received both majority glutamate and minor GABA (5%) input after three weeks (Bibel et al., 2004). In addition, using a similar method with the modification of maintaining differentiating aggregates on petri dishes with rotation, Hubbard et al. produced highly enriched glutamatergic neurons from suspension-cultured mESCs (Hubbard et al., 2012).. In conclusion, we developed an approach to obtain a homogeneous population of glutamatergic neurons differentiated from mESCs. By trypsinizing EBs in freshly produced trypsin buffer for an appropriate time, uniform neurons could be reproducibly and consistently generated. Such a method can contribute to building a physiological cellular model for investigating neuronal development/growth, and can also shed light on possible therapeutic applications in regenerative medicine.. 38.

(50) Treatment:. EB formation. +RA. 0. 4. plating. neurobasal medium.    Days:. EB formation. +RA. 8. >21. : One million of feeder-independent mES cells were seeded in a 10-cm bacterial dish and cultivated in EB medium which contained ES medium but no LIF. : A final concentration of 5 μM retinoic acid was added to the EB medium.. plating. : Determine the critical timing for EB trypsinization (7-9 min is typical). The optimal density for plating is around 1.2~1.5x105 cells/cm 2.. . :  , 24 h;  , 48 h; and. , 72 h; time after plating. Fig. 1.. 39.

(51) Fig. 1. Flow chart of experimental procedures to differentiate mESCs into neurons. mESCs were routinely cultivated in ES medium which normally contained leukemia inhibitory factor (LIF). On the day when differentiation began, 106 ES cells were cultivated on one 10-cm bacterial culture plate in which the LIF was withdrawn from the ES medium. This was designated day 0. On day 4, a final concentration of retinoic acid of 5 μM was directly added to the medium. Four days later, these embryoid bodies (EBs) were trypsinized and placed on poly-DL-ornithine/laminin-coated plates. The timing of trypsinization had to be carefully controlled as described in the text. Due to the frequent operating steps from this time point on, the time was designated hours/days after plating. Therefore, dissociated EBs were seeded onto plates at an optimal density in the range of 1.2~1.5x105 cells/cm2. The medium was replaced with fresh N2 medium at 2 and 24 h after plating. At 48 h after plating, the culture medium was switched to neural-basal-B27 medium. For the long-term culture of neuronal cells, it was necessary to partially replace the neuro-basal-B27 medium every other day. These neurons survived for at least 21 days.. 40.

(52) B Time after plating:. Time with trypsinization:. 24 h. 48~72 h. 2 min. 4 min. 6 min. 8 min. Fig. 2.. 41. Ratio of neurites length/neuronal cell numbers (Arbitrary Unit). A. 150 100 50 0 150 100 50 0 150 100 50 0 150 100 50 0. 2 min. * 4 min. * 6 min. * 8 min.

(53) Fig. 2. Effects of different timing of EBs trypsinization on neuronal differentiation. A pool of EBs on day 8 was divided into four groups and subsequently subjected to 2, 4, 6, and 8 min of trypsinization. After seeding on poly-DL-ornithine/laminin-coated plates, morphological changes were recorded at 24, 48, and 72 h after plating. It was noteworthy that all plates contained the same initial cell density of 1.35 x 105/cm2 which was also a crucial factor in successful neuronal differentiation according to the literature and our experience. These pictures were taken with inverted phase-contrast microscopy. Each photo shown is representative of one experiment that was repeated 3 times. The scale bar indicates 40 μm.. 42.

(54) Field 2. Field 1. 48~72 h after plating. Fig. 3.. 43.

(55) Fig. 3. Heterogeneous neuronal differentiation after trypsinization of whole EBs. The EBs were trypsinized and completely dissociated. This typically took 12~14 min. Cells were then counted at an optimal density (1.35 x 105/cm2) for seeding onto poly-DL-ornithine/laminin-coated plates. Morphological changes were recorded daily. The pictures shown here were taken 48~72 h after plating with two different fields. Each photo shown is representative of one experiment that was repeated five times. The scale bar indicates 40 μm.. 44.

(56) A. B. C. Fig. 4.. 45.

(57) Fig. 4. Uniform neurons differentiated from mESCs. A. Morphology of feeder-independent mESCs cultivated on gelatin-coated plates. B. EBs were observed as early as day 1 or 2 when they were cultivated as a suspension on bacterial plates. The morphology of these EBs was recorded on day 4. The inset is a magnification of one of the EBs. C. After trypsinization of the EBs with optimal timing, these isolated cells were plated onto poly-DL-ornithine- and laminin-coated plates. A population of uniform neurons was obtained and observed. The picture shown here was taken 48 h after plating. Each photo shown is representative of one experiment that was repeated four times. The scale bar indicates 40 μm.. 46.

(58) A. B. C. D. E. α Type III β tubulin α Oct-4 α Actin 1. 2. 3. Fig. 5.. 47.

(59) Fig. 5. Morphological and biochemical evidence showing that uniform neurons had differentiated from mESCs. A. Neurons were fixed on coverslips using paraformaldehyde. Immunocytochemistry was performed using type III β-tubulin as the first antibody and Alexa 488-conjugated anti-mouse immunoglobulin G (IgG) as the secondary antibody. A superimposed picture of green (type III β-tubulin) and blue (DAPI) staining is shown. White triangles indicate cells stained with DAPI only, but not type III β-tubulin. B. Neurons differentiated from mESCs in the same experiment on the other coverslip were blotted with GFAP as the first antibody and rhodamine-conjugated anti-mouse IgG as the secondary antibody. C and D. CTX TNA2 cells, an astrocyte cell line, were used to examine the specificity and efficacy of antibodies. In a paralleled experiment, these cells were stained with the same antibodies as the ones used in A and B. Therefore, type III β-tubulin staining and GFAP staining were shown in C and D, respectively. Each photo shown is representative of one experiment that was repeated three times. The scale bar indicates 40 μm. E. Undifferentiated mESCs along with the neurons differentiated from mESCs were homogenized, run onto the gel, and blotted with Oct4 and class III β-tubulin antibodies. HEK 293T cells were used as a control of the cell type. The blot is representative of one experiment that was repeated three times. An actin blot served as the loading control.. 48.

(60) A. B. C. D. α Synaptotagmin α Actin 1. Fig. 6.. 49. 2 3. 4.

(61) Fig. 6. Glutamatergic neurons were differentiated from mESCs. A. Cells differentiated at 72 h after plating were fixed in paraformaldehyde, and the immunocytochemistry protocol was performed as described in “General Materials and Methods”. Anti-vGLUT1/2 was used as the primary antibody. B. The same field was stained with DAPI. C. Merged image of A and B demonstrating that more than 95% of cells were vGLUT1/2-positive. In this field, white triangles indicate that only a few cells were not glutamatergic neurons because they were only stained with DAPI. Images were taken with a confocal microscope to scan the Z-axis from top to bottom of cells. Each photo was reconstructed by compiling all of the images together and is representative of one experiment that was repeated three times. The scale bar indicates 20 μm. D. Cell lysates harvested from another round of differentiation subjected to a Western blot analysis. Samples from HEK 293T cells, undifferentiated ES cells, and neurons differentiated from ES cells were blotted with synaptotagmin, a marker of synaptic vesicles. The blot is representative of one experiment that was repeated two times. An actin blot was used as the loading control.. 50.

(62) A Synaptotagmin. βIII-tubulin. Day 3. Day 7. B. AMPAR 1. Fig. 7.. 51. Merge.

(63) Fig. 7. Almost uniform neurons differentiated from mESCs show glutamatergic, pre-, and post-synapse markers. A. Three days (upper panel) and seven days (lower panel) after neurons differentiated from mESCs were blotted with class III β-tubulin (red) and synaptotagmin (green) as the first antibodies and DyLight 549-conjugated anti-rabbit IgG and Alexa 488-conjugated anti-mouse IgG as the second antibodies, respectively. The same field was stained with DAPI (blue). Scale bar, 10 μm. B. Lysates from neurons 3 and 9 days after differentiation from mESCs, along with controls including undifferentiated mESCs, primary neuronal culture of mice cerebral cortexes, and HEK 293T lysates were run onto the gel and blotted with antibodies that contained AMPAR 1, synaptotagmin, PSD-95, Oct4, class III β-tubulin, and actin. These photos and blots are representative of one experiment that was repeated three times.. 52.

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