Quantitative evaluation of motor function before and after engraftment of dopaminergic neurons in a rat model of Parkinson's disease

Although gait change is considered a useful indicator of severity in animal models of Parkinson's disease, systematic and extensive gait analysis in animal models of neurological deficits is not well established. The CatWalk-assisted automated gait analysis system provides a comprehensive way to assess a number of dynamic and static gait parameters simultaneously. In this study, we used the Catwalk system to investigate changes in gait parameters in adult rats with unilateral 6-OHDA-induced lesions and the rescue effect of dopaminergic neuron transplantation on gait function. Four weeks after 6-OHDA injection, the intensity and maximal area of contact were significantly decreased in the affected paws and the swing speed significantly decreased in all four paws. The relative distance between the hind paws also increased, suggesting that animals with unilateral 6-OHDA-induced lesions required all four paws to compensate for loss of balance function. At 8 weeks post-transplantation, engrafted dopaminergic neurons expressed tyrosine hydroxylase. In addition, the intensity, contact area, and swing speed of the four limbs increased and the distance between the hind paws decreased. Partial recovery of methamphetamine-induced rotational response was also noted

The infection of primary avian tracheal epithelial cells with infectious bronchitis virus

Here we introduce a culture system for the isolation, passaging and amplification of avian tracheal epithelial (ATE) cells. The ATE medium, which contains chicken embryo extract and fetal bovine serum, supports the growth of ciliated cells, goblet cells and basal cells from chicken tracheas on fibronectin- or matrigel-coated dishes. Non-epithelial cells make up less than 10% of the total population. We further show that ATE cells support the replication and spread of infectious bronchitis virus (IBV). Interestingly, immunocytostaining revealed that basal cells are resistant to IBV infection. We also demonstrate that glycosaminoglycan had no effect on infection of the cells by IBV. Taken together, these findings suggest that primary ATE cells provide a novel cell culture system for the amplification of IBV and the in vitro characterization of viral cytopathogenesis

The signals of FGFs on the neurogenesis of embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Neural induction is a complex process and the detailed mechanism of FGF-induced neurogenesis remains unclear.</p> <p>Methods</p> <p>By using a serum-free neural induction method, we showed that FGF1 dose-dependently promoted the induction of Sox1/N-cadherin/nestin triple positive cells, which represent primitive neuroblasts, from mouse embryonic stem (ES) cells.</p> <p>Results</p> <p>We demonstrated that FGF1, FGF2, and FGF4, but not FGF8b, enhanced this neurogenesis. Especially, FGF-enhanced neurogenesis is not mediated through the rescue of the apoptosis or the enhancement of the proliferation of Sox1⁺cells. We further indicated that the inactivation of c-Jun N-terminal kinase-1 (JNK-1) and extracellular signal-related kinase-2 (ERK-2), but not p38 mitogen-activated protein kinase (MAPK), inhibited the neural formation through the inhibition of ES differentiation, but not through the formation of endomesodermal cells.</p> <p>Conclusions</p> <p>These lines of evidence delineated the roles of FGF downstream signals in the early neural differentiation of ES cells.</p

腫瘤生成與幹細胞的萬能性-對人類胚幹細胞分化之神經上皮細胞其腫瘤生成的評估

Tumor formation is a major limitation for the clinical application of embryonic stem cells(ESCs). Many studies reported that the efficiency of tumorigenicity of differentiating EScells is relatively low, in contrast to the undifferentiated ES cells. However, whether thedifferentiating ES cell is really non-tumorigenic is still lacking of systemic approach. Areporting of glioneural neoplasm in a patient engrafted with embryo brain tissue raises thecaution that neural stem cell can be tumorigenic. We have established a novel and highlyefficient neural differentiation method for the human embryonic stem cells. Theundifferentiated ES cells and non-neural cells were eliminated without the requirement ofhand-picking, drug selection or cell sorting. The neuroepithelial cells (NPCs) were amplifiedand could differentiate into neurons and glia cells. Using this technique, we will examinewhether the tumor formation can be detected when the applied cell number of NPC is raisedto 106 cells in SCID mice. Transfer of pluripotent-related genes into the NPCs may convertthese cells become pluripotent and tumorigenic. Parts of engrafted hosts will be maintainedup to half year and continually be traced for the cell fates of engrafted cells, the tumorformation, biochemical analysis in blood and the influence of stem cells on the physiologyof SCID mice.腫瘤的形成是胚幹細胞應用的一大限制。雖然許多研究顯示，胚幹細胞分化後，形成腫瘤的機率極低，但目前對分化後的胚幹細胞是否會形成腫瘤的問題，仍缺乏全面且系統性的研究來證實。此外，許多研究者也證實，移植神經幹細胞可與宿主組織嵌合，而改善受傷神經組織所造成的運動功能障礙。但最近的研究發現，移植胚胎神經組織仍具有形成神經母細胞瘤的風險。本實驗室發展出一新的神經分化方法，可將人類的胚幹細胞，可高效率分化為初始神經上皮細胞，不需經過人工挑選、藥物篩選或細胞分選等方法，便可去除未分化的人類胚幹細胞及非神經細胞。經培養後，細胞可大量增生，並可分化為成熟的神經細胞與神經膠細胞。利用此一系統，我們將研究，是否在移植較高細胞數與連續投與細胞下，經三個月後，細胞的存活、分化與組織分佈，以及SCID 小鼠體內產生腫瘤的種類與機率為何。移植入小鼠胚胎後，是否仍會產生腫瘤，或者可以嵌入鼠胎腦組織，形成有功能的神經。我們也將仔細檢驗，將此神經上皮細胞經基因轉殖後，移植入SCID 小鼠的腦部與肌肉注射，是否會改變產生腫瘤的機率與體外分化的能力。部份小鼠將連續觀察，直到半年後給予解剖與進行血液的生化分析,研究移植細胞的細胞命運、腫瘤生成以及對宿主動物的正常生理的影響

Are Pluripotency and Tumorigenicity Coupled: Evaluation of the Tumorigenicity of Human Embryonic Stem Cell-Derived Neuroepithelial Cell

腫瘤的形成是胚幹細胞應用的一大限制。雖然許多研究顯示，胚幹細胞分化後，形成腫瘤的機率極低，但目前對分化後的胚幹細胞是否會形成腫瘤的問題，仍缺乏全面且系統性的研究來證實。此外，許多研究者也證實，移植神經幹細胞可與宿主組織嵌合，而改善受傷神經組織所造成的運動功能障礙。但最近的研究發現，移植胚胎神經組織仍具有形成神經母細胞瘤的風險。本實驗室發展出一新的神經分化方法，可將人類的胚幹細胞，可高效率分化為初始神經上皮細胞，不需經過人工挑選、藥物篩選或細胞分選等方法，便可去除未分化的人類胚幹細胞及非神經細胞。經培養後，細胞可大量增生，並可分化為成熟的神經細胞與神經膠細胞。利用此一系統，我們將研究，是否在移植較高細胞數與連續投與細胞下，經三個月後，細胞的存活、分化與組織分佈，以及SCID 小鼠體內產生腫瘤的種類與機率為何。移植入小鼠胚胎後，是否仍會產生腫瘤，或者可以嵌入鼠胎腦組織，形成有功能的神經。我們也將仔細檢驗，將此神經上皮細胞經基因轉殖後，移植入SCID 小鼠的腦部與肌肉注射，是否會改變產生腫瘤的機率與體外分化的能力。部份小鼠將連續觀察，直到半年後給予解剖與進行血液的生化分析,研究移植細胞的細胞命運、腫瘤生成以及對宿主動物的正常生理的影響。Tumor formation is a major limitation for the clinical application of embryonic stem cells(ESCs). Many studies reported that the efficiency of tumorigenicity of differentiating EScells is relatively low, in contrast to the undifferentiated ES cells. However, whether thedifferentiating ES cell is really non-tumorigenic is still lacking of systemic approach. Areporting of glioneural neoplasm in a patient engrafted with embryo brain tissue raises thecaution that neural stem cell can be tumorigenic. We have established a novel and highlyefficient neural differentiation method for the human embryonic stem cells. Theundifferentiated ES cells and non-neural cells were eliminated without the requirement ofhand-picking, drug selection or cell sorting. The neuroepithelial cells (NPCs) were amplifiedand could differentiate into neurons and glia cells. Using this technique, we will examinewhether the tumor formation can be detected when the applied cell number of NPC is raisedto 106 cells in SCID mice. Transfer of pluripotent-related genes into the NPCs may convertthese cells become pluripotent and tumorigenic. Parts of engrafted hosts will be maintainedup to half year and continually be traced for the cell fates of engrafted cells, the tumorformation, biochemical analysis in blood and the influence of stem cells on the physiologyof SCID mice

Establishment of Cell Lines of Tracheal Epithelial Cells for the Identification and Amplification of Avian Respiratory Viruses

目前禽類呼吸道病毒多於雞胚或雞纖維母細胞中增殖，此兩種繼代法的缺點分別有，病毒因需適應新宿主細胞常產生基因突變，以及所產生的病毒力價過低。此外，部份呼吸道病毒的毒力鑑定，如家禽支氣管炎病毒，因大都對纖維母細胞無細胞毒性，目前主要靠雞胚的生長停滯與氣管環上的纖毛擺動速率來認定，也有無法準確測出病毒力價與毒力的缺點。為解決以上問題，本計畫將藉由已建立之家禽氣管上皮細胞初代培養技術，利用腺病毒(adenovirus)與Nucleofactor，將致癌基因large T antigen, H-Ras以及c-myc送入細胞中，建立新的禽類呼吸道上皮細胞株。由於纖維母細胞容易感染慢病毒lentivirus，而呼吸道上皮細胞則否。我們將利用lentivirus帶DT-A (diphtheria toxin A)，去除繼代過程中的纖維母細胞。建立的細胞株將可提供一新穎、簡易且高效率培養禽類病毒的增殖平台。利用此初代培養細胞與所建立之各種細胞株，並可研究呼吸道病毒如何感染上皮細胞的機制。所建立的細胞株除可提供野外分離病毒之增殖與鑑定外，我們也將評估是否此細胞株可提供一穩定、低成本與高效率的流行性感冒病毒疫苗株的製造平台。In the present, most avian respiratory viruses are amplified in the chorioallantoicmembrane of embryonated eggs or chicken fibroblast cells. The viral adaptation to the newhost cells often associates with genetic mutation and low viral production. In addition, mostinfectious bronchitis viruses are non-cytopathic to fibroblast cells and their viral titers andvirulences can only be roughly estimated by the growth retention of embryos and ciliostasis oftracheal rings. To tackle these problems, our primary tracheal epithelial cell system will beapplied to establish the stable cell lines of bronchial epithelium, combined with the infectionof the recombinant adenovirus carrying the SV40 large T antigen, H-Ras and c-myconcogenes. We found that recombinant lentivirus showed high cell tropism to the fibroblastbut not to the respiratory epithelial cells. Infection with the diphtheria toxin A (DTA)recombinant lentivirus should reduce the growth of fibroblast cells and their contamination inthe transformed cell population. Primary tracheal epithelial cells and their derivative cell lineswill benefit the understanding of the mechanism of viral infection. Established cell lines willprovide a novel system for the detection, isolation and amplification of the avian viruses. Inaddition, the stable cell line may also be a stable and high efficient factory for the productionof vaccine strains of avian influenza viruses

Exploration of Early Neurogenesis by an Efficient Serum-Free ES Monoculture System

限於胚胎尺寸及採取方便，牛蛙胚胎與雞胚胎較哺乳動物更為廣泛地被運用來做早期神經發生的研究。最近研究中發現，胚胎幹細胞（ES）可代替哺乳動物胚胎做為早期發生的研究模式，且可成功模擬動物早期神經發生的誘導及晚期胚胎的神經發育。然而，目前關於神經發生早期的研究，缺乏一有效率、無須血清的分化系統以提供具高重複性的細胞研究環境。本計畫將利用人類與鼠來源之Sox1-GFP Knock-in ES，於體外誘導出最早期之神經母細胞，及建立神經發生誘導的環境，以及研究BMP、Wnt、FGF等分子對於早期神經發生的影響。本計畫也將探討神經發生的模式；是否在哺乳類中，外胚層細胞的既定分化細胞也為神經細胞，如同蛙類的外胚層一般；以及研究BMP的拮抗物在哺乳類的神經發育中是否為充分條件或必要條件。在雞胚神經早期發育時期，FGF已被證明可促進神經發生，但僅為必要條件。FGF可能活化Churchill，使得外胚層對神經誘導物，如BMP的拮抗物等，較為敏感。本計畫也將探討FGF與Churchill在哺乳類的早期神經發生過程中，與BMP/SMAD、FGF/MAPK間的相互作用。建立無血清的神經分化培養系統不只對於研究神經的早期發生甚為重要，也提供一理想的人類ES細胞的研究的平台。一再現性高、控制良好的體外分化系統也將加速日後由ES細胞產生特定神經細胞的研究及應用於細胞移植的治療

Maintenance of the Pluripotency of Sox1+-Neuroblasts

動物胚胎在原腸期(gastrula) 分化為神經細胞時， 首先藉由抑制BMP(bonemorphogenetic protein) 的路徑誘導外胚層細胞分化為初始神經母細胞(primitiveneuroblasts)。此時的神經母細胞只會分化為神經細胞(neurons)，與腦室附近的神經幹細胞(neural stem cells, NSC)。早期分化的神經包括有中腦的多巴胺神經細胞與中後腦交界的serotoninergic 神經元。在神經形成的後期，此時不表現sox1基因的神經幹細胞可再分化為大腦皮質外層的神經細胞與神經膠細胞(glia cells)。NSC的體外培養，可受EGF(epithelial growth factor)與FGF2(fibroblast growth factor 2)的刺激，而自我複製與增殖。相對於初始神經母細胞，NSC的子代則多為神經膠細胞與少部分的神經細胞，與晚期胚胎中的radial glia cells相似。雖然目前可以利用EGF與FGF2來維持NSC的神經分化能力，但如何維持早期sox1+神經母細胞的不分化則少有研究。本計畫將利用小鼠來源之sox1-GFP Knock-in胚幹細胞及建立人類sox1-GFP knock-in胚幹細胞，於體外誘導出最早期之神經母細胞， 及建立神經發生誘導的環境。利用cell sorting 與antibiotics-selection的方法純化Sox1-GFP+ cells，以及研究leukocyte inhibitory factor、Wnt3a、sonic hedgehog、anti-bone morphogenetic protein及FGF2等分子，對於維持Sox1+神經母細胞的多能性(multipotency)的影響。本計畫也將探討以上因子在小鼠與人類胚幹細胞誘導出的神經母細胞的差異。此外，目前已知一染色體licensing factor，geminin，可能與Brg1作用，共同維持神經母細胞的不分化與自我複製。藉由四環黴素誘導系統來控制geminin與Brg1兩個基因，於胚幹細胞由來之Sox1+-GFP神經母細胞中表現，觀測sox1 promoter所驅動的GFP的表現，可以進一步探討相關訊息傳遞與分子機轉。研究初始原始母細胞多能性的維持不只對於神經的早期發生的基礎研究甚為重要，臨床應用上，也可利用patterning factor的刺激，使初始神經幹細胞分化為中腦的多巴胺神經元。本研究也將加速日後由ES細胞產生特定神經細胞的研究及應用於細胞移植的治療。During vertebrate gastrulation anti-bone morphogenetic protein (BMP) signals inducethe neurogenesis of uncommitted ectoderm into neuro-ectoderm. Primitiveneuroblasts, expressing the sox1 transcriptional factor, differentiate into neurons andneural stem cells (NSCs) near the ventricular zone. These early-stage maturedneurons include the dopaminergic neurons in the midbrain and serotoninergicneurons in the midbrain-hindbrain boundary, etc. In late-stage of neurogenesis,Sox1 negative NSCs differentiate into out-layers neurons of the cortex and glia cells.In vitro clonal expansion of the NSCs and neurosphere formation are established bythe helps of epidermal growth factor (EGF) and fibroblast growth factor (FGF) -2mediated growth stimulation. Correlated to the radical glia cells in vivo, most of theprogenies of NSCs are glia cells, rather than neurons. Although EGF and FGF2signals are essential for the self-renewal of NSCs, it is still obscure for themaintenance of multipotency of Sox1+ primitive neuroblasts. In this project,Sox1-GFP knock-in mouse embryonic stem (ES) cells and Sox1-GFP knock-in humanES cells will be generated and subjected for the generation of Sox1+ neuroblasts byusing the efficient neural induction procedure and consequent FACS-sortingenrichment and antibiotics selection. Several candidate factors will be tested for theconstant expression of sox1 gene in neuroblast cells, including leukocyte inhibitoryfactor (LIF), Wnt3a, sonic hedgehog, anti-BMP factors and FGF2. The variations ofthese candidate factors between mouse and human Sox1-ES cells will be carefullyevaluated. It has been demonstrated that geminin, a chromosome licensing factor,sequesters Brg1 to maintain the multipotency of primitive neuroblasts in Xenopusembryo. A tet-on inducible system for the geminin and Brg1 expression will beestablished in Sox1 GFP knock-in mES cells and hES cells and further evaluate thesox1-driven gene expression and related proneural genes profile. The study ofmaintenance of pluripotency of sox1+ neuroblast is important for the basic braindevelopment and further clinical application, such as efficient generation of abundantdopaminergic neurons. This study will also accelerate the research for thespecification of neural fates and cell-transplantation based therapy