隔离啮齿类动物或人类的鼻嗅干细胞

Neuroscience
 

Summary

我们在这里描述的方法biopsying从大鼠和人鼻腔嗅粘膜。这些活组织切片检查或者确定脑部疾病的分子异常或隔离多能成可利用细胞移植的干细胞在脑外伤/疾病的动物模型,可用于

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Girard, S. D., Devéze, A., Nivet, E., Gepner, B., Roman, F. S., Féron, F. Isolating Nasal Olfactory Stem Cells from Rodents or Humans. J. Vis. Exp. (54), e2762, doi:10.3791/2762 (2011).

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Abstract

嗅粘膜,位于鼻腔中,负责检测气味。它也是唯一的神经组织,暴露在外部环境和方便在每一个生命的个体。因此,这个组织是唯一的,目的是查明病理大脑中的分子异常或隔离成体干细胞,细胞疗法的人。

在脑部疾病的分子异常往往是研究用神经组织样本收集的验尸报告。然而,这种材料具有很多局限性。相比之下,嗅粘膜易于访问和安全没有任何意义上的气味 1损失可以活检。因此,嗅黏膜提供一个“开放的窗口”,在成年的人,通过它可以研究发展(如自闭症, 精神分裂症 )2-4或神经退行性疾病(如帕金森,老年痴呆 ) 4,5。嗅粘膜,可用于分子要么比较研究4,6或 3,7对神经元的在体外实验。

嗅上皮也是一种神经组织,产生新的神经元每天,以取代那些污染损害的,细菌病毒感染。这个永久性的神经发生持续的祖细胞,但也干细胞在黏膜,即神经上皮和基础固有层8-10两个车厢居住。我们最近开发出一种方法来净化位于固有层中的成体干细胞,后证明,他们是密切相关的骨髓基质干细胞(BM - MSC),我们将它们命名为嗅觉ecto间质干细胞(OE海安)11。

有趣的是,当相比,BM - MSCs的,OE - MSCs的显示高增殖率,高架clonogenicity和分化成神经细胞的倾向。我们注意到这些特点优势,以执行专门推出新的候选基因,在精神分裂症和帕金森病4的研究。我们和其他OE - MSCs的12,13脊髓外伤,耳蜗损伤14后或在帕金森氏症的1516失忆的动物模型,细胞疗法是有希望的候选人,也表明。

在这项研究中,我们目前在老鼠和人类的嗅黏膜活检的方法。上皮细胞和干细胞采集后,固有层酶分离纯化使用酶或一个非酶法。纯净嗅觉干细胞,然后可以要么增加大量库存在液态氮或诱导形成球体或成神经细胞分化。这些干细胞也可以用于比较组学(基因组,转录组,表观基因,蛋白质)的研究。

Protocol

1。收集大鼠嗅黏膜

  1. 首先,准备3张35毫米的Petri菜与F12在一个干净的文化引擎盖DMEM /火腿培养基充满。
  2. 任何“安乐死”的方法,必须由该机构的动物护理和使用委员会的事先批准,由合格的专业人员进行的。大鼠戊巴比妥钠或其他如氯胺酮/甲苯​​噻嗪注射麻醉形式进入深麻醉后,斩首和消除皮肤。应避免吸入麻醉药。麻醉充足,将评估前脚趾捏斩首。取出,用剪刀下颚和咬骨钳的帮助下,消除双方的面部肌肉。
  3. 从门牙的背面,用咬骨钳的鼻腔,一次一侧的骨覆盖删除。嗅觉鼻甲进入人们的视线,橙色/棕色机关在位于鼻子后面。
  4. 娇柔与镊子丢弃的鼻甲。使用26号针头,隔离嗅粘膜,隔躺在沿着三条线切割组织:弧的垂直板,筛板和鼻腔上限。
  5. 对双方收集的活组织切片检查和转让他们在DMEM /火腿F12 -填充培养皿。这个过程不应该需要更长的时间超过10分钟从一开始安乐死。
  6. 现在,以去除黏液,转移中充满培养皿两次活组织切片检查。

2。在人类嗅黏膜收集

  1. 此过程应进行一个耳鼻喉科(ENT)操刀,根据与当地有关的伦理委员会(S),每门诊应签署知情同意书。
  2. 使用0 °或30 °刚性内窥镜(直径4 mm),两个鼻腔检查和评估假定存在的息肉或炎性病变。选择最佳的鼻腔,考虑到隔的偏差。
  3. 使用棉花撒施,申请与肾上腺素,如利多卡因局部麻醉,10分钟。
  4. 随着throughcut筛窦钳,收集了两个平方毫米活检在根中鼻甲内侧面或隔背内侧区。
  5. 然后转移的嗅觉活检,是使用消毒针头,进入无菌2毫升1毫升的DMEM / HAM F12满管。提示管倒挂,以确保活检是在培养液中浸泡。
  6. 插入管和一个冷藏集装箱运输研究实验室。在这个阶段,可用于活检本身比较分子的研究集中在特定的大脑疾病或产生干细胞的处理。

3。从人类和大鼠胃黏膜嗅觉干细胞的分离

  1. F12的DMEM /火腿洗净的活组织切片检查。在充满1毫升dispase II解决方案(2.4 IU / ml)的一个培养皿培养活检1小时,在37 ° C。
  2. 接下来,解剖显微镜下的衍射倒光,嗅觉上皮被删除从基础层,用微型抹刀固有。
  3. 嗅上皮更薄,看起来比黑色的背景相比,固有层,这是条纹的半透明橙/棕。上皮细胞在一个白色背景,看起来灰色和固有层,褐色。
  4. 纯化后,转移到培养皿用DMEM / HAM F12填补了固有层。
  5. 如果从啮齿类动物的组织,然后切成小块,与两个25表针固有层。然后,充满了1毫升胶原酶IA 15毫升管件。
  6. 在管内,用无菌塑料吸管,游离于组织。然后,管孵育10分钟,37 ° C。
  7. 要终止的解离,轻轻摇动试管,加9毫升的无钙和镁无PBS离心5分钟,在200克。
  8. DMEM /火腿F12文化与10%小牛血清,抗生素和塑料培养皿板培养基重悬细胞沉淀。
  9. 现在,如果该组织是人类,然后片成3至4件厚度从200至500微米不等的固有层。
  10. 插入到它自己的直径2厘米的培养皿每个地带,并覆盖无菌直径1.3厘米的玻璃盖玻片的组织。
  11. 然后,每个培养皿中添加500μL培养基(DMEM /火腿F12与10%小牛血清和抗生素的补充)。
  12. 对于任何一个组织的类型,更新培养液中,每2至3天。
  13. 五到七天后,干细胞就开始侵入了培养皿中,两个星期后,他们应融合。当汇合,通过转移的细胞培养瓶中。

4。圈的形成和神经Differentiation的嗅觉干细胞

  1. 要生成干细胞球,两个小时的烧瓶在37 °彗星与聚- L -赖氨酸。 (文:5 g/cm2)。
  2. 板的细胞密度在每平方厘米1.6万细胞在治疗烧瓶。
  3. 每隔两天,饲料0.2毫升每平方厘米的培养基(DMEM培养液/火腿F12补充胰岛素,转铁蛋白,硒(ITS - X,​​1%),表皮生长因子(50毫微克/毫升)和FGF2(细胞50毫微克/毫升))。
  4. 2到5天之后,收集漂浮的细胞球,要么重制版或游离移植细胞治疗的动物模型前。
  5. 嗅觉干细胞分化成神经元样细胞,培养21天Neurobasal培养基中含有的B - 27,青霉素,链霉素,谷氨酰胺和谷氨酸。
  6. 然后进行中的变化,每3天。神经元样细胞后,应该会出现两至三个星期。

5。代表性的成果:

鼻人类植体outgrowing干细胞(图2A)除以迅速汇合,可一到两周内达成。一个关键的功能,干性,nestin表达,是评估(图2B)。聚- L -赖氨酸时用无血清培养与表皮生长因子(50毫微克/毫升)和FGF2(50毫微克/毫升)的培养基中生长,嗅觉干细胞引起的领域(图2c)。在含血清培养基中生长时,新镀领域引起GFAP的表达(〜50%),微管蛋白表达的细胞(约10-15%)和O4的表达细胞(约2-5%) 9(图2D - F)的。然而,球体源性细胞的命运可以被修改。例如,当一个Neurobasal文化与B27与谷氨酸,最鼻腔嗅觉干细胞向神经元样细胞表达的β- III微管蛋白(图2G)和微管相关蛋白2(图2H)培养基中生长。

图1
图1实验的总体方案。切除大鼠或人体鼻腔嗅粘膜活检。外植体本身可用于比较,目的是查明在脑部疾病的生物标志物的分子生物学研究。隔离嗅觉干细胞,固有层和神经上皮细胞之间的相互作用与dispase二酶被破坏,45分钟后,上皮细胞是用微锅铲删除。啮齿动物的嗅觉干细胞进一步游离胶原酶IA固有层选择。对人体组织,件嗅觉固有层玻璃盖玻片培养干细胞的侵入,直到outgrowing以及整个。扩散后,用适当的培养基,嗅觉干细胞可以生成领域,或分化成神经元样细胞。嗅觉干细胞可用于I)修复脑部疾病或外伤或ii)确定中枢神经系统疾病的分子标记。插图与施维雅医药艺术的帮助。

图2
图2,文化和人类鼻嗅干细胞的分化。越来越多的固有层植(一)人类干细胞迅速分裂,在含血清培养基培养。干细胞表达的干性标记巢(二)。当镀上聚- L -赖氨酸涂层的塑料,在无血清培养与EGF和FGF2培养基栽培,嗅觉干细胞生成领域(三)。球衍生的细胞,在含血清培养基镀时,会引起GFAP的表达细胞(〜50%),微管蛋白表达细胞(10-15%)和O4的表达细胞(约2-5%) 9(DF)的。在Neurobasal文化与B27与谷氨酸培养基生长时,它们分化成神经元样细胞表达的β- III微管蛋白(G)和微管相关蛋白2(H)。

Discussion

这里介绍的技术使鼠类和人类嗅粘膜一个有用的模型,为临床研究神经发育和神经退行性疾病以及修复病态或精神创伤的脑的工具的原因。该协议是相对简单的,并可以方便地进行由经验丰富的细胞生物学家。活检和文化技术的成功率是很高的。

关键步骤

  • 对于啮齿类动物的嗅黏膜集合,它是建议不超过10分钟之间安乐死和嗅觉组织的最后切除的时间限制。
  • 通常10分钟后取得的啮齿类动物嗅觉固有层的分离。孵化胶原酶IA。如果没有,我们建议收集上清固有层浮动的未解离位,其中,离心机,200克和机械分解replating细胞在新井之前,使用巴斯德吸管的浮动层后的一天。第一口井,包含已附着的细胞充满了新鲜培养液。
  • 为人类的固有层,这是比啮齿类动物组织更紧凑,我们不建议酶解。组织切片和每个外植体之间插入的塑料盘底部和玻璃盖玻片。成功的文化,包括外植体,其厚度范围从200至500微米。

可能的修改

  • 目前的协议,可以略作修改,以产生嗅觉神经元在体外。为此,神经上皮不拆除,整个嗅黏膜切片与McIlwain斩波器(厚度为200微米)。每个外植体,接种在培养皿中,部分干一小时,然后与含有FCS的培养基水化。在第一天电镀,上皮细胞和间质细胞生长出来的外植体。然后,神经祖细胞会迁移这个细胞层的顶部,并分化成神经元。
  • 嗅觉干细胞的分离纯化,可以实现用流式细胞仪检测。从表征纸11公布的名单,可检索的特异性表面标志。
  • 用于移植的实验,它可以使用老鼠的嗅觉干细胞GFP阳性菌株。这大鼠株(只Dawley,EGFP的PGK启动子驱动)ITERT(法国南特)。在人类嗅觉干细胞的绿色荧光蛋白基因插入,我们使用基于慢病毒感染的方法。
  • 本文的重点是嗅觉ecto间质干细胞。然而,另一种兴趣,嗅鞘细胞,细胞类型,可净化来自同一组织。我们描述了一个方法的收集和净化1,17,和我们人类的鼻鞘细胞用于I期/ IIa 18例截瘫患者的临床试验。
  • OE - MSCs的间质干细胞超家族的成员,有能力,适当的培养条件下,分化成脂肪细胞,骨细胞和细胞11。

未来的应用

  • 我们已经使用了人类嗅觉活检研究自闭症,躁郁症,家族dysautonomia,帕金森氏症,阿尔茨海默氏症和精神分裂症2-7患者的细胞和分子异常。从理论上说,所有的脑部疾病,可使用鼻腔活检或外围的嗅觉干细胞研究。
  • 老鼠和人类的鼻嗅干细胞已经嫁接失忆症,帕金森氏症,耳蜗损伤和脊髓损伤 12-16的动物模型。可以想象的是这些细胞移植到阿尔茨海默氏症,多发性硬化症,脑缺血的动物模型。

Disclosures

没有利益冲突的声明。

Acknowledgments

此项工作的财政支持ANR(新电国立德拉Recherche),AFM(协会法语反对LES肌病),PACA和IRME FEDER研究所DE RECHERCHE SUR LA Moelle épinière ET L' Encéphale我们衷心感谢玛丽皮埃尔布兰查德(让罗氏研究所),她在时间的推移录制过程中的有效帮助。

Materials

Name Company Catalog Number Comments
Collection of olfactory mucosa in rats
DMEM/HAM F12 Invitrogen 31331-028
Sodium Pentobarbital
Rongeur Fine Science Tools
26 gauge needle Terumo Medical Corp. NN-2613R
Forceps
Collection of olfactory mucosa in humans
Rigid endoscope Karl Storz or Richard Wolf Medical
Lidocaine
Epinephrine
Throughcut ethmoid forceps Karl Storz or Richard Wolf Medical
Isolation of olfactory stem cells
Dispase II Roche Group 10 295 825 001
Dissecting microscope
Micro spatula Fine Science Tools
Collagenase IA Sigma-Aldrich C9891
Ca-free/Mg-free PBS Invitrogen 14190-250
Fetal calf serum Invitrogen 10270098
Glass coverslip Knittel Glaser 001/35
Sphere formation and neuronal differentiation
Poly-L-lysine Sigma-Aldrich P-1274
Insulin transferrin selenium (ITS) Invitrogen 51500056
EGF R&D Systems 236-EG
FGF2 R&D Systems 233-FB
Neurobasal medium Invitrogen 21103-049
B-27 Serum-Free Supplement Invitrogen 17504-044
Penicillin/streptomycin Invitrogen 15140122
Glutamine Invitrogen 25030024
Glutamate Sigma-Aldrich

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References

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Comments

12 Comments

  1. Hi, firstly I`d like to thank you so much to share this valuable method. I followed your procedure with 8 week old mice and found difficulty with isolation of lamina propria from the epithelium so I skipped that step and seeded whole cells. Is it possible to obtain the stem cell population from mixed cells?

    Reply
    Posted by: Yoojin S.
    January 15, 2013 - 3:05 AM
  2. It's possible to skip the separation step (removal of olfactory epithelium). At the start, you'll get an unpurified culture but olfactory neurons and progenitors will die within the next three or four days. Some basal and supporting cells will remain in your culture but, after passaging, they will disappear and you'll end up with a culture of proliferative stem cells.

    Reply
    Posted by: francois f.
    January 16, 2013 - 4:07 AM
  3. Thank you again!
    I have some more questions about the morphology of ŒSC and subculture step.
    1. In the mixed (un-seperated) culture, I found various cell types and most of them are on the process of death but few cells seem to proliferate in compact manner. They look like pebbles. Are they ŒSCs?
    ². What reagent do I have to use for subculture? (i.e trypsin or accutase....)
    I greatly appreciate for your kindness. Thank you.

    Reply
    Posted by: Yoojin S.
    January 17, 2013 - 5:34 AM
  4. Answers:
    1. All mature neurons (the vast majority of cells within the epithelium) die shortly after plating. Three main cell types survive: ensheathing cells, stem cells (both cell types display an elongated morphology) and horizontal basal cells (they look like cobblestones). Horizontal basal cells will disappear after passaging and stem cells will overwhelm ensheathing cells.
    ². We passage the cells with the usual cocktail trypsin/EDTA (5 minutes at 37°C).

    Overall, human and rat olfactory stem cells are highly proliferative. Mouse olfactory stem cells are in small number and divide less rapidly.

    All the best.

    Reply
    Posted by: francois f.
    January 17, 2013 - 7:31 AM
  5. Thanks a lot. It Did Worked, I think. While I`ve been studying about Œ-MSCs, I`m wondering if these cells can produce OSNs themselves because in my knowledge, OSNs are derived from basal cells in Œ.

    Reply
    Posted by: Yoojin S.
    January 28, 2013 - 8:52 PM
  6. It's good to hear that you succeeded in culturing Œ-MSCs. For sure, you can differentiate these stem cells into neurons but I don't know if these neurons can express olfactory receptors. It's worth a test. Good luck.

    Reply
    Posted by: francois f.
    January 30, 2013 - 9:00 AM
  7. I have one more question. I have a hard time to make healthy spheres...you`ve mentioned that ' To generate stem cell spheres, incubate the flasks for two hours at 37°C with poly-L-lysine' . Could you tell me why we should use the PLL coating dish? In neural stem cell culture, I routinely use non-coating dish to make spheres ( because they will attached to PL coating dish and start to proliferate in attached single cell state).

    Reply
    Posted by: Yoojin S.
    March 28, 2013 - 8:42 PM
  8. The sphere formation process differs from neural stem cells. A single neural stem cell gives rise to a sphere. Instead,ŒMSCs, when plated at the right density on poly-l-lysine, aggregate and, within ²-7 days, form an islet of cells that will ultimately detach from the dish and float in the culture medium. To get spheres, you need to avoid proliferation (serum is eliminated from the culture medium) and use an FGF/EGF-containing culture medium.

    Reply
    Posted by: francois f.
    March 29, 2013 - 5:13 AM
  9. hello, in the last months I have tried to obtain a culture of stem cells from mouse ... but due to my limited experience with this type of tissue, I do not separate the epithelium from the lamina propria, so I decided to do passages... my question is how many passages are enough to have a homogeneous culture and healthy stem cells from mouse???

    Morphological differences exist between mouse or human culture to make passajes?

    Thanks ....

    Reply
    Posted by: IRERI F.
    June 4, 2013 - 2:40 PM
  10. Separating the lamina propria and the epithelium are not compulsory. At the start, you'll get a mixed population of cells but after one passage, epithelial cells will disappear rapidly.
    Overall, mouse cells do not proliferate as well as human cells. In order to keep your mouse stem cells growing, I recommend to keep a 50% level of confluency after passaging. If the cell density is too low, the proliferation rate will be minimal.
    Good luck with your experiments,
    Francois Feron

    Reply
    Posted by: francois f.
    June 5, 2013 - 4:28 AM
  11. Dear Dr. Ferón

    My name is Ireri Franco and I wrote to you before asking about the features of the isolation of Olfactory Stem Cells in mouse...these cells are nestin, sox2 and beta III tubulin inmunopositive, but I have some troubles with the differentiation because I can´t get MAP2 or NeuN inmunopositive cells (antibodies works well).

    Actually I ´m working with cells on passage 1 or 2 but I don´t make the sphere formation before the differentiation, it mean when the culture is confluent, I start with the differentiation protocol ( neurobasal, B271%, glutamine 2mM, glutamate 0.025mM and antibiotic- antimycotic) I change the medium every 3 days until tha 21 day.

    I really hope that you can help me because I don´t know if I´m doing something wrong.

    Thank you.

    PD. I hope that you can understand my poor english

    Reply
    Posted by: IRERI F.
    January 13, 2014 - 2:28 PM
  12. Dear François Féron,

    I have read your insightful protocol, and would like to replicate your procedure, possibly for younger rats. Is it possible to perform this isolation procedure on P0-P2 neonatal rats?

    Also, have you directed the differentiation of the olfactory stem cells into neurons that are bipolar and express markers characteristic of mature olfactory sensory neurons? Specifically, do some of the differentiated neurons express OMP? Have you been able to direct neuron differentiation towards the formation of olfactory sensory neurons?

    Thanks for sharing your expertise.

    ~Kind regards

    Reply
    Posted by: Angela D.
    September 27, 2018 - 9:26 AM

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