Name: generation of induced neural stem cells from peripheral mononuclear cells and differentiation toward dopaminergic neuron precursors for transplantation studies
Uploaded: 2019-07-11T14:00:00
Description: Watch this Scientific Journal Video about Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studie

该课题资助项目为:干细胞与翻译国家重点项目(2016YFA0101403)、国家自然科学基金(81661130160、81422014、81561138004)、北京市自然科学基金(5142005)、北京人才基金会(201700021223TD03),北京市"十三五"期间高层次人才资助项目(CIT&TCD20180333),北京医疗系统高层次人才奖(2015-3-063),北京市卫生局基金(PXM 2018_026283_000002)、北京市百千人才基金(2018A03)、北京市医院临床医学发展专项资金支持(ZYLX201706)和皇家学会-牛顿高级奖学金(NA150482)。

所有程序都必须遵守机构人类研究伦理委员会的指导方针。采血前必须征得患者或健康志愿者的知情同意。该协议由该机构的人类研究伦理委员会批准,并根据该机构的虐待和使用动物指南执行。
1. PBMC的收集、隔离和扩展
<ol><li>PBMC 的收集
	<ol>
<li>收集10-20 mL的供体的外周静脉血,用肝素钠防腐剂小瓶进行静脉穿刺。 注:血液样本应在室温(RT)下储存或运送。在24?...

<ol>
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在这里,我们提出了一个协议,涵盖了PD模型的iNSC-DA细胞治疗的不同阶段。该协议的关键方面包括:(1) PBMC 的隔离和扩展,以及通过 SeV 感染将 PBMC 重新编程到 iNSC;(2) 将 iNSC 分化为 DA 神经元,(3) 建立单方面 6-OHDA 病变 PD 小鼠模型和行为评估,以及(4)DA前体的细胞移植和行为评估。
在此协议中,第一部分涉及在无血清介质(MNC介质)中收集和扩展PBMC,该介质优先扩展红细胞,不支持淋巴细胞?...

帕金森病 (PD) 是一种常见的神经退行性疾病,由心内脑素 (VM) 的底状尼格拉帕压实 (SNpc) 的多巴胺能 (DA) 神经元退化引起,60 岁以上人群的患病率超过 1%1,2.在过去十年中,细胞疗法,旨在取代退行性或受损的细胞,或滋养退化神经元周围的微环境,在PD3的治疗中显示出潜力。同时,重新编程技术也取得了显著进展,为替代疗法提供了有前途的细胞源。人类诱导多能干细胞(iPSCs)和胚胎干细胞(ESCs)已被证明能够分化成DA神经细胞,当移植到大鼠和非人类灵长类动物PD模型中时,这些神经细胞可以存活、成熟,并改善运动功能5 ,6,7,8.iPSC是细胞再编程技术的一个里程碑,在细胞移植方面有着巨大的潜力;然而,人们仍然担心肿瘤从不完全分化的细胞形成的风险。细胞移植的替代细胞来源是通过直接重新编程获得的系世型成人干细胞,如诱导神经干细胞(icCS),可以从不稳定的中间体中提取,绕过多能性阶段9,10,11.
iPSC 和 iNSCs 都可以从自体细胞源(如成纤维细胞、外周血单核细胞 (PBMC)和各种其他类型的细胞12、13、14)重新编程,从而减少移植细胞的免疫原性在很大程度上。此外,与iPSCs相比,iNSCs是固有的,肿瘤形成和系骨的可塑性风险降低,只能分化成神经元和胶质11。在最初的研究中,人类或小鼠iPSC和iNSCs是由从皮肤活检中获得的成纤维细胞产生的,这是一个侵入性程序14,15。在这方面,PBMNCs是一个有吸引力的起始细胞源,因为侵入性较小,并且易于在短时间内获得大量的细胞扩展时间16。初始重新编程研究采用综合输送系统,如慢病毒或抗逆转录病毒载体,这些系统在多种细胞中高效且易于实施17;然而,这些传递系统可能导致突变和残留转基因的重新激活,这提出了临床治疗目的的安全问题12。仙台病毒(SeV)是一种非整合性RNA病毒,具有负感的单链基因组,不集成到宿主基因组中,但只在受感染细胞的细胞质中复制,为重新编程提供了高效和安全的工具 ,19.可组合SeV向量,包含重新编程的因素,包括人类OCT3/4,SOX2,KLF4和c-MYC在其开放阅读帧。此外,通过引入温度敏感突变,可以进一步改善SeV病毒载体,以便在培养温度升高至39°C20时迅速去除。在本文中,我们将介绍一种使用 SeV 系统将 PBMC 重新编程到 iNSC 的协议。
许多研究已经报告从人类ESCs或iPSC衍生的DA神经元使用各种方法6,8,21。然而,在细节上描述DA神经元与iNSCs分化的协议很缺乏。在此协议中,我们将使用两阶段方法描述从 iNSC 高效生成 DA 神经元。DA神经元前体可移植到PD小鼠模型的纹状体中,用于安全性和有效性评估。本文将介绍一个详细的方案,涵盖从仙台病毒生成诱导神经干细胞,将iNSCs分化为DA神经元,建立小鼠PD模型,到将DA前体移植到纹状体的各个阶段PD 模型。使用该协议,人们可以从患者和健康捐赠者生成iNSC,并得出安全、标准化、可扩展和均匀的DA神经元,用于细胞移植目的,或用于在培养皿中建模PD和研究机制潜在的疾病开始和发展。

<table border="0" cellpadding="0" cellspacing="0" style="width:828px;" width="827">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">15-ml conical tube</td>
			<td style="width:183px;">Corning</td>
			<td style="width:153px;">430052</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">1-Thioglycerol</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">M6145</td>
			<td>Toxic for inhalation and skin contact</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">24-well plate</td>
			<td style="width:183px;">Corning</td>
			<td style="width:153px;">3337</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">50-ml conical tube&#160;</td>
			<td style="width:183px;">Corning</td>
			<td style="width:153px;">430828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">6-OHDA</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">H4381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">6-well plate</td>
			<td style="width:183px;">Corning</td>
			<td style="width:153px;">3516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Accutase</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">A11105-01</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Apomorphine</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">A4393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Ascorbic acid</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">A92902</td>
			<td>Toxic with skin contact&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">B27 supplement&#160;</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">BDNF</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">450-02</td>
			<td>Brain derived neurotrophic factor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Blood collection tubes containing sodium heparin</td>
			<td style="width:183px;">BD</td>
			<td style="width:153px;">367871</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">BSA</td>
			<td style="width:183px;">yisheng</td>
			<td style="width:153px;">36106es60</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">cAMP</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">D0627</td>
			<td>Dibutyryladenosine cyclic monophosphate</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:277px;">CellBanker 2</td>
			<td style="width:183px;">ZENOAQ</td>
			<td style="width:153px;">100ml</td>
			<td>Used as freezing medium for PBMNCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Chemically defined lipid concentrate</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">11905031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">CHIR99021</td>
			<td style="width:183px;">Gene Operation</td>
			<td style="width:153px;">04-0004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Coverslip</td>
			<td style="width:183px;">Fisher</td>
			<td style="width:153px;">25*25-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">DAPI</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">D8417-10mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">DAPT</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">D5942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Dexamethasone</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">D2915-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">DMEM-F12</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">11330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">DMEM-F12</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">11320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Donkey serum</td>
			<td style="width:183px;">Jackson</td>
			<td style="width:153px;">017-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">EPO</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">100-64-50UG</td>
			<td>Human Erythropoietin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">FGF8b</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">100-25</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Ficoll-Paque Premium</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:153px;">17-5442-02</td>
			<td>P=1.077, density gradient medium</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:277px;">GDNF</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">450-10</td>
			<td>Glial derived neurotrophic factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">GlutaMAX</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">21051024</td>
			<td>100 &#215; Glutamine stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Ham&#39;s-F12</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">11765-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">HBSS</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">14175079</td>
			<td>Balanced salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Human leukemia inhibitory factor</td>
			<td style="width:183px;">Millpore</td>
			<td style="width:153px;">LIF1010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Human recombinant SCF</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">300-07-100UG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">IGF-1</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">100-11-100UG</td>
			<td>Human insulin-like growth factor&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">IL-3</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">200-03-10UG</td>
			<td>Human interleukin 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">IMDM</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">215056-023</td>
			<td>Iscove&#39;s modified Dulbecco&#39;s medium</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Insulin</td>
			<td style="width:183px;">Roche&#160;</td>
			<td style="width:153px;">12585014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">ITS-X</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">51500-056</td>
			<td>Insulin-transferrin-selenium-X supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Knockout serum replacement</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">10828028</td>
			<td>Serum free basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Laminin</td>
			<td style="width:183px;">Roche&#160;</td>
			<td style="width:153px;">11243217001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Microsyringe</td>
			<td style="width:183px;">Hamilton</td>
			<td style="width:153px;">7653-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">N2 supplement&#160;</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">NEAA</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">11140050</td>
			<td>Non-essential amino acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Neurobasal</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">10888</td>
			<td>Basic medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">PDL</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">P7280</td>
			<td>Poly-D-lysine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">SAG1</td>
			<td style="width:183px;">Enzo</td>
			<td style="width:153px;">ALX-270-426-M01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">SB431542</td>
			<td style="width:183px;">Gene Operation</td>
			<td style="width:153px;">04-0010-10mg</td>
			<td>Store from light at -20&#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Sendai virus</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:153px;">MAN0009378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Sucrose</td>
			<td style="width:183px;">baiaoshengke</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">TGF&#946;&#8546;</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">100-36E</td>
			<td>Transforming growth factor&#160; &#946;&#8546;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Transferrin</td>
			<td style="width:183px;">R&#38;D Systems</td>
			<td style="width:153px;">2914-HT-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Triton X 100</td>
			<td style="width:183px;">baiaoshengke</td>
			<td style="width:153px;"></td>
			<td>Nonionic surfactant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Trypan blue</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Xylazine</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">X1126</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced neural stem cells from peripheral mononuclear cells and differentiation toward dopaminergic neuron precursors for transplantation studies

帕金森病 (PD) 是由心内脑素 (VM) 中基拉多巴胺能 (DA) 神经元的退化引起的。细胞替代疗法对PD的治疗大有希望。 最近,诱导神经干细胞(iNSCs)由于肿瘤形成的风险降低和可塑性增加而成为细胞替代治疗的潜在候选者。区域特定的神经元和胶质。iNSCs可以从自体体细胞源(如成纤维细胞、外周血单核细胞(PBMC)和各种其他类型的细胞)重新编程。与其他类型的体细胞相比,PBMC是一个有吸引力的起始细胞类型,因为易于访问和扩展的培养。仙台病毒(SeV),一种RNA非整合性病毒,编码重新编程的因素,包括人类OCT3/4,SOX2,KLF4和c-MYC,具有负感,单链,非分段基因组,不集成到宿主基因组,但仅在受感染细胞的细胞质中复制,为重新编程提供了高效和安全的工具。在本研究中,我们描述了一种协议,其中通过重新编程PBMC获得iNSC,并通过两阶段方法分化成专门的VM DA神经元。然后DA前体被移植到单方面的6-羟基多巴胺(6-OHDA)病变PD小鼠模型中,以评估PD治疗的安全性和有效性。该方法为研究患者特异性DA神经细胞在体外和体内的功能和治疗效果提供了一个平台。

在这里,我们报告一个协议,涵盖iNSC-DA细胞治疗的不同阶段,以治疗PD模型。首先,对PBMC进行分离和扩展,并通过SeV感染重新编程为iNSC。图1显示了使用PBMNC扩展和iNSC感应的过程的示意图。在-14日,使用密度梯度介质(材料表)对PBMC进行隔离。离心前,用PBS稀释的血液和密度梯度介质被分成两层。离心后,出现四个梯度层(从下到上):底层包含粒细胞和红细胞;第二层包含密度?...

Watch this Scientific Journal Video about Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studie

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

该协议提出了外周血单核细胞的重新编程,以诱导神经干细胞由仙台病毒感染,将iNSCs分化为多巴胺能神经元,将DA前体移植到单方面病变帕金森病小鼠模型,并评价iNSC衍生DA前体用于PD治疗的安全性和有效性。

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

该协议描述了一种将血液和单核细胞重新编程为神经干细胞的方法，可用于治疗神经退行性疾病，如帕金森病。它提供了一个自体细胞源来治疗神经退行性疾病。此外，转换到诱导神经干细胞和分化到所需的细胞类型所需的时间框架比诱导多能干细胞短。诱导神经干细胞具有良好的增殖能力，可在应用前进行增殖。由于 iNSC 可以指定到不同的区域特定的神经细胞中，如脊髓神经元或运动神经元，因此此方法可以扩展用于治疗其他神经系统疾病。整个过程需要很长时间，所有步骤都相关且关键，因此视觉演示提供了有关细胞外观的想法，并提供了一些提示，以确保成功生成所需的细胞。首先，将先前分离的外周静脉血转移到50毫升的圆锥管中，用同等体积的无菌Dulbecco的PBS稀释。在另一个50毫升锥形管中，准备15毫升的灭菌密度梯度介质。以 45 度角倾斜管，缓慢而小心地将 30 毫升稀释的外周血放在密度梯度介质上。在室温下以 800 倍 g 离心管 15 分钟，离心机制动器设置在关闭位置。吸气黄色，上等离子层，并丢弃它。使用 10 毫升移液器将含有单核细胞的白色多云薄膜层转移到新的 50 毫升锥形管中。使用 MNCs 将 30 毫升 D-PBS 添加到管中，以 600 倍 g 离心，在 4 摄氏度下 10 分钟。丢弃上经剂后，加入45毫升D-PBS，然后重新暂停细胞。在4摄氏度下以400倍g离心管10分钟后，丢弃上经剂。用五毫升D-PBS重新暂停细胞，用 trypan 蓝色排除法计算活细胞，留出扩展所需的跨国公司，并冻结剩余的细胞供将来使用。在零下14天，种子跨国公司的密度为每毫升200万至300万细胞，每井6井板与1.5毫升37摄氏度，预热的跨国公司介质。在37摄氏度，5%的二氧化碳孵育两天。在负11日，使用灭菌移液器收集细胞与介质，并转移到一个新的15毫升锥形管。在室温下以250倍g离心5分钟，丢弃上先代，将细胞重新在预加热的 MNC 介质中重新暂停。在用锥泛蓝计算可行细胞后，在六井板中以每毫升100万个细胞的密度播种。MNC在预加热的跨国公司介质中播种。在37摄氏度下孵育，三天孵育5%的二氧化碳，并在天减8天和负4天在MNC介质中重复细胞收集、离心和电镀。此程序中的仙台病毒是危险的，所有涉及仙台病毒的程序必须在安全柜中执行。所有接触仙台病毒的尖端和管子在处置前都应使用乙醇或漂白剂进行处理。在第零天，收集 MNC 介质中的细胞并转移到 15 毫升锥形管中。在200次g中离心5分钟后，吸升液，用一毫升预加热的MNC介质重新向细胞增殖。用锥盘蓝色计算活细胞，然后用预热的跨国公司介质重新暂停，在24孔板中每井20万个细胞的浓度。将含有仙台病毒的零下80摄氏度储存的管子从含有仙台病毒的储存中解冻5至10秒，然后在室温下解冻。解冻后，立即将它们放在冰上。将解冻的仙台病毒加入24孔板的井中，与跨国公司的感染率为10。为了便于细胞的附着，将1000次g的板离心30分钟，然后将板在37摄氏度、5%的二氧化碳下放在培养箱中。第一天，将带介质的细胞转移到15毫升的离心管。要进一步分离剩余的细胞，每井用一毫升的跨国公司介质冲洗井，并将带介质的细胞加入管中。在200次g中离心5分钟后，吸升液，用500微升新鲜预热的MNC介质重新悬浮细胞，并加入24井板的井。在第二天，在D-PBS中，使用以前稀释过的每微升50微克每微升50微克的一毫升每孔，在室温下将六孔板涂覆至少两个小时。从六孔板中吸出聚 D-lysine，在垂直清洁的长凳上干燥约 10 分钟。然后将以前稀释的每毫升层压素的5微克每孔1毫升加入到六孔板中，并在37摄氏度下孵育4至6小时。使用前用 D-PBS 清洗板。在第三天，在多D-赖氨酸/层压涂层六孔板上，所有仙台病毒转导MNCS在诱导神经干细胞培养基上。第5天和第7天，轻轻加入一毫升37摄氏度，预热的 iNSC 介质到六井板的每个井。从第9天到第28天，每天用新鲜的预热的 iNSC 介质更换介质。监测 iNSC 菌落的出现，并在大约两到三周内使用燃烧的玻璃移液器选择具有适当形态的菌落，并转移每个菌落到六井板的单独井中进行扩张。要建立单侧6-红霉素-多胺-病变小鼠模型，剃光麻醉，成人，雄性，SCID米色小鼠的头，并应用红霉素眼软膏。将鼠标放在立体仪器上，用切口棒固定鼠标。正确插入耳罩，以牢固和适当地定位鼠标头。用碘和异丙醇对头部进行灭菌，然后用手术刀刀片在头部皮肤上进行约1.5厘米的下垂切口，露出头骨。调整切口杆和耳杆，将啤酒和羊皮的高度差减至小于 0.1 毫米。缓慢地移动和降低微丝针的尖端朝向啤酒，并对待啤酒作为零点。将尖端移动到坐标为前 0.5 毫米且相对于啤酒的横向 2.1 毫米的位置。缩回尖端，用记号笔标记点，用电子钻头在头骨上打一个小洞。将每毫升5微克的两微升6-催眠胺溶液提取到微西林格中。将针头返回标记点，并将针头插入到后腹 3.2 毫米。以每分钟一微升的速度从注射器中注射6-hyroxydopamine溶液，将注射针留在位上再放置5分钟，然后慢慢收回。用缝合线闭合切口后，再次在眼睛上涂抹红霉素眼膏。将鼠标从立体仪器中取下，放入回收笼中，并允许获得食物和水，直到它恢复知觉。PBMNC感染SeV后，它们的形态发生变化，细胞在第五天之前经历了剧烈的死亡。INSC殖民地出现在第12天。在挑选和转移 iNSC 克隆以扩展多个段落后，它们表现出良好的形态，可以在 iNSC 介质中稳定地自我更新，无论是单层形式还是球体。经过24天的分化，iNSCs被确定为多巴胺神经元，其中大多数表示酪氨酸羟基酶，叉头盒A2，和神经元特异性III类β-图布林标记。在建立了单方6-甲状腺素-多巴胺-帕金森病小鼠模型后，进行了行为评估，以估计帕金森病的症状。接受细胞移植的小鼠的运动功能明显改善。通过骨质肉、中前脑束和亚斯坦蒂尼格拉帕西坦的TH死后免疫荧光染色，验证了6-甲状腺多胺诱发病变的程度。TH阳性信号在植入细胞的线状体中恢复极大，在SN也轻微恢复。移植三个月后，在存活的细胞中，约13.84%为TH阳性多巴胺能神经元。约91.72%的TH阳性细胞表示孤立的核受体，约86.76%表示FOXA2，约98.77%与G蛋白耦合向式整流器钾共同标记。将30密耳稀释的外周血铺设到密度梯度介质上时，请缓慢小心地进行，不要干扰梯度界面。应注意避免仙台病毒的反复解冻和冻结，以确保病毒活性高。当重新编程外周血单核细胞诱导神经干细胞时，请每天密切观察细胞的形态。此外，6-羟基多巴胺对光和温度敏感。小心保护溶液免受光线的利用，并在使用前将溶液放在冰上。按照这个程序，一个人可以继续研究疾病机制使用家庭PD患者衍生多巴胺神经元培养。人们还可以继续研究移植的细胞如何影响受损的神经元回路。

Research

JoVE Journal

Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

从人外周T细胞无饲养条件下，使用仙台病毒诱导多能干细胞的生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

犬卵巢间充质干细胞的提取与分化

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...