The authors have no conflicts of interest to disclose.

富集造血祖1.从成人全血细胞注:造血祖细胞或CD34 +细胞可以从各种来源，包括脐带血，白细胞分离材料和全血使用密度梯度离心为基础的方法衍生的外周血单核细胞（PBMC）进行纯化。这里列出的方法使用全血作为例子。 <ol><li>孤立于全血的PBMC: <ol><li>添加4.5毫升淋巴细胞分离介质向SepMate管通过在刀片的中心孔吹打它。 </li><li>稀释血液样品与含有2％人血清的无菌DPBS等体积（体积/体积）。例如，稀5毫升样品用5ml DPBS + 1％的人血清。 </li><li>吹打下来管的侧面添加的稀释试样。小心不要直接通过中心孔倒出样品。 </li><li>离心在1200 xg离心在室温10分钟。 </li><li>小心地取出PMBC层以上的上清液。 </li><li>收集的PBMC层（混浊）到一个新的试管中。 </li><li>添加含有2％人血清入管和离心机15毫升Dulbecco氏磷酸盐缓冲盐水（DPBS）中，在150×g离心在室温10分钟的制动切断。 </li><li>弃上清，悬浮颗粒在15毫升含有2％人血清的DPBS。计数细胞的数目。 </li><li>离心在690×g离心在室温10分钟。除去从管的所有上清液。 </li><li>重悬的细胞以每15ml中Iscove氏改性Dulbecco氏培养基（IMDM）含1％抗生素（体积/体积）和10％人血清1×10 7个细胞（体积/体积）。 </li><li>分离的CD34 +细胞之前孵育细胞O / N在37℃在5％CO 2培养箱中培养。 </li></ol></li><li> CD34 +细胞的使用的CD34 MultiSort套件的PBMC阳性选择: 注:工作速度快，使用预先冷却解决方案，使细胞感冒，防止封盖的细胞表面上的和非特异性的细胞标记每试剂盒的指令的抗体。 <ol><li>收集在50毫升管中的所有PMBCs和计数细胞总数中的介质的数量。 </li><li>离心细胞，在300×g离心10分钟。彻底清除所有上清。 </li><li>悬浮高达每300微升10 8个细胞在珠缓冲液（DPBS含有0.5％人血清（体积/体积）和2mM EDTA）中。 </li><li>加入100微升Fc受体阻断剂，并加入100微升CD34 MultiSortMicroBeads的。 </li><li>拌匀，孵育在冰箱30分钟（2 - 8°C）。 </li><li>加入2毫升每10 8个细胞珠缓冲液和离心机在300×g离心在RT 10分钟洗涤细胞。彻底去除上清。 </li><li>悬浮高达10 8个细胞在500μl缓冲液中。 </li><li>放置MACS LS柱在MACS分离器的磁场和通过用3ml缓冲液珠漂洗制备柱。 </li><li>应用细胞悬液到CENTEr中柱。未标记的细胞将流经可如果愿意被收集。 </li><li>洗涤塔三次用3ml缓冲液珠。 </li><li>从分离器中取出的列并将其放置在一个15毫升的管中。 </li><li>加入5毫升珠缓冲到柱上，并立即冲洗出磁性标记的细胞通过推动柱塞入列。 </li><li>加入20微升的MultiSort释放试剂每1ml细胞悬液。拌匀孵育在冰箱10分钟（2 - 8℃）。 </li><li>加入1 - 2毫升每10 7个细胞珠缓冲液和离心将细胞在300×g离心在室温10分钟。彻底去除上清。 </li><li>重悬的细胞在50μl每10 7个细胞珠缓冲器。 </li><li>添加30微升MultiSort停止试剂的每10 7个细胞，并孵育在冰箱15分钟。 </li><li>加入5毫升珠缓冲液和离心将细胞在300×g离心在室温10分钟。弃去上清液。 </li></ol></li></ol>从CD34 +细胞2.推导诱导神经干细胞<ol><li> CD34 +细胞的培养: 注:新鲜制备介质应在7天的时候贮存在冰箱中。 24小时后，可以观察到显著CD34 +细胞的损失，但细胞的增殖显著应该引人注目，特别是在5天如果细胞数连续降低或不存在的增殖，它意味着介质或CD34的细​​胞的任一质量差，并将细胞不应该被用于下一步骤。 <ol><li>重悬并培养的CD34 +细胞中CD34的维持培养基（StemSpan SFEM介质含有人血小板生成素（TPO，100纳克/毫升），fms样酪氨酸激酶3（FLT-3）的配体（100毫微克/毫升）和干细胞因子（SCF， 100毫微克/毫升），白介素-6（IL-6，20纳克/毫升）和白细胞介素-7（IL-7，20纳克/毫升））在一个24孔板（高达3×10 5个/孔在1毫升在5％CO 2培养箱中，每孔培养基），在37℃的。 </li><li>自动对焦之三24小时，收集漂浮细胞和离心将细胞在110×g离心在RT除掉任何附着的细胞和细胞碎片。弃去上清液，并在37℃下在5％CO 2培养箱中重悬细胞中加入1ml新鲜的CD34维持培养基和种子在一个新的24孔板（高达1×10 5个/孔），用于附加的5天。 </li><li>通过仔细吸上清的上半部分，而CD34 +细胞都浮于孔中（ 图1A）的底部替换一半的每个培养基中的其他日子。 </li></ol></li><li>仙台病毒转染: <ol><li>第5天，收集CD34细胞和离心细胞在170 XG在室温下10分钟。弃去上清液。 </li><li>重悬在1×10 5个/孔在24孔板在1ml培养基中的细胞在新鲜培养基中。 </li><li>通过首先浸渍管的底部在37℃水浴中，持续10秒解冻cytotune-的iP​​S重编程仙台试剂盒，然后完全解冻吨RT。短暂离心管中，并把它们放在冰上。通过混合的分析证书（COA）的相应大量列出的每个病毒的推荐量准备仙台病毒混合。 15 MOI建议。 </li><li>添加仙台病毒组合，以CD34的细​​胞的每个孔中。轻轻摇晃混合井。孵育所述细胞在37℃在5％CO 2培养箱中培养。 </li><li> 24小时后观察转染效率。注意，细胞聚集和球形成是指标成功转染（ 图1B）。 </li><li>通过仔细转移介质的上半部分到另一个井改变一半的培养基。如果有，在新井细胞球，加入新鲜培养基的量相同。 </li><li>重复步骤2.2.6改变中的每一天。 </li></ol></li><li>神经干细胞诱导: 注:根据电池的条件下，转染后约5天或7天，单层贴壁细胞将达到30 -50％汇合（ 图1C）。 <ol><li>除去含有漂浮细胞和球体上清液，然后加入1 ml的神经祖培养基（Dulbecco氏改良的Eagle培养基:营养混合物F-12（DMEM / F12），含有1×N2添加剂，0.1％（重量/体积）牛血清白蛋白（ BSA），1％（V / V）的抗生素，碱性成纤维细胞生长因子20纳克/毫升（bFGF）的，和表皮生长因子（EGF））20纳克/毫升到贴壁细胞。 </li><li>任选地，轻轻吹打分离的细胞球和离心将细胞在170×g离心10分钟。除去上清液和重悬细胞于1ml神经祖介质的在一个新的24孔板涂覆有每指令基质胶作为备份。 </li><li>改变介质隔日直到细胞达到60％ - 80％汇合，一般1周后。 </li><li>弃去上清液，并添加1毫升的神经干细胞培养基（无血清培养基，NSC SFM）的。通过使用细胞刮随后解离的细胞通过非常轻柔吹打。 </li><li>除去细胞和种子个个到6孔板的一个孔中。添加另一个1毫升神经干细胞培养基中，以使2毫升介质每个孔中。孵育所述细胞在37℃在5％CO 2培养箱中培养。 </li><li>改变中的每一天。 </li><li>当细胞达到60％汇合时，解离和replate的细胞以1:3的比例在以下2.3.4和2.3.6的步骤。 </li></ol></li><li>冷冻神经干细胞的: <ol><li>通过加入20％二甲亚砜（DMSO）成神经干细胞培养基中制备的神经干细胞冷冻培养基。置于冰上冻结中。 </li><li>解离的细胞在6孔板中，用细胞刮棒，随后轻轻吹打。计数细胞。离心细胞，在170×g离心在室温10分钟。弃去上清液。 </li><li>重悬的细胞在神经干细胞培养基中以1×10 6个 / ml。加冷冻培养基与细胞的相同体积。 </li><li>加入1毫升（5×1细胞在离心管的0 5个细胞）。冻结使用Froster先生冷冻集装箱继指令的单元。 </li></ol></li></ol> 3.神经细胞分化<ol><li>神经细胞分化: <ol><li>当神经干细胞达到80％汇合时，用一个橡皮分离的神经干细胞，然后轻轻吹打分离。 </li><li>计数细胞和神经干细胞培养基中，调至浓度为1×10 5个 / ml。 </li><li>对神经元的分化，板在聚-D-赖氨酸/层粘连蛋白包被盖的细胞以1×10 5个/孔滑倒在24孔板中。孵育所述细胞在37℃在5％CO 2培养箱中培养。 </li><li>与神经分化培养基替换培养基（DMEM衍生含有1x N2添加剂，1X B27添加剂，300纳克/毫升的cAMP，0.2mM的维生素C，10纳克/毫升的脑源性神经营养因子（BDNF）和10ng / ml的神经胶质细胞/ F12神经营养因子（GDNF））AF器24小时。孵育所述细胞在37℃在5％CO 2培养箱中培养。 </li><li>一个星期2星期改变神经分化培养基两次。 </li></ol></li><li>星形胶质细胞分化: <ol><li>通过移液分离的神经干细胞。 </li><li>计数细胞并调节细胞浓度至5×10 4个细胞/ ml。种子上24孔板的细胞。孵育所述细胞在37℃在5％CO 2培养箱中培养。 </li><li>更换培养基的DMEM / F12，用10％胎牛血清（FBS）和在5％CO 2培养箱孵育所述细胞在37℃。 </li><li>每周更换培养基两次持续2周。如果细胞前2周达到汇合，replate以下步骤3.2.1和3.2.2的细胞。 </li></ol></li><li>少突胶质细胞分化: <ol><li>通过移液分离的神经干细胞。 </li><li>计数细胞和种子1×10 5个细胞在聚L-鸟氨酸涂布的24孔板在1ml少突胶质differenti的ATION介质包括DMEM / F12的含N2添加剂，血小板衍生生长因子-AA（PDGF-AA）为10纳克/毫升，对神经营养因子-3（NT-3）2纳克/毫升，音猬蛋白2纳克/毫升（ SHH），以及为3nM的甲状腺素（T 3）。孵育所述细胞在37℃在5％CO 2培养箱中培养。 </li><li>每周更换培养基两次持续2周。 </li><li>替换用DMEM / F12与1xN2补充和T3为3nM和培养所述细胞额外周髓鞘碱性蛋白（MBP）的生产培养基中。 </li></ol></li></ol> 4.免疫荧光染色<ol><li>用4％多聚甲醛（PFA）替换培养基。孵育在室温10分钟。 </li><li>丢弃的PFA和洗涤用PBS含有0.1％的Triton X-100（PBS-T）3次，每次5分钟。 </li><li>通透细胞用PBS含有0.5％的Triton X-100在室温下10分钟。 </li><li>阻断细胞用PBS-T含有4％山羊血清20分钟，在室温。 </li><li>孵育细胞与相应的主antibo模具稀释在含有0.1％BSA的2小时，在室温或O / N在冷室中的PBS-T。 </li><li> 3次用PBS-T，每次5分钟洗涤细胞。孵育细胞与相应加上1用荧光染料的二抗:400稀释在含有0.1％BSA进行1小时在黑暗中的摇床PBS-T。 </li><li>洗涤细胞用含有4&#39;，6-二脒基-2-苯基吲哚（DAPI）在室温10分钟的PBS-T。 </li><li>用PBS-T，每次5分钟，在RT下洗涤细胞两次。 </li><li>观察用显微镜标记的细胞并拍摄照片（ 图3）。 </li></ol>

<ol>
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The study is funded by NIH intramural funds.

提供了一种详细的协议，以直接产生从外周造血祖细胞的神经干细胞。 相比成纤维细胞，人外周血是更容易获得。使用所提出的协议，大于1×10 5个造血祖细胞，或CD34阳性细胞中，可以在10毫升的全血被富集。虽然污染血小板通常不是可预防的，它可以容易地通过低速离心降低，它不与神经干细胞的产生干扰。然而，CD34 +细胞的质量和数量将决定神经干细胞的最终生产。 CD34 +细胞的质量可以通过其响应于CD34的培养基中大体判断。一个积极的回应，这意味着CD34细胞的快速增殖培养基，是为后来成功转型的关键。如果CD34 +细胞未能增殖，甚至经受显著细胞损失，则TRANSFO细则第十五将变得困难，从而导致更少的增殖性细胞。因此，建议只开始转型以优良的品质CD34细胞。虽然1×10 5个CD34 +细胞是足够用于一个有经验的研究人员，也可使用对于初学者3×10 5个细胞。 使用仙台病毒提供了一种非融合，引入转录因子进入靶向细胞14，15高效和方便的方式。本来用于产生的iPSC，山中的因素也被成功地用于直接从成纤维细胞11产生神经干细胞。在使用所提出的协议的先前报告，也可以使用含有山因子的仙台病毒，以产生从脐带血或成人外周血造血血细胞的神经干细胞。利用皮肤活检培养成纤维细胞相比，血液更容易，方便获得。此外，据报道，造血祖细胞也表达某些神经特异性标志物16，使其产生神经干细胞是更好的选择。事实上，仙台病毒感染后，大部分附着单层细胞表达巢蛋白，神经干细胞标记物，除了更凝集紧凑菌落，其中的一些可能会引起的iPSC在稍后的时间点。从而细胞的附着单层被收集并进一步培养在神经祖细胞培养基，它已被广泛应用在初级神经祖细胞培养，以促进细胞的神经干细胞的命运。 有在维持神经干细胞的微妙平衡。一方面，优选的是该细胞具有更好的增殖能力，将传代多次越好。另一方面，便于它们分化为神经元的也很关键。两个不同的媒体被用来实现这种平衡。目前广泛使用的神经祖介质被选择的n个eural干细胞诱导标识/选择，这是摆在首位的关键过程。它也可以用来在后来的通道，以加强神经元分化的能力。然而，神经干细胞是非常脆弱和敏感，使得长期暴露在神经祖细胞培养基可大大抑制细胞增殖，并导致过量的分化。因而神经干细胞培养基是用来帮助细胞恢复，并促进细胞的扩增。所述神经祖介质用于神经诱导在通道1中，当细胞汇合。如果过度抑制细胞增殖和毒性注意到，有必要用神经干细胞培养基中早期更换介质。然而，神经干细胞的培养基是不太有效的神经选择;因此随着时间的推移，尤其是与过度汇合的神经干细胞或延迟传代，污染非神经细胞或缺乏神经分化的可能发生。因此，信元需要定期传代，当小于60％汇合，并且每周至少一次。神经选择与神经祖细胞培养基有可能在以后的段落重新建立神经的身份。影响的神经干细胞的干性的另一个因素是在组织培养板的涂层。涂层与基质胶或聚-D-赖氨酸可以帮助细胞附着在第一情况下，细胞是难以replating期间附着。但长期暴露于涂层导致较少的通路号码。这是因为该涂层增强了细胞的分化过程，并且可能导致解离过程中对细胞的伤害。 作为一个总结，本协议使用含有山因素直接3内获得人神经干细胞的仙台病毒 - 从访问的外周血样品获得成年造血祖细胞4周。的神经干细胞可进一步分化为神经元和神经胶质细胞。这种方法绕过目前使用的冗长和复杂的iPSC产生过程，并且可以被用于在体外细胞培养模型关于各种神经病学病症，其可以更好地代表在特定疾病的遗传差异，尤其是来自个体患者的样品。

在体外的神经元培养物已被用作用于神经性疾病的研究中的基本工具。主动物（大多数啮齿类）神经培养1,2和从胶质瘤或其他肿瘤衍生的人神经细胞系是最常用的此类研究。但是，人们已经认识到，有啮齿动物和人类细胞间显著差异。基于啮齿动物的许多研究结果不能被转换为人类。此外，在分析海量基因组信息和比较容易的基因编辑和全基因组测序的迅速发展，这种趋势越来越侧重发现疾病容易发生基因和划分职能作用的特定疾病，这使得一些人的神经细胞系只有有限的用法。从理论上说，从病人的神经系统的样品得出人类的主要神经文化是最好的选择，但他们都无法获得;因此替代甲基ODS是必要的。近年来，一些方法已被追求的，有两个是最区分。下列产生诱导性多能干细胞（IPSC），使用小鼠和人类体细胞的技术的发展3，4，神经细胞可进一步分化，从他们5-7。然而，产生和表征IPSC的要求密集型劳动，技术和时间的输入，有时甚至令人望而却步。不久之后，另一种方法是开发可直接由体细胞8,9变换神经元细胞。作为所得到的神经元都是非增殖性，它限制了它在深入细致的研究和药物筛选，这需要大量的细胞的应用。取这两种技术，神经干的直接推导的优点/由体细胞祖细胞已探索由几组10-12，它绕过的iPSC产生和表征，但仍PROVI的繁琐过程DES神经干细胞后神经分化的一个体面的数量。我们以前曾表明，在采用新的山中转录因子的成造血祖细胞，神经干细胞可以直接使用神经祖细胞在选择培养基中13产生。这里，我们报告了详细的方法。

<table><tbody><tr><td>Lymphocyte separation medium</td><td>Lonza</td><td>17-829E</td><td>1x</td></tr><tr><td>SepMate</td><td>Stemcell Technologies</td><td>15415</td><td>15 ml</td></tr><tr><td>Human serum</td><td>Invitrogen</td><td>34005-100</td><td></td></tr><tr><td>Antibiotics</td><td>Gibco</td><td>15240-062</td><td>1%</td></tr><tr><td>CD34 MultiSort Kit</td><td>Miltenyi Biotec</td><td>130-056-701</td><td></td></tr><tr><td>EDTA</td><td>Cellgro</td><td>46-034-Cl</td><td>2 mM</td></tr><tr><td>MACS LS column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>StemSpan SFEM medium</td><td>Stemcell Technologies</td><td>9650</td><td>1x</td></tr><tr><td>IMDM</td><td>Quality Biologicals</td><td>112-035-101</td><td>1x</td></tr><tr><td>TPO</td><td>Peprotech</td><td>300-18</td><td>100 ng/ml</td></tr><tr><td>Flt-3</td><td>Peprotech</td><td>300-19</td><td>100 ng/ml</td></tr><tr><td>SCF</td><td>Peprotech</td><td>300-07</td><td>100 ng/ml</td></tr><tr><td>IL-6</td><td>Peprotech</td><td>200-06</td><td>20 ng/ml</td></tr><tr><td>IL-7</td><td>Peprotech</td><td>200-07</td><td>20 ng/ml</td></tr><tr><td>IPS Sendai Reprogramming Kit</td><td>Life Technologies</td><td>A1378001</td><td></td></tr><tr><td>Cell scraper</td><td>Sarstedt</td><td>83.183</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>CryoTube vials</td><td>Thermo</td><td>368632</td><td></td></tr><tr><td>Mr. Frosty container</td><td>Thermo</td><td>5100-0001</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>12400-024</td><td>1x</td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502-048</td><td>1x</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2934</td><td>0.1% (w/v)</td></tr><tr><td>bFGF</td><td>Peprotech</td><td>100-18B</td><td>20 ng/ml</td></tr><tr><td>EGF</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>17504-044</td><td>1x</td></tr><tr><td>NSC serum free medium</td><td>Life Technologies</td><td>A1050901</td><td>1x</td></tr><tr><td>Poly-D-lysine/laminin coated cover slips</td><td>BD Bioscences</td><td>354087</td><td></td></tr><tr><td>cAMP</td><td>Sigma</td><td>A9501</td><td>300 ng/ml</td></tr><tr><td>Vitamin C</td><td>Sigma</td><td>A0278</td><td>0.2 mM</td></tr><tr><td>BDNF</td><td>Peprotech</td><td>450-02</td><td>10 ng/ml</td></tr><tr><td>GDNF</td><td>Peprotech</td><td>450-10</td><td>10 ng/ml</td></tr><tr><td>Poly-L-ornithine</td><td>Sigma</td><td>P4957</td><td>1x</td></tr><tr><td>PDGF-AA</td><td>Peprotech</td><td>100-13A</td><td>10 ng/ml</td></tr><tr><td>NT-3</td><td>Peprotech</td><td>450-03</td><td>2 ng/ml</td></tr><tr><td>Shh</td><td>Peprotech</td><td>1314-SH/CF</td><td>2 ng/ml</td></tr><tr><td>T3</td><td>Sigma</td><td>T6397</td><td>3 nM</td></tr><tr><td>PFA</td><td>Sigma</td><td>P6148</td><td>4%</td></tr><tr><td>PBS</td><td>Quality Biological</td><td>119-069-101</td><td>1x</td></tr><tr><td>Goat serum</td><td>Sigma</td><td>G9023</td><td>4%</td></tr><tr><td>TritonX-100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Mouse monoclonal anti-Nestin</td><td>Millipore</td><td>AB5922</td><td>1:1,000 dilution</td></tr><tr><td>Anti-SOX2 antibody</td><td>Applied Stemcell</td><td>ASA0120</td><td>Ready to use</td></tr><tr><td>Mouse anti-&amp;beta;III-tubulin antibody</td><td>Promega</td><td>G712A</td><td>1:1,000 dilution</td></tr><tr><td>Rabbit anti-GFAP antibody</td><td>Sigma</td><td>G4546</td><td>1:100 dilution</td></tr><tr><td>Anti-O4 antibody</td><td>R&amp;amp;D Systems</td><td>MAB1326</td><td>IgM; 1 ng/ml</td></tr><tr><td>Alexa Fluor 594 goat anti-rabbit antibody</td><td>Life techniologies</td><td>A11012</td><td>1:400 dilution</td></tr><tr><td>Alexa Fluor 488 goat anti-mouse antibody</td><td>Life techniologies</td><td>A11001</td><td>1:400 dilution</td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgM antibody</td><td>Life techniologies</td><td>A21042</td><td>1:250 dilution</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>1 &amp;mu;g/ml</td></tr></tbody></table>

direct induction of human neural stem cells from peripheral blood hematopoietic progenitor cells

人类疾病的特定的神经元的文化是产生的体外模型人类神经系统疾病至关重要。然而，由于缺乏获得初级成人神经文化提出了独特的挑战。在诱导多能干细胞的最新发展（IPSC）提供了另一种方法，通过患者特定的iPSC以导出从皮肤成纤维细胞的神经培养物，但是这个过程是劳动密集的，需要特殊的专业知识和大量资源，并且可能需要数月。这可以防止该技术的广泛应用，以神经系统疾病的研究。为了克服其中的一些问题，我们已经开发出一种方法，可直接从人成人外周血衍生的神经干细胞，从而绕过的iPSC推导过程。从成人外周血富集造血祖细胞在体外培养，并用含有转录因子Sox2的，十月仙台病毒载体3/4，Klf4和c-Myc的。转染的结果，其中通过使用人神经祖介质含有碱性成纤维细胞生长因子（bFGF）和血管内皮生长因子（VEGF）进一步选择在细胞形态学变化。将得到的细胞的特征在于为神经干细胞标记物，如巢蛋白和Sox2的表达。这些神经干细胞可进一步分化为神经元，星形胶质细胞和在指定的分化培养基少突胶质细胞。使用便利的人外周血样品中，该方法可以被用来推导神经干细胞进一步分化为神经细胞的体外模型神经障碍并可以前进与这些疾病的发病机制和治疗的研究。

<html> CD34 +细胞的质量为神经干细胞转导的成功是至关重要的。在第一夫妇培养天高品质的CD34细胞增殖，并出现非贴壁，均匀的圆形细胞，浮动只是培养容器（ 图1A）的底部上方。成功感染导致细胞的聚集体，它扩展随时间（ 图1B），而细胞培养过程中，可能会出现的非特异性的细胞聚集体，即扩大和关联松动。贴壁细胞通常会出现周围转染后三到五天;他们大多是双极和会激增，使单分子膜（ 图1C）。后与神经祖细胞培养基的选择大部分粘附细胞是巢蛋白和SOX2阳性但OCT4阴性（ 图2）。诱导神经干细胞可进一步分化为神经元和神经胶质细胞（ 图3）。 ENT"FO:保持together.within页="总是"&gt; <img alt="图1" src="/files/ftp_upload/52298/52298fig1.jpg" /> 图1.形态学仙台病毒转染后CD34 +细胞的变化。（A）的 CD34 +细胞的显着数量可在转染时被观察到。 （B）的球样细胞集合体可以仙台病毒转染而在时间扩展后观察24小时。 （C）多云双极贴壁细胞出现5天后转染。 （D和E）的神经干细胞的典型形态诱导神经祖介质和膨胀之后被示出。比例尺:200微米的（A）及（B），400微米为其他<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52298/52298fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2.诱导神经干细胞表达神经干细胞标记物。神经干细胞表达巢蛋白和Sox2，但没有OCT4。比例尺:200微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52298/52298fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52298/52298fig3.jpg" /> 图3.神经干细胞分化的神经干细胞可以分化为细胞的神经元标记物（A）中，星形胶质细胞标志物GFAP（B）和少突胶质细胞标记物O4（℃）。比例尺:100微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52298/52298fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells at JoVE.com

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

的方法的开发是为了直接从外周血细胞富集的造血祖细胞衍生的人神经干细胞。

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 ...

direct-induction-human-neural-stem-cells-from-peripheral-blood

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.4K 次观看.  National Institutes of Health. 的方法的开发是为了直接从外周血细胞富集的造血祖细胞衍生的人神经干细胞。 

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

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

科研

发育生物学

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

从血细胞使用仙台病毒和离心诱导多能干细胞代

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

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

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

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 ...

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

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

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

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

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

人脐带血衍生CD34的泛骨髓分化–造血干细胞和祖细胞

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

神经科学

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

生物学

Generation of Neuronal Cells from Blood-Derived Pluripotent Stem Cells

Source: Becker-Koji&#263;, Z. A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61634">GM-Free Generation of Blood-Derived Neuronal Cells.</a> J. Vis. Exp. (2021).
This video demonstrates a technique to generate neuronal cells from blood-derived pluripotent stem cells (BD-PSCs). The BD-PSCs are obtained via reprogramming blood progenitor cells. The undifferentiated BD-PSCs are cultured on cell culture substrate-coated coverslips using a neural induction medium and a neural differentiation medium, triggering differentiation into cells with a neuronal phenotype.

从血源性多能干细胞生成神经元细胞

Ethic approvals were obtained when performing the experiments.
1. Isolation of human peripheral blood mononuclear cells (PBMNCs)

	Ensure that all donors ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

Generation of Induced Neural Stem Cells (iNSCs) from Peripheral Blood Mononuclear Cells (PBMNCs): A Sendai Virus-based Procedure to Reprogram PBMNCs into iNSCs

Source: Zheng, W. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59690" target="_blank">Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates the reprogramming of peripheral blood mononuclear cells (PBMNCs) into induced neural stem cells (iNSCs) using Sendai virus. Sendai virus is a non-integrating virus that offers an efficient and safe vehicle for reprogramming cells.

人类神经干细胞从外周血造血祖细胞直接诱导

摘要

探索更多视频

此视频中的章节

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

从血细胞使用仙台病毒和离心诱导多能干细胞代

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

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

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

人脐带血衍生CD34的泛骨髓分化^–造血干细胞和祖细胞

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

从血源性多能干细胞生成神经元细胞

从外周血单核细胞（PBMNCs）产生诱导神经干细胞（iNSC）:一种基于仙台病毒的程序，将 PBMNCs 重编程为 iNSC

人类神经干细胞从外周血造血祖细胞直接诱导

摘要

探索更多视频

此视频中的章节

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

从血细胞使用仙台病毒和离心诱导多能干细胞代

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

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

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

人脐带血衍生CD34的泛骨髓分化–造血干细胞和祖细胞

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

从血源性多能干细胞生成神经元细胞

从外周血单核细胞 （PBMNCs） 产生诱导神经干细胞 （iNSC）:一种基于仙台病毒的程序，将 PBMNCs 重编程为 iNSC

人脐带血衍生CD34的泛骨髓分化^–造血干细胞和祖细胞

从外周血单核细胞（PBMNCs）产生诱导神经干细胞（iNSC）:一种基于仙台病毒的程序，将 PBMNCs 重编程为 iNSC