与三机构的干细胞的倡议（斯塔尔基金会），这项工作是由来自瑞士国家科学基金会通过从NYSTEM（C026447 C026446）资助先进的研究人员的研究奖金支持。

1，文化传媒，涂层餐具和hPSCs维护的制备 1.1介质的制备注：过滤所有媒体灭菌和储存在4℃下在黑暗中长达2周。试剂名称，公司和产品编号列在材料表 。 <ol><li> DMEM/10％FBS：结合885毫升的DMEM 100毫升FBS，将10毫升青霉素/链霉素和5ml L-谷氨酰胺。 </li><li> HES-介质：联合800毫升DMEM/F12，200毫升KSR，加入5ml L-谷氨酰胺，将5ml青霉素/链霉素，将10ml的MEM最小必需氨基酸溶液，加入1ml的β-巯基乙醇。加入10毫微克/毫升过滤介质的FGF-2之后。 注意：β-巯基乙醇有毒，避免吸入，食入和皮肤接触。 </li><li> KSR-分化培养基：联合820毫升敲除DMEM中，加入150ml KSR，将10毫升L-谷氨酰胺，将10毫升青霉素/链霉素，将10ml的MEM最小必需氨基酸溶液和1ml的β-巯基乙醇。 </li><li> N2-differentiati对中：将12克DMEM/F12粉980毫升DH 2 O，加1.55克葡萄糖，2克小苏打和100毫克的APO人转。混合2毫升DH 2 O和25毫克人胰岛素和40微升1N NaOH中，加入溶解液的培养基中。加入100μl腐胺盐酸盐，60微升亚硒酸钠，100微升孕激素，并把音量调高至1 L DH 2 O。 </li></ol> 1.2涂层的培养皿<ol><li>基质胶涂层：解冻1毫升吹打19毫升DMEM/F12在等分，直到它已冻结溶解基质胶等分。通过一个40微米的细胞过滤器和板8 ml/10厘米盘子传递给它移除团块。孵育菜1小时在室温。立即电镀细胞前吸出基质胶。 注：与基质胶快的工作，因为它可以聚集在温度高于4℃。 </li><li> PO /林/ FN涂层：添加含有15微克/毫升聚-L鸟氨酸氢溴酸盐到10 cm培养皿10毫升1X PBS中。孵育菜过夜，在37°C。用1X PBS洗涤板一次，并添加1×PBS中含有2微克/毫升小鼠层粘连蛋白-I和2μg/ ml的纤维连接蛋白10 ml/10厘米的菜。孵育菜过夜，在37°C。电镀前的细胞中，完全除去溶液，并让平板站在它们到组织培养罩壁而不在盖彻底干燥在室温15-20分钟。 注：该菜是完全干燥，并准备细胞接种时，人们可以看到表面（肉眼可见）的晶体结构。所述板可保持在室温数小时在这个干燥状态。 PO /林/ FN菜必须提前准备时间2天。在紧急情况下，林先生/ FN可以培养只有2-4小时。然而，这也许会引起不理想的分化/生存结果。 </li></ol> 1.3维护hPSCs的注：hPSCs维持在0.1％明胶和有丝分裂灭活小鼠胚胎成纤维细胞（MEFS）在HES-培养基中添加10毫微克/毫升如前10,12所描述的FGF-2。这些细胞应该分开每6-8天。 <ol><li>外套10 cm培养皿用8ml 0.1％明胶（在1x PBS中没有镁或钙），在室温下5分钟。 </li><li>在37℃水浴中快速解冻冷冻的MEF。新增100万的MEF〜10毫升DMEM/10％FBS。 </li><li>吸出明胶和板的细胞。在37°C至少6小时。 </li><li>从准备MEF板吸出DMEM/10％FBS，用1X PBS洗涤板一次，并加入10 ml的HES-培养基中添加10μMY-27632二盐酸盐。允许介质进行预热，在37℃20分钟。 注意：对于hPSCs的手工分割层流罩具有嵌入显微镜使用。然而，所述细胞可以使用适当的替代方法进行传代。 </li><li>在层流罩与嵌入式显微镜，使用细胞升降机分离独立的殖民地，并让他们自由浮动。 </li><li>使用1ml注射器吸出漂浮的殖民地和派遣他们到了清新，温暖的MEF板。将大约四分之一的细胞的新菜。在37°C。 </li><li>每天用新鲜的HES中等进给单元格。 </li></ol> hPSCs 2。电镀的分化注：hPSCs应该分开或镀分化时，菌落较大，但仍然有尖锐的边缘与尽可能少的分化细胞在它们的边界（ 见图1B）。当细胞使用手动传代保持的菌落应足够大，以容易地通过眼睛看到。为了得到正确的感觉这个时间点可以维护一个单独的HPSC菜了两周，没有传代，观察细胞到达和经过的理想时间点传代/区分它们。 <ol><li>准备10厘米菜基质胶1小时开始分化之前。成功的基质胶涂层可以是以4倍的放大倍率验证。 </li><li>当hPSCs准备进行分割，吸出培养基并加入4毫升的0.05％胰蛋白酶-EDTA细胞中。 </li><li>水平摇动培养皿2分钟剧烈直到1可以看到的MEF为单细胞，在显微镜下剥离所述板。该HPSC殖民地保持连接的殖民地。 </li><li>立即，彻底，吸出胰蛋白酶，让站在板在室温下2-4分钟。 </li><li>加入2ml HES-介质的板块和使用P1000的移液管，取下电池。 注意：如果细胞不能轻易提起离开板面，该板可以用于另一个2-3分钟孵育空在RT。 </li><li>细胞转移到8毫升HES-培养基中添加10μMY-27632二盐酸盐。 </li><li>从预先准备的10cm皿吸出基质胶。板上的细胞以1:1或1:2的比例（例如：从一个10cm皿将细胞分成两10厘米菜）到基质胶盘。在37°℃过夜。 注：此电镀，就会导致约100,000个细胞/ cm 2。 </li></ol> 3，诱导神经分化注意：分化可以被启动（0天）时，细胞是90-100％汇合（ 见图1C），通常第二天。如果未达到准确汇合的是，该细胞可每日用HES培养基喂养，直到他们准备好差异化。或者，细胞镀的初始数量可以增加。 <ol><li>在第0天至3，每天喂细胞10 ml/10 cm培养皿中含有0.1微米LDN193189和10μMSB431542 KSR-分化培养基。 </li><li> 4日及5饲料的细胞用75％KSR-分化培养基和25％N2-分化培养基均含有LDN193189和SB431542。 </li><li>在第6天及7进料的细胞，用50％KSR-分化培养基和50％N 2 - 分化培养基中均含有LDN193189和SB431542。 </li><li>在第8天及9进料的细胞，用25％KSR-分化培养基和75％N 2 - 分化培养基中均含有LDN193189和SB431542。 </li><li>在9日和10准备PO /林/ FN菜肴在1.2.2所示的第11天细胞replating。 </li><li>每天上含10进细胞N2-分化培养基LDN193189和SB431542。 </li></ol> 4，在液滴数控规格Replating <ol><li>在一天11吸蓝/ FN从先前准备的板，让他们完全干燥在室温20-30分钟，如1.2.2所述。 </li><li>从分化细胞移除培养基，用1×PBS洗一次细胞加入8毫升accutase/10厘米的菜。孵育20分钟，在37℃下</li><li>使用细胞起苗，分离细胞和悬浮在其中的Accutase用5毫升吸管。该悬浮液转移到15毫升管。 </li><li>加入5 ml的1×PBS中，并旋转该细胞5分钟，在114×g下。 </li><li>重悬细胞于10ml 1x PBS中。为确保单细胞悬浮液，通过40微米的细胞滤网过滤它们并使用血细胞计数器或等同技术计数细胞。 </li><li>旋转细胞向下，重悬它们在含有200μM的抗坏血酸（AA），20纳克/毫升的BDNF，100纳克/毫升FGF8，20毫微克/毫升SHH，10μMY-27632二盐酸盐以100,000的浓​​度N 2 - 分化培养基-150,000 cells/10微升。 </li><li>使用重复移液器，板10微升滴接近对方没有他们接触到干燥的PO /林/ FN15厘米菜肴。 注：分化细胞的一个10 cm培养皿通常会导致大约2-3倍15厘米菜肴或约100×10微升droplets/15厘米的菜。 </li><li>让水滴站立在室温20-30分钟，然后很认真（不打扰滴）加到30毫升N2/AA/BDNF/FGF8/SHH/Y和孵化，在37°C。 </li><li>第12天，精心喂养细胞20 ml/15 cm培养皿N2/AA/BDNF/FGF8/SHH。 </li><li>从大Ÿ14-17养活细胞每隔第二或第三天与N2/AA/BDNF/FGF8。 </li><li>在16日和17准备PO /林/ FN板FACS分选后replate数控细胞。 </li></ol> 5，荧光激活细胞分选的NC细胞（FACS） 注：该细胞流式细胞仪的编制需要约2小时。 <ol><li>在18天删除介质，用1×PBS洗一次细胞在培养皿并添加12毫升的Accutase。于室温孵育20分钟，在37℃下</li><li>使用细胞挺杆，从表面分离细胞，并让它们自由浮动。吸取细胞进入暂停使用5毫升吸管，将它们转移到50毫升的试管，加入30毫升的1X PBS中。旋转细胞5分钟，在114×g下。 </li><li>使用P1000移液器，悬浮在1ml 2％FBS / HBSS（在HBSS中含有15mM的HEPES）的细胞。加19毫升的2％FBS / HBSS中，并通过一个40微米的细胞滤网过滤细胞，以确保单细胞悬浮液。冰上孵育的细胞15米在对抗体阻断，然后用血细胞计数器计数。 </li><li>旋下来的细胞，重悬在他们1000万细胞/ 2％FBS / HBSS毫升的浓度。 </li><li>预留100万单元，每个单元为未染色，单染（HNK-1只和p75只）和二级抗体染色只（APC只和488只），流式细胞仪控制。 </li><li> 1毫升悬液加入5微升的HNK-1和5微升的p75的主要抗体每10万个细胞，以适当的样品孵育20分钟冰。 </li><li>在10ml的1×PBS（离心5分钟，在114×g离心）洗涤细胞，重悬细胞于1ml的2％FBS / HBSS中。在1ml的悬浮液中加入2μl的APC和1μl的488次级抗体，每10万个细胞，以适当的样品，孵育20分钟在冰上于黑暗中。 注意：APC及488在这里用于HNK-1和p75染色，分别二次抗体。 </li><li>在10ml的1×PBS洗涤细胞两次，重悬千万细胞于500μl2％FBS / HBSS含DAPI（在0.5纳克/微升），或另一种合适的活细胞染色来排除死细胞的FACS分析。 </li><li>转移样品到适当的FACS管。 </li><li>制备FACS管用0.5毫升的2％FBS / HBSS进行细胞收集。 注：细胞和集合管中的排序时间保持在冰上，在黑暗中。 </li><li>用细胞分选机的激光器，可以检测DAPI，APC和488排序DAPI-/HNK-1 + / P75 +双阳性细胞。 注：制备时间和处理细胞的导致细胞死亡的一个很小的比例，DAPI染色死细胞仅从而允许这些细胞被排除在所划分的人口。任何替代活/死染色同样适用于这一目的。 DAPI本身不引起细胞毒性的活细胞群。 </li></ol> 6。Replating排序细胞，数控维护和扩展注：流式细胞仪分选细胞应该是手与领导的特殊照顾，以确保最佳的生存。让他们在冰上，直到replating他们。不要旋涡振荡或吸取他们严厉。该细胞可以通过轻弹管再悬浮。 <ol><li>流式细胞仪分选后，算上数控细胞，然后旋下来，重悬在N2分化培养基中，在适当的音量10毫微克/毫升FGF2，20 ng / mL的EGF和10μMY-27632二盐酸盐，以replate他们在30,000细胞/ 10微升滴。 </li><li>彻底干燥先前准备的PO /林/ FN板和每10 cm培养皿板约50-70滴。让站在菜在室温20-30分钟。 </li><li>非常小心地加入N2/FGF2/EGF/Y-27632 20 ml/10厘米的菜，而不会干扰液滴。孵育的细胞在37℃下</li><li>喂细胞每2-3天用N2/FGF2/EGF。 注：NC细胞膨胀或传代大约每隔4-5天，或当他们开始液滴内堆放。 </li><li>要通过数控细胞，去除培养基，WASH细胞一次用1×PBS加入8毫升accutase/10厘米的菜。孵育20分钟，在37℃下</li><li>吸取单元的电源使用5毫升吸管板，并将它们转移到15毫升管。加6毫升的1X PBS。 </li><li>旋转细胞5分钟，在114×g下。 </li><li>重悬细胞中的N2-分化培养基中，以便有20000 cells/10微升液滴补充有10纳克/毫升FGF2，20毫微克/毫升的EGF和10μM的Y-27632二盐酸盐。 </li><li> Replate细胞在10微升滴干上PO /林/ FN板。让站在菜在室温20-30分钟。 </li><li>小心加入N2/FGF2/EGF/Y-27632 20 ml/10厘米的菜。在37°C。 注：NC细胞可以维持或扩大长达2周为相对均匀的祖细胞群。较长的文化可能会导致它们分化成各种子代细胞类型。 </li></ol>

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M., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+neural+crest+induction,+melanocyte+specification,+and+disease-related+pigmentation+defects+in+hESCs+and+patient-specific+iPSCs.">Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs.</a> Cell Rep. 3, 1140-1152 (2013).</li><li>Molofsky, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi-1+dependence+distinguishes+neural+stem+cell+self-renewal+from+progenitor+proliferation.">Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation.</a> Nature. 425, 962-967 (2003).</li></ol>

作者没有利益冲突的披露。

对于R-NC细胞的人类胚胎干细胞从/人iPS细胞成功分化以下考虑应作出。这是至关重要的无菌培养条件下工作在任何时候。特别是，它要定期检测HPSC文化的支原体污染，因为这种污染会阻碍成功的差异化是重要的，但还不能轻易地在HPSC文化视觉检测。在R-NC分化应在90-100％的细胞密度被启动;低细胞密度的影响细胞存活和R-NC分化的效率。重要的是要凭经验验证最佳浓度和一些试剂的批号，特别是KSR支...

人类胚胎干细胞（胚胎干细胞）和人类诱导多能干细胞（hiPSC）显示巨大的潜力，特别是人类疾病对没有良好的动物模型也主要组织提供的调查和今后的治疗。对人类胚胎干细胞/ hiPSC技术应用实例如下：可从人类胚胎干细胞/人iPS细胞生成的再生医学在无限数量1特别感兴趣的细胞。细胞可以从携带特定疾病的患者被制造，以及用于建立体外疾病模型2,3。这种疾病模型，然后可以在追求新的药物化合物4和现有药物的疗效和毒性5的测试被用于大规模药物筛选， 体外疾病模型可以导致新的疾病机制的鉴定。对于人类胚胎干细胞/ IPSC的技术的所有应用中的具体工作是很重要的，良好定义受影响的权益性疾病ð细胞类型。因此，固体和可重复的体外分化协议的可用性是对人类胚胎干细胞/ hiPSC技术的所有应用是至关重要的。协议是可取的，显示最小的变异性，时间牺牲，努力，其中胚胎干细胞/ hiPSC线和不同的研究者难度和成本，以及最大可再现性。 在表皮和神经上皮之间的脊椎动物神经胚形成神经嵴（NC）的细胞出现。它们增殖和迁移广泛地在整个发育中的胚胎和产生子代细胞类型的令人印象深刻的多样性，包括骨/软骨时，颅面骨，感觉神经，雪旺氏细胞，黑素细胞，平滑肌细胞，肠神经元，自主神经元，嗜铬细胞，心脏室间隔细胞，牙齿和肾上腺/甲状腺腺细胞6。因此，NC细胞是一个有吸引力的细胞类型的干细胞领域和对于重要的各种疾病，如先天性巨结肠7，家族性自律神经失调8以及癌症，如神经母细胞瘤9建模。此外，他们提供学习的体外人类胚胎发育的某些方面的可能性。 当前可用的和广泛应用的体外分化方案的数控细胞从人类胚胎干细胞10,11的推导需要长达35天分化的，它涉及到神经诱导的基质饲养细胞如MS5细胞，因而根据定义不明确的条件下进行。虽然它可以是按比例放大，以产生大量的NC细胞，例如需要用于高通量药物筛选4，这是劳动和成本密集型的。此外，它涉及神经花环，可以是难以再现的手动传代并且当它被施加于多种人类胚胎干细胞或hiPSC的因而是受总体变异性，特别是线。这里，示出了NC细胞在18天的协议，它是不含饲养细胞的逐步推导。这个方法是更短，比目前使用的协议更明确。此外，它是在生成的NC细胞不同hiPSC线中非常健壮。重要的是，它表明，数控细胞由双方协议产生出现在的神经花环（以下称为花环-NC或R-NC）的边界。无论是使用的两个协议所产生的细胞形态看起来是相同的，他们表达了同样的数控标记和聚集在一起的微阵列分析。使用新的协议（R-NC）衍生的NC细胞是功能性的，类似于使用旧协议（MS5-R-NC），使得它们可以迁移并进一步分化成神经元衍生的NC细胞。因此，这些细胞可以同时使用与MS5-R-NC细胞。数控细胞从人类胚胎干细胞/ IPSC的推导在R-NC细胞的协议将是对人类胚胎干细胞/ IPSC的技术涉及数控谱系的所有应用程序非常有用。&lt;/ P&gt;

<table border="0" cellpadding="0" cellspacing="0" style="width:897px;" width="897">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Regents</td>
			<td style="width:219px;">company</td>
			<td style="width:160px;">cataloge number</td>
			<td style="width:165px;">comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12</td>
			<td>Gibco-Life Technologies</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout Serum Replacement</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td>Lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicinlin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM minimum essential amino acids solution&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-Mercaptoethanol&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant human FGF basic (FGF2)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12 powder&#160;</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">APO human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human insulin&#160;</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Putriscine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-L Ornithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dispase in Hank&#39;s Balanced Salt Solution 5U/ml</td>
			<td>Stem Cell Technologies</td>
			<td>7013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic Acid&#160;</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BDNF</td>
			<td>R&#38;D Systems</td>
			<td>248-BD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FGF8</td>
			<td>R&#38;D Systems</td>
			<td>423-F8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse recombinant sonic hedgehog (SHH)</td>
			<td>R&#38;D Systems</td>
			<td>464SH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco-Life Technologies</td>
			<td>14170-112</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Gibco-Life Technologies</td>
			<td>1563-080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant EGF</td>
			<td>R&#38;D Systems</td>
			<td>236EG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti HNK-1/N-Cam (CD57) mIgM</td>
			<td>Sigma</td>
			<td>C6680-100TST</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-p75 mIgG1 (Nerve Growth factor receptor)</td>
			<td>Advanced Targeting Systems</td>
			<td>AB-N07</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">APC rat anti-mIgM</td>
			<td>BD Parmingen</td>
			<td>550676</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 488 goat anti-mouse IgG1&#160;</td>
			<td>Invitrogen</td>
			<td>A21121</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti Oct4 mIgG2b (used at 1:200 dilution)</td>
			<td>Santa Cruz</td>
			<td>sc-5279</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Material/Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-6105M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture dishes: 10cm and 15 cm plates, centrifuge tubes, FACS tubes,&#160; pipetts, pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass hematocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture incubator (CO2, humidity and temperature controlled)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture laminar flow hood with embedded microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture biosafety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell sorting machine, i.e. MoFlo</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inverted microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml TB syringe 27Gx1/2</td>
			<td>BD Biosciences</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell lifter Polyethylene</td>
			<td>Corning Incorporated</td>
			<td>3008</td>
			<td></td>
		</tr>
	</tbody>
</table>

feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells

人类多能干细胞（hPSCs）对研究人类胚胎巨大的发展潜力，在盘模拟人类疾病和移植细胞的疾病或意外事故后再生的应用程序的来源。神经嵴（NC）的细胞是前体种类繁多的成年体细胞，例如从外周神经系统和神经胶质细胞，黑素细胞和间充质细胞的细胞。它们是细胞来研究人类胚胎发育，包括细胞命运规范和迁移方面的重要来源。数控祖细胞进一步分化成终末分化的细胞类型提供模拟人类疾病的体外 ，研究疾病的机理及产生细胞再生医学的可能性。本文介绍了目前在体外分化协议数控细胞从hPSCs推导的适应。这个新的协议需要18天迪菲的rentiation，是无饲养，易于扩展和高重现性之间的人类胚胎干细胞（hESC细胞）线，以及人类诱导多能干细胞（hiPSC）线。旧的和新的协议得到平等的身份数控细胞。

在R-NC协议在MS5-R-NC协议11的两个最重要的改进是无饲养层，定义的分化条 ​​件和时间要求的整体缩短。 MS5料器单元13是已被证明可以从人胚胎干细胞14支持神经分化的鼠骨髓衍生的基质细胞。人类胚胎干细胞培养的MS5饲养层细胞密度低的形式上皮结构和神经花环15，在其外围的数控细胞出现10，从而模拟人类早期神经发育。然而，目前还不清楚什么信号分...

Watch this Scientific Journal Video about Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells at JoVE.com

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

从人类多能干细胞（HPSC）源性神经嵴（NC）细胞具有模拟人类发展和疾病的细胞替代疗法的巨大潜力。这里，一个无饲养适应的目前广泛使用的<em&gt;在体外</em&gt;分化方案用于从hPSCs NC单元的推导给出。

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

feeder-free-derivation-neural-crest-progenitor-cells-from-human

Research

JoVE Journal

Neuroscience

14.1K 次观看.  Sloan-Kettering Institute for Cancer Research. 从人类多能干细胞（HPSC）源性神经嵴（NC）细胞具有模拟人类发展和疾病的细胞替代疗法的巨大潜力。这里，一个无饲养适应的目前广泛使用的在体外分化方案用于从hPSCs NC单元的推导给出。

视频: Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

从人类毛囊神经嵴干细胞的分离和培养的

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

从人类多能干细胞的规范在前脑谷氨酸能神经元

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

人类神经的聚集体cGMP的适用展开法干细胞和祖细胞来源从多能干细胞或胎儿脑组织

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

从人多能干细胞定向多巴胺能神经元分化

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

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 ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...