这项工作是由来自乔安娜M.尼古拉基金会研究员黑色素瘤的奖学金和健康的露丝下L. Kirschstein国家研究服务奖F31的国家机构提供支持。这项工作是通过从NYSTEM和三机构干细胞的倡议（斯塔尔基金会）资助的进一步支持。

注意:这里列出的黑素细胞协议最早是由云母等证明。 1.培养基的制备，涂层餐具和hPSCs维护<ol><li>介质制备工艺 注:存储在黑暗中长达2周，在4℃，所有的网上平台。过滤消毒所有的网上平台。 <ol><li>制备的DMEM / 10％FBS中。混合885毫升的DMEM，将100ml的FBS，将10毫升青霉素/链霉素和5ml L-谷氨酰胺。过滤消毒。 </li><li>准备人类胚胎干细胞中。混合800毫升的DMEM / F12加入200ml KSR，加入5ml L-谷氨酰胺，将10ml的MEM最小必需氨基酸溶液，将1mlβ巯基乙醇，和5ml青霉素/链霉素。过滤加入10毫微克/毫升FGF-2之后。 </li><li>制备KSR-分化培养基:混合820毫升敲除DMEM，加入150ml KSR，将10毫升L-谷氨酰胺，将10毫升青霉素/链霉素，将10毫升的MEM最小必需氨基酸溶液和1mlβ巯基乙醇。过滤消毒。 </li><li>准备N2-分化培养基。溶解12克DMEM / F12粉我ñ980毫升卫生署2 O.添加1.55克葡萄糖，2克碳酸氢钠和100mg载脂蛋白人转。混合2毫升的dh 2 O 25毫克的人胰岛素和40微升1N的NaOH;一旦溶解，将该混合物加入到培养基中。加入100微升腐胺，60微升亚硒酸钠，100微升黄体酮。过滤前将最终体积1升与卫生署2 O。 </li><li>准备全黑素细胞中。结合50％Neurobasal培养基，30％的低葡萄糖DMEM中，20％MCDB201。这个插件:0.8％的+，250纳米L-谷氨酰胺，100μM抗坏血酸（L-AA），50纳克/毫升霍乱毒素，50纳克/毫升SCF，0.05μM地塞米松，100nM的EDN3，4纳克/毫升FGF2 。无菌过滤器再加入其余试剂:2％B27补充，25纳克/毫升BMP4，3μMCHIR99021，500微米阵营。 </li></ol></li><li>培养皿的涂料<ol><li>开展利用胶质蛋白如基质胶涂层。开盘后，分装，冷冻基质胶在1ml部分，以免重复冻融Çycles。解冻并重新暂停与19毫升DMEM / F12 1毫升冷冻等分。板5毫升到10cm皿。孵化的菜在室温下1小时。吸出胶状蛋白质立即电镀细胞之前，用DMEM / F12洗涤。 </li><li>用聚L-鸟氨酸氢溴酸盐/鼠标层粘连蛋白I /纤维连接蛋白（PO /林/ FN）进行涂层。大衣菜含有PBS 15微克/毫升的聚L-鸟氨酸氢溴酸盐。在湿润的培养箱中孵育O / N在37℃。吸出溶液，并用含1μg/ ml的小鼠层粘连蛋白I和2微克/毫升纤连蛋白的PBS涂布之前用PBS三次洗平板。在加湿培养箱中孵育碗碟O / N在37℃。 <ol><li>之前电镀细胞，吸出溶液，并让所述板彻底干燥而不会在组织培养罩盖。允许将板干燥约10-15分钟。 注意:菜肴干燥并准备好细胞电镀时的晶体结构显示由眼的表面上。该开发平台ES可以保持用含有PBS林/ FN在孵化两周，只要该液体不会蒸发。所述板可以保持在室温下干燥几个小时。 </li></ol></li></ol></li><li> hPSCs保养 注:hPSCs保持在人类胚胎干细胞培养基中0.1％的明胶和有丝分裂灭活的小鼠胚胎成纤维细胞（MEF）。细胞应每6-8天分裂。 <ol><li>外套10cm皿用在PBS中0.1％的明胶在室温下5分钟。 </li><li>在37℃水浴迅速解冻的MEF。 </li><li>吸出明胶和板的细胞。板的MEF在DMEM / 10％FBS的的〜50,000个细胞/ cm 2的密度。加入hPSCs之前孵育在37℃CO / N的MEF。 </li><li>吸从制备MEF板的DMEM / 10％FBS洗涤板用PBS和加入10 mL人类胚胎干细胞的培养基与hPSCs。通道以1:1的比例hPSCs:视密度传代之前5/10。 注意:如果hPSCs正在解冻或传代的单细胞，SUPplement用10μM的Y-27632二盐酸盐（ROCKi），直到第一通道。 </li><li>日采食细胞新鲜10毫升人类胚胎干细胞培养基。 </li><li>在此之前开始分化删除看起来包含分化细胞，边界不规则，或透明的中心28多能干任何殖民地。机械地去除并用解剖显微镜在层流罩下的吸移管吸出不规则菌落。 </li></ol></li></ol> 2. hPSCs的电镀分化注意:分化条件10 cm的培养皿说明。 <ol><li>如步骤1.3.1描述开始分化前准备10厘米基底膜菜肴。 </li><li>当用于分化电镀hPSCs开始的密度准备通道（〜80％铺满）吸出人类胚胎干细胞的培养基，用PBS洗并加入3毫升0.05％胰蛋白酶-EDTA的向细胞hPSCs的板。 </li><li>大力摇晃菜horizo​​ntally表示2分钟，同时在显微镜下可视化，直到该MEF升空为单细胞。该HPSC殖民地应保持连接的殖民地。 </li><li>吸出胰蛋白酶该MEF已经解除后，但在hPSCs菌落分离之前。 </li><li>添加10含有10μM的Y-27632二盐酸盐向板的hESC培养基中，并通过上下吹打在菌落分离细胞。 </li><li>吸在步骤2.1中制备的10cm皿的基质胶溶液中，用DMEM / F12洗涤以除去团块。 2比到基质胶盘:在1板hPSCs。加入含10μMY-27632盐酸达至10ml人类胚胎干细胞培养基。在37°CO / N。 注意:此电镀，就会导致约100,000个细胞/厘米2。 </li></ol> 3.神经诱导分化注意:分化应开始（0天）时hPSCs 80％汇合。细胞可以每日用人类胚胎干细胞的培养基被供给含10μMY-27632盐酸盐，直到开始分化。 <ol><li>在第0天与第1天，喂细胞用10毫升含有100nM的LDN193189和10μMSB431542 KSR-分化培养基。 </li><li>第2天，进料将细胞与10毫升含有100nM的LDN193189，10μMSB431542和3微米CHIR99021 KSR-分化培养基。 </li><li>在第3天，进料将细胞与10毫升含10μMSB431542和3微米CHIR99021 KSR-分化培养基。 </li><li>上4和5天进料将细胞用15ml 75％KSR-分化培养基和含有3μMCHIR99021 25％N 2的分化培养基。 注意:不要惊慌，看到漂浮细胞方面有大量的细胞死亡。这是正常的，可以预期的。 </li><li>天6和7进料的细胞用15ml的50％KSR-分化培养基中，既含有3微米CHIR99021，25纳克/毫升BMP4，和100nM EDN3 50％N 2的分化培养基。 </li><li>在8天和9馈送的细胞用20ml 25％KSR-分化培养基中，既含有3微米CHIR99021，25纳克/毫升BMP4，和100nM EDN3 75％N 2的分化培养基。 </li><li>天9和10制备的PO /榄/ FN菜作为细胞在第11天重新电镀在1.2.2指示。 </li><li>第10天进料细胞用20毫升含有3微米CHIR99021，25纳克/毫升BMP4，和100nM EDN3 N2-分化培养基。 </li></ol> 4. Replating液滴数控规范<ol><li>在11天吸蓝/ FN从准备PO /林/ FN板和完全干燥。 </li><li>从第11天的细胞中取出，用PBS洗净，加4 ml的细胞分离液，如按的Accutase 10cm皿。在37℃下孵育25分钟。 </li><li> 5毫升全黑素细胞培养基添加到盘和通过手动上下吹打用10毫升吸管重悬细胞直到所有的细胞已经抬离板。转移悬浮液到15毫升管。 </li><li>旋细胞向下在200 xg离心5分钟，重悬的细胞以每毫升2×10 6个细胞中全黑素细胞培养基。计数上使用血球或等效技术的台盼蓝排除细胞。 </li><li>板10微升液滴彼此接近（没有它们触碰）到干燥PO /榄/ FN 10 cm的培养皿。如果盘已被充分干燥，液滴应该明确的边缘和不运行。 注意:这为细胞，replating细胞时，在保持菜的扩张房间内是重要的高密度当地的环境。 </li><li>允许该液滴到在RT静置10-20分钟，以允许细胞附着，然后缓慢（不干扰附着的细胞）加入10 mL全黑素细胞培养基。移动盘的孵化器。 </li></ol> 5.扩大黑素细胞祖细胞<ol><li>继续与全黑素细胞培养基饲养，每2至3天。 注意:色素沉着应该开始可见瓦特由第一周结束ithin细胞群，并成为在第二周很清楚。查看板在白色背景诸如一张纸来辨别这些早期的小的，暗簇。细胞将逐渐变得随着时间的推移更色素，直到整个板被均匀着色（参见图2B）。 </li><li>在〜1的比例传代细胞每周一次:6（镀以液滴不再需要在该阶段）。使用的Accutase来分离细胞和黑素细胞完全重新中等镀之前用普通Neurobasal培养基清洗两次。维持对PO /林/ FN板细胞。 注意:我们已经发现，全黑素细胞培养基是最适合于保持和扩大的hESC和源自iPSC的黑素细胞。然而，细胞可以是在市售M254培养基短暂培养，但简化的媒体增加垂死和剥离所述板的细胞的风险。 </li></ol>

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K., 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=Dominant+role+of+the+niche+in+melanocyte+stem-cell+fate+determination.">Dominant role of the niche in melanocyte stem-cell fate determination.</a> Nature. 416, 854-860 (2002).</li><li>Zur Nieden, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Embryonic stem cell therapy for osteo-degenerative diseases : methods and protocols. , (2011).</li><li>Lee, J. H., 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=high-content+screening+for+novel+pigmentation+regulators+using+a+keratinocyte/melanocyte+co-culture+system.">high-content screening for novel pigmentation regulators using a keratinocyte/melanocyte co-culture system.</a> Exp dermatol. 23, 125-129 (2014).</li></ol>

作者没有利益冲突的披露。

用于从hPSCs黑素细胞的成功分化以下建议，应考虑。首先，它是必不可少的在任何时候都无菌培养的条件下工作。此外，为了开始与多能性，完全未分化hPSCs是很重要的;如果起始群体包含分化细胞的产量将不可避免地下降，因为污染物不能对黑素细胞被引导，甚至可能进一步扰乱了正常分化细胞。 为了确保细胞保持多能照顾坚持干细胞维持既定规则;定期饲料和通道和新郎文化传代之前删除分化细胞。当传代到用于分化凝胶状蛋白是很重要的，该盘被正确涂覆以防止细胞分化过程中起重关闭。 在黑素细胞的不同entiation应该在80％左右的细胞密度来启动;过高的密度会导致更多的细胞死亡，而过低的密度差异会产生负面影响。传代时，细胞应洗涤两次电镀前除去任何细胞脱离溶液。为了最大限度地提高在第11天的生存，它是通过重要的细胞成高密度，良好的间距液滴是触及20分钟，使细胞聚集和粘附。 这个协议是有效的产生大量的黑素细胞，并研究有限数量的人类患者样品时将是有价值的。另外，作为PSC衍生的黑素细胞群体是有选择性的和可膨胀的黑素细胞群体可以相乘以非常大量的细胞即使分化产量低的数字或黑素细胞的百分比。同的iPSC发起协议打开了研究发育疾病和患者特定样本的可能性。一华里这个协议的mitation实际上是由于黑素细胞被导出为单一不存在的所有其它细胞类型构成在体内正常利基。此外，该协议要求在HPSC文化和分化的技术专长。然而，随着在IPSC的磁场产生hPSCs最近的发展已经变得越来越便于管理和用于培养hPSCs方法已成为常规。迄今为止，该协议已经被用在我们的实验室，以产生从H9人胚胎干线从5个不同的供体产生的黑素细胞，以及从14的iPS线。 云母等。该协议成功地用于从人胚胎干细胞和iPS细胞6黑素细胞的推导。纸张表明患者衍生的色素沉着的缺陷的iPSC可以分化成黑素细胞和协议忠实产生与疾病相关的表型的大小和数量的黑素。 在W扫示出的用于与使用源自iPSC的黑素细胞的用于研究疾病机制的协议的许多可能的应用之一，并推出扩大生产用于药物筛选6,29的可能性。重要的是，纸表现出的鲁棒性和协议的再现性，从一大组基因不同hiPSC线产生的黑素细胞。

人多能干细胞（hPSCs）提供一个平台，以模仿正常分化以可伸缩的方式用于疾病建模，药物筛选和细胞替代疗法1-6。特别感兴趣的，hPSCs开拓各种途径，为研究难以分离或在患者样本稀少罕见/短暂性的细胞类型。此外，诱导多能干细胞（iPS细胞）使研究人员研究发育和疾病建模在患者中特定的方式解开独特机制1,2,7-11。用于从hPSCs黑色素细胞的分化的先前公布的协议需要长达6周分化的和涉及培养细胞从L- Wnt3a的单元12的条件培养基。该协议最初由云母等人提出，并说明这里在三周内产生色素细胞和消除歧义和条件培养基相关的不一致。 黑素细胞ARE从神经嵴，独特的脊椎动物细胞的流动人口的。神经嵴是原肠胚形成过程中定义，并在神经板的边缘代表细​​胞群体，神经和非神经外胚层之间接壤。在神经胚形成，神经组织由神经板的发展，形成神经褶，其中汇聚在导致神经管13,14背中线。 神经嵴细胞的神经管的屋顶板出现，脊索对面，并迁移远离以产生分化细胞的不同群体之前经历上皮至间质转变。的嵴细胞的命运是部分地由沿胚的体轴顶板的解剖位置界定。神经嵴细胞衍生物包括谱系两者中胚层的特征（平滑肌细胞，成骨细胞，脂肪细胞，软骨细胞）和外胚层细胞（黑素细胞，雪旺氏Ç厄尔，神 ​​经元）14。神经嵴干细胞上调转录因子SOX10，可以通过荧光激活细胞用针对P75和HNK1排序来分离。 注定要成为黑素细胞穿过一个黑素细胞阶段和上调KIT和MITF（小眼球相关的转录因子）6,21 MITF是黑素细胞发展的主要调节并是一种转录因子，负责控制多黑素细胞发展的神经嵴细胞22- 24。人黑素细胞迁移到它们驻留无论是在毛发凸起或由角质形成细胞在表皮包围（形成色素单位）作为前体的成熟的，着色的黑素细胞表皮的基底层。黑素细胞的分化和成熟成颜料黑素发生伴随毛球的定植和表达的黑色素生产途径（TYRP1，TYR，OCA2的和PMEL）25,26。 从患者中分离的人类黑色素细胞和黑素细胞是昂贵的，困难的，在数量上的限制。该协议使研究人员能够区分hPSCs（诱发或胚胎）为黑素细胞或黑素细胞前体在一个定义良好的，快速的，可重复的，可扩展的，廉价的无细胞分选方法。协议以前用于从患者的色素沉着紊乱分化的iPSC时标识特定疾病的缺陷。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>apo human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid (L-AA)</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>B27 (B27 Supplement)</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-bp</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>4423</td>
			<td>6 mM</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>50 mg/ml</td>
		</tr>
		<tr>
			<td>cAMP (cyclicAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D2915-100MG</td>
			<td>50 &#956;M</td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco-Life Technologies</td>
			<td>11985-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 - Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Gibco-Life Technologies</td>
			<td>1133--032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 powder</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
		</tr>
		<tr>
			<td>EDN3 (Endothelin-3, human)</td>
			<td>American Peptide Company</td>
			<td>88-5-10B</td>
			<td>100 &#956;M</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td>200 &#956;g/ml</td>
		</tr>
		<tr>
			<td>gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td>0.1% in PBS</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Human insulin</td>
			<td>Sigma</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS+ Universal Culture Supplement Premix&#160;</td>
			<td>BD Biosciences</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR (Knockout Serum Replacement)</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Invitrogen</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Dissolve 1:20 in DMEM/F12</td>
		</tr>
		<tr>
			<td>MCDB201 Medium</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM minimum essential amino acids solution</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-8105M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Poly-L Omithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>15 mg/ml&#160;</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>0.032g in 100ml 100% ethanol</td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 (Recombinant human FGF basic)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1814</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF (Stem Cell Factor, recombinant Human)&#160;</td>
			<td>Peprotech Inc.</td>
			<td>300-07</td>
			<td>50 &#956;g/ml</td>
		</tr>
		<tr>
			<td>TYRP1 (G-17) Antibody</td>
			<td>Santa Cruz</td>
			<td>10443</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>TYRP2 Antibody</td>
			<td>Abcam</td>
			<td>74073</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

feeder-free derivation of melanocytes from human pluripotent stem cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

这个协议提供了用于从在体外无饲养，成本高效的，和可重复的方式hPSCs导出充分着色，成熟的黑素细胞的方法。与此相反的先前建立的Fang等协议为HPSC衍生的黑素细胞，所述概述协议不需要调节的培养基，并降低了时间要求。方舟子等人的协议从WNT3A生产的小鼠细胞系利用条件培养基，并采取了长达6周的可视化色素沉着6,12。到偏向黑素细胞命运的神经嵴干细胞（黑素细胞），我们的早期脉冲后除去BMP和TGFβ抑制剂（分别LDN和SB），随后引入外源性BMP4和EDN3六一天，而维持的Wnt激活信令（CHIR） 6。所得黑素细胞可以通过KIT和MITF的表达来表征，以及维护SOX10 expr的分裂国家6。可以在培养皿形成由天11培养（ - C 图1B）的脊区域进行可视化SOX10的表达。 以下黑素细胞的诱导，将细胞传代到PO /林/ FN板并用全黑素细胞培养基进料。这是一个具有作为选择性的黑素细胞群体，从而消除了任何荧光激活细胞分选6需要额外的好处极其丰富的介质。期间黑素细胞的成熟，细胞上调的黑色素合成基因TYR和TYRP1同时保持许多黑素细胞的基因，包括TYRP2。 Day25干细胞适当衍生的黑素细胞染色用于黑色素生产蛋白质TYRP1和TYRP2（ 图2）。这个协议是健壮并已用于与H9胚胎干细胞系和在多个iPSC系。最近，该协议被用来忠实地再现了UL<html>使用特定患者的iPSC系6色素沉着性疾病trastructural功能。 <img alt="图1" src="/files/ftp_upload/53806/53806fig1.jpg" /> 图1中的hESC衍生的黑素细胞分化方案的关键步骤。（A）中的分化的协议方案。 KSR:KSR-分化培养基，N2:N2-分化培养基，LDN:LDN193189，SB:SB431542，CHIR:CHIR99021，BMP4:骨形态发生蛋白4，EDN3:内皮素3，ROCKi:Y-27632盐酸乙 - ℃。示出了明场（B）和分化的GFP荧光（C）的图像上使用的转基因pSOX10 replating之前11天:GFP的人类胚胎干细胞系。在明图像中可见的黑脊富集SOX10 +细胞会产生黑素细胞和黑素细胞最终。<html>上传/ 53806 / 53806fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53806/53806fig2.jpg" /> 图2.黑素细胞和中间阶段。（ 一 ）色素日25格（右）可以很容易地可视化和尊贵日11个细胞（左）沉淀时。 （B）×20天协议的文化将同时包含未着色，成熟和黑素细胞完全成熟，色素的黑素细胞。 （C - D）的黑素细胞可在明亮的现场可视化，并为黑素细胞/黑色素细胞标记TYRP2成熟，完全色素的黑素细胞染色两种。 （E - F）只有色素色斑的黑色素细胞对晚期黑素细胞标记TYRP1。得到="_空白"&gt;点击此处查看该图的放大版本。

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

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

从人多能干细胞的黑素细胞的无饲养推导

这个工作描述了一种在体外分化协议经由神经嵴，以产生自人多能干细胞色素，成熟的黑素细胞，并使用一个无饲养25日实验方案黑素细胞的中间阶段。

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

feeder-free-derivation-melanocytes-from-human-pluripotent-stem

Nanyang Technological University

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JoVE Journal

Developmental Biology

10.2K 次观看.  Memorial Sloan-Kettering Cancer Center. 这个工作描述了一种在体外分化协议经由神经嵴，以产生自人多能干细胞色素，成熟的黑素细胞，并使用一个无饲养25日实验方案黑素细胞的中间阶段。 

视频: Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

视网膜色素上皮细胞的干细胞高效的推导

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

从人类诱导多能干细胞使用小分子调制分化和随后的葡萄糖饥饿高度纯化心肌推导

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

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 ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

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 ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...