Name: feeder-free derivation of melanocytes from human pluripotent stem cells
Uploaded: 2016-03-03T15:00:00.000+00:00
Duration: 12 min 21 s
Description: Watch this Scientific Journal Video about Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells at JoVE.com

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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M., 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=KIT+expression+reveals+a+population+of+precursor+melanocytes+in+human+skin.">KIT expression reveals a population of precursor melanocytes in human skin.</a> J invest dermatol. 106, 967-971 (1996).</li><li>Li, L., 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=Human+dermal+stem+cells+differentiate+into+functional+epidermal+melanocytes.">Human dermal stem cells differentiate into functional epidermal melanocytes.</a> J cell sci. 123, 853-860 (2010).</li><li>Nishimura, E. 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><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>&amp;beta;-Mercaptoethanol</td><td>Gibco-Life Technologies</td><td>21985-023</td><td>10 mg/ml</td></tr><tr><td>BMP4</td><td>R&amp;amp;D Systems</td><td>314-bp</td><td></td></tr><tr><td>CHIR99021</td><td>Tocris-R&amp;amp;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 &amp;mu;M</td></tr><tr><td>DMEM - Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco-Life Technologies</td><td>11985-092</td><td></td></tr><tr><td>DMEM/F12 - Dulbecco&#039;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 &amp;mu;M</td></tr><tr><td>Fibronectin</td><td>BD Biosciences</td><td>356008</td><td>200 &amp;mu;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&amp;nbsp;</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&amp;amp;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&amp;nbsp;</td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td>0.032 g in 100 ml 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&amp;amp;D Systems</td><td>233-FB-001MG/CF</td><td>10 mg/ml</td></tr><tr><td>SB431542</td><td>Tocris-R&amp;amp;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)&amp;nbsp;</td><td>Peprotech Inc.</td><td>300-07</td><td>50 &amp;mu;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&amp;amp;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

Research

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

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

科研

发育生物学

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

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 ...

神经科学

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 ...

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

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

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 ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

在无馈体和化学定义的培养条件下，使用人类多能干细胞的后多能感感神经元的有效分化

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

将小鼠成纤维细胞直接重编程为黑素细胞

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

免费接驳适应，文化和传代使用完整的基因敲除血清替代馈线中的人类iPS细胞

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

生物学

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

从小鼠心脏中分离主黑素细胞样细胞

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...