Name: feeder-free derivation of melanocytes from human pluripotent stem cells
Uploaded: 2016-03-03T15:00:00
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 K...

<ol>
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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?...

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...

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 ...

Research

JoVE Journal

Developmental Biology

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 ...

科研

发育生物学

Efficient Derivation of Retinal Pigment Epithelium Cells 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

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

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

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

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 ...

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

分化软骨细胞与外周血源性人诱导多能干细胞

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

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

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) ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver 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

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 ...