Name: feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells
Uploaded: 2014-05-22T14:45:00
Description: Watch this Scientific Journal Video about Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells at JoVE.com

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...

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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;

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			<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>
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			<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

从人多能神经嵴前体细胞的无饲养推导干细胞

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。

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

Research

JoVE Journal

Neuroscience

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

小鼠神经嵴干细胞的培养鸡胚提取物的生产

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

科研

神经科学

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

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). ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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

Live-cell Imaging of Sensory Organ Precursor 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

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

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

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

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

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 ...