Name: the specification of telencephalic glutamatergic neurons from human pluripotent stem cells
Uploaded: 2013-04-14T14:00:00
Description: Watch this Scientific Journal Video about The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells at JoVE.com

作者想感谢Y.笹井，慷慨提供FOXG1抗体。这项工作是由康涅狄格州干细胞研究资助（08-SCB-UCHC- 022和11-SCB24）和痉挛性截瘫基金会。

1。代的人类多能干细胞聚集体（D1-D4） <ol><li>人类多能干细胞对小鼠胚胎成纤维细胞（MEF）的送料器，在胚胎干细胞的存在下与碱性成纤维细胞生长因子（bFGF，4纳克/毫升）的培养基中培养。经过5-7天的培养时，菌落大，但仍然未分化的，他们是为下一步做好准备。 </li><li>应先来制备的酶溶液。在一个50毫升的试管中，溶解在1毫克/毫升的浓度成DMEM/F12培养基分散酶（或胶原酶）。由于这些解决...

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在神经细胞分化过程中有几个关键的步骤。重要的是要确保人的产品分成合同，因为否则的细胞是多能干细胞，可能已经偏向成为非神经元谱系。这可以通过以下的确认染色与多能性的标记物，如即Oct4，Sox2基因，Nanog基因，和的Tra-1-60 1-3的抗人类安全公司。如果人类安全公司不重视很好后传代，ROCK抑制剂（Y27632）可以被添加到帮助。对于那些有困难，保持其细胞多能性，一些潜在的问题...

人多能性干细胞（PSCs），包括人类胚胎干细胞（胚胎干细胞），诱导式多能性干细胞（iPS细胞），具有的能力，以产生在体内的每一个细胞类型，包括神经元1-3。定向分化为各种神经元亚型人分成合同持有这些细胞在再生医学中的应用的关键。在开发过程中的功能神经元亚型的产生是一个复杂的过程，涉及诱导的神经系的区域沿rostro尾鳍轴祖细胞，该规范，并从区域4,5祖细胞的有丝分裂后的神经元类型的分化。从2001年开始，一些系统已建立了从人类胚胎干细胞，它提供了一个平台，为后续神经元亚型6,7代产生神经系。根据发展原则，一些神经元类型，如8-12脊髓运动神经元，脑DOP13日至15日 ，胺类神经元和神经视网膜细胞16,17已有效地从人的产品分成合同中指定。这样做是通过施加临界形态发生素体内发育过程中的规格，这些神经元类型是重要的。其他协议也得到了发展，促进人类胚胎干细胞分化成神经元可以使用其他的因素，如小分子或与其他类型的细胞共培养，以18-20帮助促进花芽分化21。 人类的大脑新皮质是高度发达的，包含许多类型的细胞，包括谷氨酸能神经元在学习，记忆和认知功能的22,23，其中扮演了重要的角色。产生谷氨酸能神经元在培养的第一个步骤是指定端脑的祖细胞。笹井芳树的组首次报道了端脑前体定向分化的小鼠胚胎干细胞（mESCs）使用无血清悬索桥的N培养物的存在下，DKK1（抑制Wnt信号）以及LeftyA（抑制节点信令）24。随后，几个研究小组还报告了包括我们在内的人类在无血清培养基25日至27日的物业服务端脑前体的规范。并不需要外源的形态发生和效率产生这些前体的使用是远高于从mESCs 26,27的端脑前体从人类安全公司代。在这里，神经诱导，以及设立的张的基7的化学定义的系统已被描述。此协议没有此外外源性caudalizing因素，有效地产生端脑前体从人类安全公司27。通过调节Wnt信号和Sonic Hedgehog（SHH）的信令，这些祖细胞可以分化成背侧或腹侧祖细胞，，背祖细胞进一步分化成谷氨酸能神经元Ëfficiently 27。此外，该协议还可以很好地用于谷氨酸能神经元的产生，从人类iPSCs 28，这允许针对具体患者的神经元的产生，可以利用机制的探索行动，以及潜在的治疗疾病的大型阵列。此外，我们的系统还提供了一个平台，探索不同的前脑的神经元类型的发展和规范。

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Supplier</td>
			<td>Catalog #</td>
		</tr>
		<tr>
			<td>Dulbecco's modified eagle medium with F12 nutrient mixture (DMEM/F12)</td>
			<td>Gibco</td>
			<td>11330-032</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>Gibco</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030</td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>Gibco</td>
			<td>1140-050</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (14.3 M)</td>
			<td>Sigma</td>
			<td>M-7522</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587-010</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (polyornithine)</td>
			<td>Sigma</td>
			<td>116K5103</td>
		</tr>
		<tr>
			<td>Laminin (human)</td>
			<td>Sigma</td>
			<td>L-6274</td>
		</tr>
		<tr>
			<td>Laminin (mouse)</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini</td>
			<td>100-106</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A-7906</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Invitrogen</td>
			<td>17104-019</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
		</tr>
		<tr>
			<td>ROCK Inhibitor</td>
			<td>Stemgent</td>
			<td>04-0012</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Stemgent</td>
			<td>04-0024</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2, bFGF)</td>
			<td>Invitrogen</td>
			<td>13256-029</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075</td>
		</tr>
		<tr>
			<td>0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
		</tr>
		<tr>
			<td>Foxg1 antibody</td>
			<td>Dr. Y. Sasai</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoxb4 antibody (1:50)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>I12</td>
		</tr>
		<tr>
			<td>Pax6 antibody (1:5000)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>PAX6</td>
		</tr>
		<tr>
			<td>Nkx2.1 antibody (1:200)</td>
			<td>Chemicon</td>
			<td>MAB5460</td>
		</tr>
		<tr>
			<td>Tbr1 antibody (1:2000)</td>
			<td>Chemicon</td>
			<td>AB9616</td>
		</tr>
		<tr>
			<td>vGLUT1 antibody (1:100)</td>
			<td>Synaptic Systems</td>
			<td>135302</td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-02</td>
		</tr>
		<tr>
			<td>Glial derived neurotrophic factor (GDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-10</td>
		</tr>
		<tr>
			<td>Insulin growth factor 1 (IGF1)</td>
			<td>PrepoTech Inc.</td>
			<td>100-11</td>
		</tr>
		<tr>
			<td>Cyclic AMP (cAMP)</td>
			<td>Sigma</td>
			<td>D-0260</td>
		</tr>
		<tr>
			<td>Sonic hedgehog (SHH)</td>
			<td>R&amp;D</td>
			<td>1845-SH</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Becton Dickinson (BD)</td>
			<td>352098</td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>BD</td>
			<td>352097</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>BD</td>
			<td>353046</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>BD</td>
			<td>353047</td>
		</tr>
		<tr>
			<td>T25 flasks (untreated)</td>
			<td>BD</td>
			<td>353009</td>
		</tr>
		<tr>
			<td>T75 flasks (untreated)</td>
			<td>BD</td>
			<td>353133</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Chemiglass Life Sciences</td>
			<td>1760-012</td>
		</tr>
		<tr>
			<td>6 cm Petri dishes</td>
			<td>BD</td>
			<td>353004</td>
		</tr>
		<tr>
			<td>9'' glass pipetes</td>
			<td>Fisher</td>
			<td>13-678-20D</td>
		</tr>
		<tr>
			<td>Steriflip filters (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
		</tr>
		<tr>
			<td>Stericup filters 1,000 ml (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGPU10RE</td>
		</tr>
		<tr>
			<td>Phase contrast microscope (Observer A1)</td>
			<td>Zeiss</td>
			<td>R2625</td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator (Hera Cell 150)</td>
			<td>Thermo Electron Corporation</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biosafety hood (Sterilgard III Advance)</td>
			<td>The Baker Company</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge (5702 R)</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

the specification of telencephalic glutamatergic neurons from human pluripotent stem cells

这里，已经描述了用于高效地生成从人多能性干细胞（PSCs）端脑谷氨酸能神经元的一个逐步的过程。的分化过程开始向上舍入到时形成聚集体的悬浮培养细胞被放置在成团块通过打破人类安全公司。聚集在人类胚胎干细胞培养基中生长1-4天，以允许自发分化。在这段时间内，将细胞有能力成为任何三种胚层。从5-8天，将细胞放置在一个神经诱导培养基中，推入神经谱系。第8天左右，细胞被允许连接到6孔板上，并分化期间的时间的神经上皮细胞形式。这些神经上皮细胞，可以隔离17天。这些细胞可以保持神经球，直到他们准备好盖玻片的培养皿上。使用碱性介质中没有任何caudalizing因素，神经上皮细胞specifieD插入端脑前体，然后可以进一步区分有效地背端脑祖细胞和谷氨酸能神经元的。总的来说，我们的系统提供了一个工具来生成人体谷氨酸能神经元的研究人员研究这些神经元疾病，影响到他们的发展。

在这里，一个协议来区分人类安全公司到端脑谷氨酸能神经元通过几个关键步骤：PSC的聚集体的形成，神经上皮细胞的诱导，在前脑祖细胞的生成，这些祖细胞的终末分化的成端脑的神经元（ 图1）具有被描述。此系统是稳健的和有效率的，在端脑祖细胞和谷氨酸能神经元的生成。作为一个例子（ 图2），未经此外caudalizing因素，胚胎干细胞分化为神经系27。在24天...

Watch this Scientific Journal Video about The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells at JoVE.com

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

此过程产生在前脑的​​神经元通过检查站，这是人类发展过程中所观察到的类似。细胞可自发地分化，暴露因素推动对神经系，是孤立的，并镀上盖玻片，以便终末分化和成熟。

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 ...

Research

JoVE Journal

Neuroscience

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 ...

科研

神经科学

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 ...

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

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

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

从人多能干细胞定向多巴胺能神经元分化

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

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

分化后再蛋白的人类多能干细胞衍生神经元，用于高含量的内尿成生和突合性成熟筛查

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

人类诱导多能干细胞脑微血管内皮细胞的衍生、扩张、冷冻保存和特征化

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...