ES细胞源性神经上皮细胞培养
Summary
从胚胎干细胞（ES）细胞基质细胞衍生诱导活性（SDIA）上皮前体的推导。
Abstract
胚胎干细胞有潜力分化成所有胚层，这使得他们的新疗法的发展有吸引力的工具细胞。在一般情况下，ES细胞的分化如下的概念，首先生成不成熟的祖细胞，然后就可以繁殖成成熟的细胞表型分化。这也适用于ES细胞源性神经元的神经细胞的发展，其中以下两个主要步骤：首先，推导和扩大，不成熟的神经上皮前体和第二，其分化为成熟的神经细胞。从ES细胞产生的神经前体细胞胚体（EB）的形成，从而揭示了所有胚层，包括神经外胚层细胞的分化是基于一个共同的方法。另一种更有效的方法诱导上皮细胞的发育，使用基质细胞衍生诱导活性（SDIA），可实现与颅骨骨骨髓来源的基质细胞（1）共培养的ES细胞。两个EB形成和SDIA，揭示发展的莲座状结构，这被认为是类似于神经管和/或神经嵴祖。可以分离出神经前体，扩大和进一步分化成特定的神经元和神经胶质细胞，使用定义的文化条件。在这里，我们描述的一代在与基质细胞MS5（2-5）共培养的实验和隔离等花环。
Protocol
第1步板丝裂霉素C ggrowth抑制（10微克/毫升为2.5小时）MS5细胞明胶包30分钟（0.01％）6孔板中的α- MEM媒体的密度在70,000 /平方厘米。 当细胞附着并已经形成了单层（过夜增长），切换至SRM。 手动隔离ES细胞克隆胚胎干细胞的使用注射器文化与27 ½ G针。 1毫升的蓝色提示和低密度板MS5细胞（通常为2-3％菌落6孔板）仔细Tritrurate殖民地。 注：胚胎…
Discussion
此协议表明，在生成和隔离上皮细胞从人类胚胎干细胞使用SDIA的不同步骤。这种方法的应用是多方面的，在许多协议，并已用于生产特定的神经元（如1，2，5-9）。花环被认为是像神经管细胞与前表型（2，5，10），还含有神经嵴祖细胞（11，12）。此外，他们还保留一定程度的可塑性，因为它们可以在规定的培养条件下的具体因素的图案。因此，SDIA源性神经前体细胞可以引起多种细胞类型，从中央和外周神经系?…
Disclosures
The authors have nothing to disclose.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| L-glutamine | Gibco | 25030 | ||
| alpha-MEM | Gibco | 12571 | ||
| penicillin/streptomycin | Gibco | 15140 | ||
| Knockout-DMEM | Gibco | 10829 | ||
| Knockout serum replacement | Gibco | 10828 | ||
| MEM non-essential amino acid solution | Gibco | 12383 | ||
| DMEM/F12 | Gibco | 11330 | ||
| N2-A supplement | Stem Cell Technologies | 07152 | ||
| mitomycin-C | Sigma | M0503 | ||
| gelatine type-A | Sigma | G1890 | ||
| poly-L-ornithine | Sigma | P4957 | 0.01 % solution | |
| laminin | Sigma | L-2020 | ||
| fibronectin | Sigma | F2006 | ||
| basic fibroblast growth factor (bFGF) | Invitrogen | 13256 | ||
| 1 ml syringe with 27 1/2 G needle | Becton Dickinson | 309623 | ||
| N2-A media | medium | DMEM/F12 + 1% N2-A supplement | ||
| Serum replacement media (SRM) | medium | Knockout-DMEM + 20 % Knockout serum replacement +1% MEM non-essential amino acid solution + 2 mM L-glutamine | ||
| α-MEM media | medium | α-MEM + 10 % FBS + 2 mM L-glutamine + 1%penicillin/streptomycin | ||
| MS5 | cell line | stromal cells | ||
| Microscope | ||||
| 6 well plates | for tissue culture |
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Tags
ES CellsNeuroepithelial Cell CulturesDifferentiationGerm LayersTherapiesProgenitor CellsCellular PhenotypesNeurogenesisNeuroepithelial PrecursorsNeural CellsEmbryoid Body FormationNeuroectodermStromal Cell-derived Inducing Activity (SDIA)Co-culturingSkull Bone Marrow-derived Stromal CellsRosette-like StructuresNeural Tube-like ProgenitorsNeural Crest-like ProgenitorsIsolationExpansionSpecific NeuronsGlia Cells
Cite This Article
Karki, S., Pruszak, J., Isacson, O., Sonntag, K. C. ES Cell-derived Neuroepithelial Cell Cultures. J. Vis. Exp. (1), e118, doi:10.3791/118 (2006).