从废弃的人类胎儿大脑皮质组织神经干细胞的生成
Summary
描述一个简单和可靠的方法是从丢弃的人类胚胎皮层组织的神经干细胞的分离和培养。从已知的人类神经系统疾病的病理细胞和分子过程的特性，可用于文化，以及提供一个平台，以评估药理药效。
Abstract
神经干细胞（NSCs）居住在沿脑室区神经上皮的皮质板的发展。这些早期的祖细胞，最终上升到中间的祖先，后来，大脑皮层的各种神经元和神经胶质细胞亚型。发电量和扩大人类神经干细胞从废弃的正常胎儿组织（所谓的神经球）提供了一种手段，用以直接研究人体正常的国科会发展 1-5的功能方面。这种方法也可以直接向从已知的神经系统疾病的神经干细胞的生成，从而给予的机会，以确定疾病的进程，改变祖细胞的增殖，迁移和分化6-9。我们一直专注于识别人类唐氏综合征的神经干细胞可能有助于加速老年痴呆症的疾病表型10,11的病理机制。无论在体内还是在体外培养的小鼠模型，可以复制的基因位于人类21号染色体上的相同的剧目。
这里我们使用一个简单而可靠的的方法隔离综合征从流产的人类胚胎皮层神经干细胞，并在文化中成长。该方法提供有限的解剖标志，细胞分选，电镀和人类神经干细胞传代收获组织，解剖的具体方面。我们还提供一些基本的协议更有选择性的细胞亚型的人类神经干细胞诱导分化。
Protocol
1。神经干细胞培养的清扫和维护的解决方案和材料的制备提前准备100毫升夹层中（KNOCKOUT的DMEM/F12，Invitogen）和冷藏。 Prepare100毫升培养液（干临NSC可持续森林管理，Invitrogen公司），并保持在37 ° C在水浴。 准备长期长期冷冻保存的细胞的细胞冷冻培养基（KNOCKOUT的DMEM/F12 +10％胎牛血清+ 5％DMSO）。 如果需要，准备为4％多聚甲醛（PFA）组织固定。 消毒，灭菌镊子…
Discussion
是对文化的新鲜组织和人类细胞系生产的各种方法。从历史上看，已经收获和新鲜组织培养，立即在中枢神经系统中产生的各种细胞类型。然而，这种做法显然是有限的，可这在人类样本的情况下，通常是相当小的样本数量。鉴于操纵的最小程度，新鲜培养的神经细胞提供最可靠的实验系统，通过限制延长培养潜在的工件。供应有限导致的第二种方法产生人体细胞线，已通过插入各种癌基因12…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是支持部分由美国国立卫生研究院：HD054347和NS063997 – 01到VLS的。通过国务院卫生合同＃C024324到VLS的纽约帝国州干细胞基金中的一部分，这项工作也得到了支持。这里所表达的意见仅为作者个人的，不一定反映帝国干细胞，纽约州卫生署，纽约州的。 VLS的是一个多丽丝公爵临床科学家发展奖得主。我们也感谢他的反O1，反物理学抗体的礼物教授蒂莫西Vartanian。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| KNOCKOUT DMEM/F12 | Invitrogen | 12660-012 | Dissociation medium |
| Stem Pro NSC SFM | Invitrogen | A10509-01 | Culture medium |
| Fetal Bovine Serum | Invitrogen | 10091-148 | Frozen medium |
| Hanks solution (-Ca2+, -Mg2+) | Invitrogen | 14175-095 | Dissociation medium |
| DMSO | Sigma-Aldrich | D2650 | Frozen medium |
| EDTA | Sigma-Aldrich | 431788 | Dissociation medium |
| Paraformaldehyde | Sigma-Aldrich | 158127 | Fixation solution |
| bFGF | R&D | 234-FSE | Differentiation medium |
| SHH | R&D | 1845-SH | Differentiation medium |
| PDGF-AA | R&D | 221-AA | Differentiation medium |
| B27 | Invitrogen | 17504-044 | Differentiation medium |
| Mouse Anti-MAP2 | Sigma-Aldrich | M2320 | 1:200 |
| Rabbit Anti-DCX | Cell signaling | 4604s | 1:200 |
| Rabbit Anti-GFAP | DAKO | Z0334 | 1:200 |
| Rabbit Anti-S100B | DAKO | Z0311 | 1:200 |
| Rabbit Anti-O1 | gifts of Professor Timothy Vartanian* | 1:50 | |
| Rabbit Anti-O4 | Gifts of Professor Timothy Vartanian* | 1:50 | |
| 40μm cell strainer | BD Falcon | 352340 |
* Timothy Vartanian, MD, PhD, Department of Neurology and Neuroscience, Weill Cornell Medical College, New York, USA
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Tags
Neural Stem CellsNSCsNeuroepitheliumCortical PlateIntermediate ProgenitorsNeuronal CellsGlial CellsCerebral CortexNormal Human NSC DevelopmentNeurological DisordersProgenitor ProliferationMigrationDifferentiationDown Syndrome NSCsAlzheimer’s Disease PhenotypeGenes Located On Human Chromosome 21DissectionCell SortingPlatingPassaging Of Human NSCsDifferentiation Of Human NSCs
Cite This Article
Lu, J., Delli-Bovi, L. C., Hecht, J., Folkerth, R., Sheen, V. L. Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue. J. Vis. Exp. (51), e2681, doi:10.3791/2681 (2011).