This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.
Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.
神经元由我们1 体外分化自人类诱导多能干细胞(人iPS细胞)和其他2,3的基因表达的研究表明,神经元hiPSC类似于胎儿,而不是成人脑组织。目前,hiPSC为基础的模型可能更适合于易感性的研究,而不是最近的功能,神经系统疾病。我们以前曾报道,精神分裂症hiPSC衍生的神经元的基因标记的显著馏分保守精神分裂症hiPSC源性神经祖细胞(NPC的),这表明的NPC可能是一种有用的细胞类型用于研究分子途径促成精神分裂症1 。我们和其他人已经报道异常迁移,增加的氧化应激和活性氧,灵敏度到亚阈值的环境压力和在精神分裂症受损线粒体功能hiPSC的NPC 1,4-6,以及减小神经Çonnectivity和精神分裂症hiPSC神经元突触5,7-10功能。如果分子因素在精神分裂症hiPSC的NPC有助于异常迁移和/或氧化胁迫也背后精神分裂症hiPSC衍生的神经元的减少神经连接性,NPC的可能是一个强大的和高度复制神经元群,用以研究负责疾病的机制。此外,由于可以迅速地产生大量的细胞,并且不需要等待数周或数个月的神经元成熟,鼻咽癌基测定法适用于较大的患者群的研究,并且更适合于高通量筛选。我们相信,hiPSC的NPC可以作为可能造成疾病的发病机制的发育途径的代理,如已被证明在疾病等不同精神分裂症1和亨廷顿氏病11。
从人iPS细胞,在最初的神经分化的NPCduction由双SMAD抑制(0.1mM的LDN193189和10毫SB431542)12完成。通过拮抗骨形态发生蛋白和TGF与这些小分子信令,内胚层和中胚层规范被阻塞,加速神经元分化,并导致的可见神经花环形成电镀的一个星期内。神经形成发生在这一过程的早期,在神经花环形成,随后立即期间大概。在没有其他线索,这些原始神经细胞承担前端脑样的命运13。紧随其后的神经花环形成,并在整个NPC不断扩大,前脑的NPC与FGF2 8,14培养。它们具有双重谱系的潜力,并且可以分化为70-80%III微管蛋白阳性的神经元和20%-30%胶质纤维酸性蛋白(GFAP)阳性的星形胶质细胞( 图1)的神经种群。大多数脑hiPSC神经元是VGLUT1阳性等等都是推测谷氨酸,尽管神经元的大约30%是GAD67阳性(GABA能)8。
NPC的例行传代在体外的十倍以上,同时保持一致分化型材,和没有积累核型异常。组报道传代的NPC超过40倍15,然而,我们发现,超过10通道,NPC的显示增加倾向星形胶质细胞分化。 NPC的良好耐受多次冷冻 – 解冻,并且可以转换通过简单地在培养非粘附板成长为神经球。的NPC用病毒载体转导的效率,从而使遗传扰动的分子和细胞后果快速评估,并易于扩展,以产生足够的材料用于生化研究。另外,因为病毒载体容许鲁棒过度表达和/或与疾病相关的基因敲除,在对照或患者来源neur人的细胞,可以使用这个平台来测试对这些操作的遗传背景的影响。虽然不适合突触或基于活动的检测需要成熟的神经元,NPC的可能是来自患者的神经细胞的许多简单的分子或生化分析一个实际的选择。
我们已经描述了通过该分化人iPS细胞的NPC成,神经细胞类型,其中hiPSC衍生的神经元的基因标记的显著分数是保守,并且可以作为一个代理可能造成疾病的发病机制8的发育途径的方法, 11。此外,正如我们已经详细介绍,NPC的是一个稳健的复制和转导容易神经元群,我们相信这可能是用于疾病易感性的分子和生化研究。
虽然我们已经详细方法分化和培养前脑?…
The authors have nothing to disclose.
Kristen Brennand is a New York Stem Cell Foundation – Robertson Investigator. The Brennand Laboratory is supported by a Brain and Behavior Young Investigator Grant, National Institute of Health (NIH) grant R01 MH101454 and the New York Stem Cell Foundation.
Name of Material/ Equipment | Company | Catalog Number | Comments |
DMEM/F12 | Life Technologies | #11330 | for HES media |
DMEM/F12 | Life Technologies | #10565 | for neural media |
KO-Serum Replacement | Life Technologies | #10828 | Needs to be lot tested |
Glutamax | Life Technologies | #35050 | |
NEAA | Life Technologies | #11140 | |
2‐mercaptoethanol (55mM 1000x) | Life Technologies | #21985-023 | |
N2 | Life Technologies | #17502-048 | Needs to be lot tested |
B27-RA | Life Technologies | #12587-010 | Needs to be lot tested |
FGF2 | Life Technologies | #13256-029 | Resuspend in PBS + 1% BSA |
LDN193189 | Stemgent | #04-0074 | |
SB431542 | Stemgent | #04-0010 | |
BDNF | Peprotech | #450-02 | Resuspend in PBS + 0.1% BSA |
GDNF | Peprotech | #450-10 | Resuspend in PBS + 0.1% BSA |
Dibutyryl cyclic-AMP | Sigma | #D0627 | Resuspend in PBS + 0.1% BSA |
L-ascorbic acid | Sigma | #A0278 | Resuspend in H20 |
STEMdiff Neural Rosette Selection Reagent | Stemcell Technologies | #05832 | |
Accutase | Innovative Cell Technologies | AT-104 | |
Collagenase IV | Life Technologies | #17104019 | |
CF1 mEFs | Millipore | #PMEF-CF | |
Poly-L-Ornithine | Sigma | P3655 | |
Laminin, Natural Mouse 1mg | Life Technologies | #23017-015 | |
BD Matrigel | BD | #354230 | Resuspend on ice in cold DMEM at 10mg/ml, use 1mg per two 6-well plate equivalent |
Tissue culture treated 6-well plates | Corning | 3506 | |
Ultra low attachment 6-well plates | Corning | 3471 | |
goat anti-Sox2 | Santa Cruz | sc17320 | use at 1:200 |
mouse anti-human Nestin | Millipore | MAB5326 | use at 1:200 |
rabbit anti-βIII-tubulin | Covance | PRB435P | use at 1:500 |
mouse anti-βIII-tubulin | Covance | MMS435P | use at 1:500 |
mouse anti-MAP2AB | Sigma | M1406 | use at 1:200 |
Plate centrifuge | Beckman Coulter | Beckman Coulter Allegra X-14 (with SX4750 plate carrier) |