从人多能干细胞定向多巴胺能神经元分化
Summary
We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson’s disease.
Abstract
Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson’s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.
Introduction
多巴胺（DA）神经元可以在几个脑区域，包括脑，下丘脑，视网膜，和嗅球中找到。在A9多巴胺神经元在黑质致密部（黑质）控制由突出到前脑，纹状体和形成锥体外运动系统的行为和动作。 A9多巴胺神经元变性导致帕金森氏病（PD），这是第二个最常见的人类神经变性疾病，目前无法治愈的。细胞替代疗法是最有前途的策略为PD 1的处理中的一个，因此，出现了在由人多能干细胞（hPSCs）导出A9多巴胺神经元的极大兴趣，包括人类胚胎干细胞（胚胎干细胞）和最近的人类诱导多能干细胞（iPS细胞）。
许多研究都试图通过各种方法hPSCs得到A9的多巴胺神经元。通过神经网络的最早报道都产生多巴胺的神经元莲座祖阶段。通过合作，培养与MS5 2或3-5尼龙6间质细胞有或没有外部生长因子2-4周，L的Studer和同事成功地诱导人类胚胎干细胞产生神经花环其他三个组。然后，他们丰富了这些玫瑰花机械清扫或酶消化进一步分化。在其他报告中，研究人员通过生成的胚体悬浮培养分化6-9神经花环（EB）。后来，研究人员建立了基于单层分化方法10,11，在那里它们镀人类胚胎干细胞和人iPS细胞的细胞外基质，添加于不同生长因子的培养以诱导hPSCs分化为多巴胺神经元，其模拟了体内胚胎DA神经元的发展。虽然所有这些研究中得到的酪氨酸羟化酶（TH）表达细胞与多巴胺神经元的某些特征，整个分化过程中的时间和劳动消耗，通常效率低，而且更重要的是，这些神经元的A9身份，除了一个与LMX1a异位表达12多数研究都没有表现出来。最近，一种新的地板板（FP）的协议被开发13-16，其中，首先由活化的音猬和经典Wnt信号转导途径的过程中分化的早期阶段产生的FP的前体与多巴胺神经元的电势，然后这些计划生育细胞进一步指明DA能神经元。虽然这个协议是更有效的，但仍存在一些问题;例如，在整个分化过程需要很长的时间（至少35天），是饲养细胞依赖的15，或者是EB依赖性16或A9身份没有被证实14。
在此基础上，从体内胚胎多巴胺神经元发育等研究人员公布业绩的知识，我们优化培养条件为EFFicient代来自人类胚胎干细胞和iPS细胞多巴胺神经元。我们首先生成的FP的前体细胞通过激活Wnt信号传导的小分子CHIR99021和音猬蛋白与小分子SAG和嘌吗啡胺的信令。这些计划生育细胞表达FOXA2，LMX1a，CORIN，OTX2和巢。然后，我们指定了这些计划生育细胞对多巴胺神经元的生长因子，包括BDNF，GDNF 等 。生成的多巴胺神经元的A9细胞类型，因为它们是阳性GIRK2而负钙结合蛋白17。该协议是饲养细胞和EB独立，高效和可重复性。通过此协议，一个也可以，或者从PD患者的体外模型的PD或测试潜在的治疗药物为PD iPS细胞在不到4周的胚胎干细胞或正常人的细胞移植研究iPS细胞获得多巴胺神经元。
Protocol
Representative Results
Discussion
人多能干细胞（包括胚胎干细胞和人iPS细胞）可以在体外分化而产生大部分，如果不是全部，我们的身体，包括多巴胺神经元，这已被证明在以前的研究中19的细胞类型。这里，基于从胚胎多巴胺能神经元的发展20知识和公布的协议从其他实验室14,15，我们优化了培养条件对从胚胎干细胞和iPS细胞多巴胺神经元的产生。这个协议是有效的，可重复的，我们已经成功地?…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank members of the Renee Reijo Pera laboratory for help during development of this protocol and during preparation of this manuscript. This work is supported by California Institute for Regenerative Medicine (CIRM) shared laboratory (CL-00518).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| DMEM | Life Technologies | 10569-010 | |
| FBS | Life Technologies | 26140 | |
| penicillin/ampicillin | Life Technologies | 15140-122 | |
| DMEM/F12 | Life Technologies | 10565-018 | |
| KSR | Life Technologies | 10828-028 | |
| Non-essential amino acid | Life Technologies | 11140-050 | |
| b-mercaptoethanol | Millipore | ES-007-E | |
| bFGF | R&D Systems | 233-FB-025 | |
| mTesR1 | STEMCELL Technologies | 5850 | |
| Collagenase IV | Life Technologies | 17104-019 | |
| Gelatin | Sigma-Aldrich | G9391 | |
| N2 supplement | Life Technologies | 17502-048 | |
| B27 supplement | Life Technologies | 17504-044 | |
| Neurobasal | Life Technologies | 21103-049 | |
| Glutamax | Life Technologies | 35050-061 | |
| PBS | Life Technologies | 10010-023 | |
| Growth factor reduced matrigel | BD Biosciences | 354230 | |
| Accutase | MP Biomedicals | 1000449 | |
| Thiazovivin | Santa Cruz Biotechnology | sc-361380 | |
| SB431542 | Tocris Bioscience | 1614 | |
| LDN-193189 | Stemgent | 04-0074 | |
| SAG | EMD Millipore | 566660-1MG | |
| Purmorphamine | Santa Cruz Biotechnology | sc-202785 | |
| FGF8b | R&D Systems | 423-F8-025 | |
| CHIR99021 | Cellagen Technology | C2447-2s | |
| BDNF | R&D Systems | 248-BD-025 | |
| GDNF | R&D Systems | 212-GD-010 | |
| TGF-beta3 | R&D Systems | 243-B3-002 | |
| Ascorbic acid | Sigma-Aldrich | A4034 | |
| cAMP | Sigma-Aldrich | D0627 | |
| Mouse anti human NESTIN antibody | Santa Cruz Biotechnology | sc-23927 | 1/1000 dilution |
| Rabbit anti human OTX2 antibody | Millipore | AB9566 | 1/2000 dilutiion |
| Goat anti human FOXA2 antibody | R&D Systems | AF2400 | 1/200 dilution |
| rabbit anti human LMX1a antibody | Millipore | AB10533 | 1/1000 dilution |
| Rabbit anti human TH antibody | Pel Freez | P40101 | 1/500 dilution |
| Chicken anti human TH antibody | Millipore | AB9702 | 1/500 dilution |
| Mouse anti human TUJ1 antibody | Covance | MMS-435P | 1/2000 dilution |
| Rabbit anti human GIRK2 antibody | Abcam | ab30738 | 1/300 dilution |
| Rabbit anti human Calbindin antibody | Abcam | ab25085 | 1/400 dilution |
| Centrifuge | Eppendorf | 5804 |
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