本研究介绍并描述了得出两种特定人类神经器官的规程,作为研究人类神经器官内人类胶质细胞瘤发育的相关和准确的模型,2)神经元多巴胺能分化产生三维有机体。
缺乏相关的体外神经模型是神经病理学医学进展的重要障碍。建立相关的细胞模型对于更好地了解这些疾病的病理机制和确定新的治疗目标和策略至关重要。为了相关,体外模型必须复制人类疾病的病理特征。然而,在神经退行性疾病的背景下,相关的体外模型应提供神经细胞替代作为宝贵的治疗机会。
这种模型不仅允许筛选治疗性分子,还可用于优化神经协议分化[例如,在帕金森病(PD)的移植环境中]。本研究描述了人类神经器官(NO)和2)神经元多巴胺能(DA)分化内人类胶质细胞瘤发育的两个体外方案,产生三维(3D)有机体。为此,建立了一个标准化良好的协议,允许生产从人类胚胎干细胞(hESC)分化中提取的经过校准的大小的神经球。第一个模型可用于揭示神经器官内胶质母细胞瘤发育过程中发生的分子和细胞事件,而DA有机体不仅代表了帕金森病中细胞治疗的DA神经元的合适来源,而且还可以用于药物测试。
世界卫生组织(世卫组织)将星形细胞瘤归类为低年级(一至二年级)或高年级(三级和四级)。胶质母细胞瘤多毛病(GBM)是星形细胞瘤IV级,最致命的原发性脑肿瘤,是抵抗所有目前形式的治疗1。尽管包括神经外科、化疗和放疗在内的标准护理治疗,GBM仍然致命,15个月的整体存活率在过去15年中没有显著变化2。要在了解 GBM 发病机制方面取得显著进展,使用相关模型是关键。到目前为止,GBM的研究已经依赖于细胞系,啮齿动物器官切片,和异种移植患者衍生细胞到小鼠或转基因小鼠发展自发肿瘤3,4。虽然这些模型对研究脑转移和肿瘤攻击性非常有用,但它们受到物种间差异的限制,因此得出的结论可能错误地转化为人体组织。此外,现有的人体细胞模型也受到缺乏宿主组织/肿瘤相互作用3,4的限制。实验模型对于从基础科学到治疗靶点的转换至关重要。因此,描述与GBM启动细胞(GIC)共同培养的体外人类神经器官生成方案可以提供一个模拟GBM发育的形态和功能特征的相关系统。该系统再现了GBM发展的一些体内特征,如入侵细胞和坏死区的扩散迁移,突出与肿瘤生物学相关的基因表达。如前文所述,一些关键微RNA在3D神经组织5、6的GIC发育过程中被诱导。
PD是一种主要的神经退行性疾病,与多个神经元亚型的退化7相关。即使症状的逐渐出现(例如,血症、不对称的休息震颤、僵硬和姿势不稳定)是该病的特征,其确切病因也没有明确确定。事实上,许多研究都强调了主要风险因素可能由遗传和环境因素共同作用的证据。帕金森症状与亚斯坦西亚尼格拉(SN)中多巴胺能神经元的双边退化有关,导致多巴胺基(DA)的斧状消失,并投射到纹状体8,9。因此,纹状多巴胺水平的降低与PD患者运动功能障碍的进展相关。多巴胺能神经元含有酪氨酸羟基酶(TH),这是一种合成儿茶胺能神经递质的关键酶,可将氨基酸L-酪氨酸转化为L-3,4-二羟基苯甲氨酸(L-DOPA,多巴胺前体)转化为多巴胺10。TH活性的早期丧失,随后TH蛋白表达下降是PD的一个标志。
本研究描述了使用人类神经器官的两种方案,其中一种专门针对富含TH阳性细胞的中脑样表型。
该协议最重要的方面之一包括维持hESC多能性在细胞培养和密切监测球体和神经器官形态。hESCs非常敏感,每次操作都可能导致早期不受控制的分化以及细胞死亡。为了提高实验的可重复性,避免异常型骨型事件的发生,建议在验证其染色体稳定性后,在最低通道下冷冻保存几批hESCs。此外,建议为每个实验解冻一个新的小瓶,并检查细胞每天的行为。如果球体折射率较低,尺寸异常较大,则它们可能会开始聚集和死亡。
该系统的一个改进是灌注或实现血管化系统(通过添加内皮细胞或在3D流体微芯片内)12,13。然而,控制神经有机体的厚度(±300 μm)可以有效地被动灌注氧气和营养,并防止坏死。另一个改进是引入免疫细胞(微胶质)。考虑到这些限制,神经器官加上GIC系统可能是一个相关的工具,有几个原因。首先,该系统允许药物筛选,以监测治疗化合物如何影响器官或肿瘤细胞。其次,可以研究细胞与细胞之间的相互作用,可以可视化和探索个体和集体入侵背后的微环境决定因素5、6、13。
在帕金森病的背景下,在DA神经元中富集的神经有机体可以代表一个相关而准确的3D模型来研究疾病的发展。在以前的研究中,帕金森氏症患者衍生的诱导多能干细胞分化为DA神经元已被用来研究受影响的神经元亚型。值得注意的是,一些与疾病有关的表型,如β-核糖核酸的积累和对氧化应激的敏感性已经观察到14,15。此外,神经器官可用作筛选治疗分子的工具。然而,应该建立具体和相关的读出,以评估DA神经元的生存和功能,如多巴胺生产和电生理活动。总之,该协议提供了两种标准化和准确的基于干细胞的方法来生成神经器官。
The authors have nothing to disclose.
作者感谢癌症协会(瑞士日内瓦)、ISREC基金会(瑞士洛桑)和克莱顿研究基金会(美国得克萨斯州休斯敦)的财政支持。此外,作者感谢HES-HO和怀斯中心的财政支持。我们感谢克劳斯实验室的有益讨论和支持,并感谢Halah Kutaish博士的校对。
6-well plate (6-well plate) | Falcon / Corning | 07-201-588 | |
ABI Prism 7900 HT detection system (Real-Time PCR detection systems) | Applied Biosystems | Discontinued | |
Aggrewell 400 (Microwell culture plates ) | StemCell Technologies | 34421 | |
Amplifier (W2100-HS32) (Amplifier) | Multi Channel Systems | ||
Anti-EGFR (phospho Y1101) antibody | Abcam | ab76195 | 1/100 dilution |
Anti-GFAP Antibody | Dako | Z334 | 1/1000 dilution |
Anti-Nestin, Human Antibody | Millipore | ABD69 | 1/400 dilution |
Anti-Synapsin I Antibody | Chemicon | AB1543P | 1/500 dilution |
B27 supplements (B27) | Life Technologies / Invitrogen | 1238 | For both protocol, stock solution 100x, final solution 1x |
Brain-derived neurotrophic factor (BDNF) | Cell Guidance | GFH1-2 | For both protocol, stock solution 100 µg/mL in pure H2O, final solution 20 ng/mL |
CHIR-99021 (GSK-3β inhibitor ) | Axon Medchem | ct99021 | For Dopaminergic protocol, stock solution 7.5 mM in DMSO, final solution 3 µM |
Compound E a γ–secretase inhibitor (γ–secretase inhibitor) | Calbiochem | CAS 209986-17-4 | For both protocol (gamma-secretase inhibitor XXI), stock solution 5 mM in DMSO, final solution 1 µM |
Coulochem III (Coulometric detector parameters) | Thermo scientific | ||
Dibutyryl cyclic-AMP (Dibutyryl cAMP) | Sigma | D0627 | For Dopaminergic protocol, stock solution 0.5 M in DMSO, final solution 0.5 mM |
Dimethyl Sulfoxide Pure (DMSO) | Sigma-Aldrich | C6164 | Compounds solvent, ready to use |
Dulbecco's Modified Eagle Medium (DMEM) | Life Technologies | 12491-015 | For cell culture, ready to use |
Dulbecco's Modified Eagle Medium Mixture F-12 (DMEM-F12) | Gibco | 11320033 | For cell culture, ready to use |
EDTA 0.1 mM (EDTA) | Life Technologies | AM9912 | For cell culture, ready to use |
Epidermal Growth Factor (EGF) | Gibco | PHG0313 | For GIC culture, stock solution 100 µg/mL in pure H2O, final solution 10 ng/mL |
Fibroblast Growth Factor 20 (FGF20) | Peprotech | 100-41 | For Dopaminergic protocol, stock solution 100 µg/mL in pure H2O, final solution 5 ng/mL |
fibroblast growth factor 8 (FGF8) | Peprotech | GFH176-5 | For Dopaminergic protocol, stock solution 100 µg/mL in pure H2O, final solution 100 ng/mL |
Fibroblast growth factor-basic (bFGF) | Gibco | PHG0024 | For GIC culture, stock solution 100 µg/mL in pure H2O, final solution 10 ng/mL |
G5 supplements (G5) | Invitrogen | 17503012 | For GIC culture, stock solution 100x, final solution 1x |
Glial cell-derived neurotrophic factor (GDNF) | Cell Guidance | GFH2-2 | For both protocol, stock solution 100 µg/mL in pure H2O, final solution 20 ng/mL |
Hydrophilic polytetrafluoroethylene membrane (PTFE membrane) | BioCell-Interface | Discontinued | |
LDN-193189 (BMP inhibitor) | Axon Medchem /Stemgen | 04-0072-02 /1509 | Dual/Smad, stock solution 5 mM in DMSO, final solution 0.5 µM |
L-glutamine (L-glutamine) | Gibco | 25030081 | L-Glutamine (200 mM), stock solution 200 mM, final solution 2 mM |
Matrigel (extracellular matrix) | BD Biosciences | 354277 | hESC-qualified Matrix, stock solution 18-22 mg/mL, final solution 180-220 µg/mL |
Millicell-CM Culture plate insert (0.4 µm) (Culture plate insert) | Millipore | PICM03050 | |
Monoclonal Anti-β-Tubulin III antibody | Sigma | T8660 | 1/1000 dilution |
MS Orbital Shaker, MS-NOR-30 (Orbital shaker) | Major Science | MS-NRC-30 | |
N2 supplements (N2) | Invitrogen | 17502-048 | For GIC culture, stock solution 100x, final solution 1x |
Nanodrop (Nanodrop) | Thermo Fisher Scientific | Discontinued | |
Neurobasal (Neurobasal) | Life Technologies / Gibco | 21103049 | Maintenance and maturation embryonic neuronal cell populations , ready to use |
Non-Essential Amino Acids (NEAA) | Gibco | 11140 | Non-essential Amino Acids 100X, stock solution 100x, final solution 1x |
Nurr1 Antibody (M-196) | Santa Cruz | Sc-5568 | 1/100 dilution |
Nutristem (hESC medium ) | Biological Industries | 05-100-1A | Stem cell media, ready to use |
Penicilin / Streptomycin (Penicilin / Streptomycin ) | Life Technologies / Gibco | 15140122 | For cell culture, stock solution 5 mg/mL, final solution 50 µg/mL |
Perchloric acid 0.1N (HCLO4) | Merck | 100519 | For HPLC, ready to use |
Phosphate Buffered Saline without Ca2+/Mg2+ (PBS without Ca2+/Mg2+ ) | Life Technologies | 14190250 | For cell culture, ready to use |
PrimeScript RT-PCR Kit (Reverse transcription kit) | Takara | RR014A | |
Purmorphamin (smoothened agonist) | Calbiochem | SML0868 | For Dopaminergic protocol, stock solution 10 mM in DMSO, final solution 2 µM |
Rho-associated Kinase Y-27632 (ROCK) | Abcam Biochemicals | ab120129-1 | Rock Inhibitor, stock solution 50 mM in DMSO, final solution 10 µM |
RNeasy mini kit (RNA extraction kit ) | Qiagen | 74104 | |
SB-431542 (TGFβ/Activin/Nodal inhibitor ) | Ascent | Asc- 163 | Dual-Smad, stock solution 50 mM in DMSO, final solution 10 µM |
Sonic Hedgehog (SHH) | Cell Guidance | GFH168-5 | For Dopaminergic protocol, stock solution 100 µg/mL in pure H2O, final solution 100 ng/mL |
StemPro Accutase (hESC enzymatic solution) | Gibco | A11105-01 | hESC enzymatic solution, ready to use |
Symmetry C-18,5 mm (4.6 150mm2) (Reversed-phase column) | Waters Corporation | ||
T150 flask (T150 flask) | Falcon | 08-772-1F | |
TH Antibody (F-11) | Santa Cruz | Sc-25269 | 1/200 dilution |
Transforming Growth Factors beta 3 (TGFβ3) | Cell Guidance | GFH109-2 | For Dopaminergic protocol, stock solution 100 µg/mL in pure ethanol , final solution 1 ng/mL |
Trichostatine A (inhibitor of histone deacetylase ) | Sigma | T8552 | For Dopaminergic protocol, stock solution 100 µM in DMSO, final solution 20 nM |
TrypLE (recombinant enzymatic solution) | Invitrogen | 12604021 | recombinant enzymatic solution, ready to use |
Trypsin 0.25% (enzymatic solution) | Life Technologies | 15050065 | enzymatic solution, ready to use |
W2100, Multi Channel Systems (Data acquisition system ) | WAT045905 | ||
X-vivo (serum free medium) | Lonza | BE04-743Q | serum free medium, ready to use |