利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元
1Department of Biology and Biotechnology Charles Darwin,Sapienza University of Rome, Italy, 2Center for Life Nano Science,Istituto Italiano di Tecnologia, Italy, 3Laboratory of Stem Cell biology and Molecular Embryology,The Rockefeller University, USA, 4Department of Physiology and Pharmacology,Sapienza University of Rome, Italy
Summary
该协议允许诱导多能干细胞快速有效地转换为具有脊柱或颅内身份的运动神经元, 通过从诱导的 piggyBac 载体的转录因子的异位表达。
Abstract
我们这里描述了一种方法来获得功能性脊椎和颅内运动神经元从人类诱导的多能干细胞 (Ipsc)。直接转化为运动神经元是通过转录因子的替代模块, 即 Ngn2、Isl1 和 Lhx3 (NIL) 或 Ngn2、Isl1 和 Phox2a (NIP) 的异位表达而获得的。NIL 和 NIP 分别指定脊柱和颅内运动神经元的身份。我们的协议从生成修改后的 iPSC 线开始, 在这些线路中, NIL 或 NIP 通过 piggyBac 转座子载体稳定地集成到基因组中。然后由多西环素诱导转基因基因的表达, 并在5天内导致 Ipsc 转化为 MN 祖细胞。随后的成熟, 7天, 导致脊柱或颅内 Mna 的同质种群。与以前的协议相比, 我们的方法有几个优点: 它非常快速和简化;它不需要病毒感染或进一步 MN 分离;它允许产生不同的 MN 亚群 (脊椎和颅骨) 与显著的成熟程度, 这证明了火训练的动作电位。此外, 大量的运动神经元可以获得没有纯化从混合人群。ipsc 衍生的脊髓和颅内运动神经元可用于肌萎缩侧索硬化症和运动神经神经元的其他神经退行性疾病的体外建模。均匀运动神经元群可能是细胞类型特异性药物筛选的重要资源。
Introduction
运动神经元 (MN) 变性在肌萎缩侧索硬化症 (ALS) 和脊髓肌萎缩 (SMA) 等人类疾病中起着致病作用。建立合适的体外细胞模型系统, 总结人类 MN 的复杂性, 是朝着发展新的治疗方法迈出的重要一步。诱导多能干细胞 (ipsc) 具有显著的多系分化特性, 目前已从一些受运动神经元疾病 1,2 的患者中提取。从控制 “健康” 多能干细胞3开始, 基因编辑产生了更多携带与 MN 疾病相关的致病突变的 iPSC 系.这些生产线是体外疾病建模和药物筛选的有用工具, 前提是有适当的 iPSC 分为 Mn 的方法。这种方法发展的基本原理是为对 MN 疾病感兴趣的科学界提供快速有效的分化协议, 从而产生成熟的功能 Mn。此方法的第一个优点是其执行时间范围。另一个相关的强项来自于任何净化步骤的消除。最后, 该协议可用于生成两个不同的运动神经元群。
生成不同子类型的 mn 的可能性与 MN 疾病的建模特别相关。并非所有的 MN 亚型在 ALS 和 SMA 中都同样脆弱, 不同运动单位的症状出现对预后有很大影响。在 ALS 中, 脊柱出现症状开始于上肢和下肢, 导致死亡约 3-5年 4.相反, 球囊发作, 从颅骨 MNs 的退化开始, 有一个最坏的预后。此外, rna 结合蛋白 FUS 和 TDP-43 突变患者的球状发病百分比明显高于 SOD1 突变5患者。几乎所有的替代 mn 分化协议依赖于维甲酸 (ra) 的活性, 这赋予了脊椎字符分化 ipsc6,7,8。这限制了研究内在因素的可能性, 而内在因素可能对特定的 mn 亚型9、10 具有保护作用。
与以前在小鼠胚胎干细胞11中的研究一致, 我们最近已经表明, 在人类 Ipsc 中, Ngn2、isl1 和 Lhx3 (nil) 的异位表达诱导了脊柱 mn 的身份, 而 Ngn2 和 isl1 加 phox2a (nip) 则指定了颅内 mn12。因此, 我们开发了一个高效的协议, 导致在12天的周转时间内生产具有功能特性的人类 MNs。这种方法的目的是在很短的时间内, 在不需要纯化的情况下 (例如, 通过流式细胞仪), 获得具有脊柱或颅内身份的 MNs 高度丰富的细胞群。
Protocol
1. 人类 Ipsc 的维护 基质涂层板的制备 在4°C 下连夜解冻一个5毫升的基质 (见材料表)。原始的基质库存在不同的库存浓度下, 并根据数据表上显示的稀释系数进行。重要的是要保持小瓶和管冰凉, 以防止过早的胶凝矩阵。在冰上预先冷却的低温管中, 将基质分配到等价物中。在-20°c 时冻结未使用的脂肪。 将一个放在冰上大约2小时才能解冻。 <li…
Representative Results
图 1显示了微分方法的示意图描述。人体 Ipsc (WT I线 3) 通过 epB-Bsd-TT-NIL 或 EpB-Bsd-TT-NIL 进行转染, 在选择无菌性细胞线12后生成稳定和诱导细胞系, 以下分别称为 Ipsc-nil 和 ipsc-nip。对分化细胞的多能性标记 OCT4 和泛神经元标记 TUJ1 的表达进行了表征。免疫染色分析显示, 在没有 TUJ1 阳性的情况下, 在第0天, OCT4 在所有细胞中的表达均匀 (<strong c…
Discussion
由于谱系特异性转录因子的异位表达, 该协议可以有效地将人的 Ipsc 转化为脊柱和颅内运动神经元。这些转基因是由多西环素诱导和稳定地集成在基因组中感谢一个 piggyBac 转座子基载体。在混合人群中, 一个或多个携带 Bac 载体的副本将随机整合到单个细胞的基因组中, 从而增加基因组完整性改变的风险。此外, 随着时间的推移, 可能会逐步选择 iPSC 亚克隆, 可能会对分化以及疾病和控制细胞系的比?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者希望感谢意大利纳米科学研究所生命纳米科学中心的成像设施提供的支持和技术咨询。我们感谢生命纳米科学中心的成员进行了有益的讨论。这项工作得到了 AriSLA (2016年 “StressFUS” 试点赠款) 向 AR 提供的赠款的部分支持。
Materials
| 5-Bapta | Sigma-Aldrich | A4926-1G | chemicals for electrophysiological solutions |
| Accutase | Sigma-Aldrich | A6964-100ML | Cell dissociation reagent |
| anti-CHAT | EMD Millipore | AB144P | Anti-Choline Acetyltransferase. Primary antibody used in immunostaining assays. RRID: AB_2079751; Lot number: 2971003 |
| anti-goat Alexa Fluor 488 | Thermo Fisher Scientific | A11055 | Secondary antibody used for immonofluorescence assays. RRID: AB_2534102; Lot number: 1915848 |
| anti-mouse Alexa Fluor 647 | Thermo Fisher Scientific | A31571 | Secondary antibody used for immonofluorescence assays. RRID: AB_162542; Lot number: 1757130 |
| anti-Oct4 | BD Biosciences | 611202 | Primary antibody used in immunostaining assays. RRID: AB_398736; Lot number: 5233722 |
| anti-Phox2b | Santa Cruz Biotechnology, Inc. | sc-376997 | Primary antibody used in immunostaining assays. Lot number: E0117 |
| anti-rabbit Alexa Fluor 594 | Immunological Sciences | IS-20152-1 | Secondary antibody used for immonofluorescence assays |
| anti-TUJ1 | Sigma-Aldrich | T2200 | Primary antibody used in immunostaining assays. RRID: AB_262133 |
| B27 | Miltenyi Biotec | 130-093-566 | Serum free supplement for neuronal cell maintenance |
| Bambanker | Nippon Genetics | NGE-BB02 | Cell freezing medium, used here for motor neuron progenitors |
| BDNF | PreproTech | 450-02 | Brain-Derived Neurotrophic Factor |
| Blasticidin | Sigma-Aldrich | 203350 | Nucleoside antibiotic that inhibits protein synthesis in prokaryotes and eukaryotes |
| BSA | Sigma-Aldrich | A2153 | Bovine Serum Albumin. Blocking agent to prevent non-specific binding of antibodies in immunostaining assays |
| CaCl2 | Sigma-Aldrich | C3881 | chemicals for electrophysiological solutions |
| Clampex 10 software | Molecular Devices | Clampex 10 | Membrane currents recording system |
| Corning Matrigel hESC-qualified Matrix | Corning | 354277 | Reconstituted basement membrane preparation from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Used for adhesion of iPSC to plastic and glass supports |
| CRYOSTEM ACF FREEZING MEDIA | Biological Industries | 05-710-1E | Freezing medium for human iPSCs |
| D-Glucose | Sigma-Aldrich | G5146 | chemicals for electrophysiological solutions |
| DAPI powder | Roche | 10236276001 | 4′,6-diamidino-2-phenylindole. Fluorescent stain that binds to adenine–thymine rich regions in DNA used for nuclei staining in immonofluorescence assays |
| DAPT | AdipoGen | AG-CR1-0016-M005 | Gamma secretase inhibitor |
| Dispase | Gibco | 17105-041 | Reagent for gentle dissociation of human iPSCs |
| DMEM/F12 | Sigma-Aldrich | D6421-500ML | Basal medium for cell culture |
| Doxycycline | Sigma-Aldrich | D9891-1G | Used to induce expression of transgenes from epB-Bsd-TT-NIL and epB-Bsd-TT-NIP vectors |
| DS2U | WiCell | UWWC1-DS2U | Commercial human iPSC line |
| E.Z.N.A Total RNA Kit | Omega bio-tek | R6834-02 | Kit for total extraction of RNA from cultured eukaryotic cells |
| GDNF | PreproTech | 450-10 | Glial-Derived Neurotrophic Factor |
| Gibco Episomal hiPSC Line | Thermo Fisher Scientific | A18945 | Commercial human iPSC line |
| Glutamax | Thermo Fisher Scientific | 35050038 | An alternative to L-glutamine with increased stability. Improves cell health. |
| Hepes | Sigma-Aldrich | H4034 | chemicals for electrophysiological solutions |
| iScript Reverse Transcription Supermix for RT-qPCR | Bio-Rad | 1708841 | Kit for gene expression analysis using real-time qPCR |
| iTaqTM Universal SYBR Green Supermix | Bio-Rad | 172-5121 | Ready-to-use reaction master mix optimized for dye-based quantitative PCR (qPCR) on any real-time PCR instrument |
| K-Gluconate | Sigma-Aldrich | G4500 | chemicals for electrophysiological solutions |
| KCl | Sigma-Aldrich | P9333 | chemicals for electrophysiological solutions |
| L-ascorbic acid | LKT Laboratories | A7210 | Used in cell culture as an antioxidant |
| Laminin | Sigma-Aldrich | 11243217001 | Promotes attachment and growth of neural cells in vitro |
| Laser scanning confocal microscope | Olympus | iX83 FluoView1200 | Confocal microscope for acquisition of immunostaining images |
| Mg-ATP | Sigma-Aldrich | A9187 | chemicals for electrophysiological solutions |
| MgCl2 | Sigma-Aldrich | M8266 | chemicals for electrophysiological solutions |
| Mounting Medium | Ibidi | 50001 | Mounting solution used for confocal microscopy and immunofluorescence assays |
| Multiclamp patch-clamp amplifier | Molecular Devices | 700B | Membrane currents recording system |
| Na-GTP | Sigma-Aldrich | G8877 | chemicals for electrophysiological solutions |
| NaCl | Sigma-Aldrich | 71376 | chemicals for electrophysiological solutions |
| NEAA | Thermo Fisher Scientific | 11140035 | Non-Essential Amino Acids. Used as a supplement for cell culture medium, to increase cell growth and viability. |
| Neon 100 μL Kit | Thermo Fisher Scientific | MPK10096 | Cell electroporation kit |
| Neon Transfection System | Thermo Fisher Scientific | MPK5000 | Cell electroporation system |
| Neurobasal Medium | Thermo Fisher Scientific | 21103049 | Basal medium designed for long-term maintenance and maturation of neuronal cell populations without the need for an astrocyte feeder layer |
| NutriStem-XF/FF | Biological Industries | 05-100-1A | Human iPSC culture medium |
| Paraformaldehyde | Electron Microscopy Sciences | 157-8 | Used for cell fixation in immunostaining assays |
| PBS | Sigma-Aldrich | D8662-500ML | Dulbecco s Phosphate Buffer Saline w Calcium w Magnesium |
| PBS Ca2+/Mg2+ free | Sigma-Aldrich | D8537-500ML | Dulbecco s Phosphate Buffer Saline w/o Calcium w/o Magnesium |
| Penicillin/Streptomycin | Sigma-Aldrich | P4333-100ML | Penicillin/Streptomicin solution used to prevent cell culture contamination from bacteria. |
| poly-ornithine | Sigma-Aldrich | P4957 | Promotes attachment and growth of neural cells in vitro |
| SU5402 | Sigma-Aldrich | SML0443-5MG | Selective inhibitor of vascular endothelial growth factor receptor 2 (VEGFR-2) |
| Triton X-100 | Sigma-Aldrich | T8787 | 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether. Used for cell permeabilization in immunostaining assays |
| Upright microscope | Olympus | BX51VI | Microscope for electrophysiological recording equipped with CoolSnap Myo camera |
| Y-27632 (ROCK inhibitor) | Enzo Life Sciences | ALX-270-333-M005 | Cell-permeable selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK). Increases iPSC survival |
| μ-Slide 8 Well | Ibidi | 80826 | Support for high–end microscopic analysis of fixed cells |
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Citer Cet Article
Garone, M. G., de Turris, V., Soloperto, A., Brighi, C., De Santis, R., Pagani, F., Di Angelantonio, S., Rosa, A. Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors. J. Vis. Exp. (147), e59321, doi:10.3791/59321 (2019).