建立区域特异性人多能干细胞衍生星形胶质细胞和神经元模拟ALS的电生理平台
Summary
我们描述了一种区分脊髓人诱导的多能源星形胶质细胞和神经元及其共培养以进行电生理记录的方法。
Abstract
人多能干细胞衍生的星形胶质细胞（hiPSC-A）和神经元（hiPSC-N）为 体外肌萎缩侧索硬化症（ALS）病理生理学建模提供了强大的工具。多电极阵列（MEA）记录是一种记录大量神经元的电场电位并分析网络活动随时间变化的方法。先前已经证明，与没有hiPSC-A或在存在啮齿动物星形胶质细胞的情况下培养的hiPSC-A相比，使用促进脊髓星形胶质细胞表型的技术分化的hiPSC-A的存在改善了区域特异性脊髓hiPSC-运动神经元（MN）的成熟和电生理活性。这里描述的是一种将脊髓hiPSC-A与hiPSC-MN共培养并使用MEA记录记录电生理活性的方法。虽然这里描述的分化方案特定于星形胶质细胞和脊髓区域特异性的神经元，但共培养平台可应用于星形胶质细胞和神经元，这些分化技术特定于其他命运，包括皮质hiPSC-A和hiPSC-N。这些协议旨在提供电生理测定，以告知神经胶质神经元相互作用，并为测试ALS中具有治疗潜力的药物提供平台。
Introduction
人类多能干细胞衍生的星形胶质细胞（hiPSC-A）和神经元（hiPSC-N）是体 外 模拟肌萎缩侧索硬化症（ALS）病理生理学的强大工具，并为药物发现策略提供了转化范式1。研究人员已经证明，hiPSC-A 与 hiPSC-N 的共培养增强了两种细胞类型的形态、分子、电生理和药理成熟，产生复杂的神经元网络和星形胶质细胞-神经元相互作用，类似于 体内 对应物2，3。类似的共培养实验可以概括ALS病理生物学的特征，例如星形胶质细胞介导的神经毒性4，5 和神经元过度兴奋性6。此外，随着分化方案的进步，人诱导多能干细胞（hiPSC）可以分化为区域特异性神经亚型，包括皮质和脊髓hiPSC-A和hiPSC-N7，8。这些策略为模拟ALS中的皮质和脊髓运动神经元病理学以及星形胶质细胞对两者的影响提供了潜力。然而，这需要有可重现的功能测定来确定这些影响。
最近研究表明，多电极阵列（MEA）记录特别适用于神经元-星形胶质细胞共培养物的电生理表征2。与单细胞电生理分析相反，这些高密度电极阵列被动记录来自大量神经元的细胞外场电位，而不会破坏培养条件并保持细胞膜的完整性。这些平台对于记录培养物随时间推移的细胞和网络活性以及对药物操作的响应特别有用。最后，当星形胶质细胞的存在是一个培养变量时，MEA记录可以提供星形胶质细胞-神经元双向相互作用的功能见解2，9。
这里介绍的是将hiPSC分化为脊髓hiPSC-A和hiPSC运动神经元（MN）的优化方案，该方案先前已经过验证2。脊髓 hiPSC-A 分化方案一致导致星形胶质细胞培养，分别在多达 80%、50% 和 90% 的细胞中对 S100 钙结合蛋白 B （S100β）、神经胶质纤维酸性蛋白 （GFAP） 和同源盒 B4 （HOXB4） 呈阳性，表明神经胶质细胞和脊髓规格成熟2，10.hiPSC-MN 分化方案产生的神经元胆碱乙酰转移酶 （ChAT） 呈 >90% 阳性，提示成熟的 α 运动神经元身份2。此外，该协议描述了用于生成hiPSC-A / MN共培养的技术，与没有星形胶质细胞或啮齿动物星形胶质细胞的神经元培养物相比，先前通过Scholl分析和免疫荧光显微镜证明导致神经元具有增强的形态复杂性2。虽然这些描述特定于脊髓hiPSC-A和hiPSC-N，但一个独特的优势是，星形胶质细胞和神经元的初始独立培养，然后在以后的时间点进行共培养的步骤可以转化为研究来自其他特定区域以及疾病特异性细胞的神经元 – 星形胶质细胞相互作用的影响7，8.最后，该协议描述了如何在MEA平板上培养这些培养物，以便随着时间的推移，可以通过操纵细胞组成和培养条件的能力来研究作为共培养组成因子的功能活性。
这些协议的目标是提供一种功能测定，以研究星形胶质细胞 – 神经元相互作用，检查疾病特异性变化，并测试在ALS领域具有治疗潜力的药物。为该协议最具挑战性的步骤提供了视频说明。
Protocol
1. 细胞培养基制备 使用 表1中提到的组合物制备单个细胞培养基。 在500mL过滤瓶中混合并无菌过滤培养基，并在4°C避光下储存长达2周。 2. 维持和传代非融合人诱导多能干细胞 （hiPSC） 将基底膜基质（在-80°C下等分储存）在2-8°C冰箱中解冻过夜。 在冷磷酸盐缓冲盐水（PBS）或Dulbecco的改良鹰培养基（DMEM）中以1：100（v / v）稀释?…
Representative Results
用于生成hiPSC-MN和脊髓hiPSC-A的脊髓模式方案如图 1所示。在该协议中，hiPSCs作为非融合集落维持和传代（图2A）。通过添加LDN193189和SB431542的双重SMAD抑制启动神经发生（神经诱导），分别灭活骨形态发生蛋白（BMP）和转化生长因子-β（TGF-β）途径。此步骤使用基于单层的方法，其中将hiPSC接种到贴壁基质（即基底膜基质）上，以产生神经上皮样2D培养物?…
Discussion
迄今为止，基于hiPSC和MEA的星形胶质细胞-神经元共培养电生理记录方法在ALS6领域的应用有限，并且仍然没有在完全人类平台上应用，相比之下，它们更广泛地用于癫痫9的体外建模。然而，该平台有可能解决ALS研究中的病理生理学相关问题，例如神经元过度兴奋的机制，星形胶质细胞对神经毒性的贡献或网络活动在疾病进展中的作用。此外，该平台允许?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
该手稿得到以下支持：2019 MSCRFF 5119 （AT）。K08NS102526 NIH/NINDS （CWH），2020 年多丽丝杜克慈善基金会临床科学家职业发展奖 （CWH）。1R01NS117604-01NIH/NINDS （NJM）， DOD ALSRP W81XWH2010161 （NJM）， 2019 MSCRFD 5122 （NJM）.我们感谢Raha Dastgheyb博士和Norman Haughey博士提供MEA平台和数据分析软件，我们用它来验证所描述的电生理平台。我们要感谢Khalil Rust在协议演示和拍摄方面的协助。
Materials
| 10 cm sterile culture plates | Falcon | 353003 | |
| 25 cm2 sterile culture flasks | Falcon | 353136 | |
| 2-Mercaptoethanol (β-ME) | Thermofisher | 21985023 | Working concentration 110 µM |
| 500 mL 0.2 µm CA Filter System | Corning | 430769 | |
| 5 mL pipette | Falcon | 357543 | |
| 6 well sterile culture plates | Falcon | 3046 | |
| Amphotericine B | Gibco | 15290018 | Working concentration 2.0 μg/mL |
| Anionic detergent with protease enzyme – Terg-A-Zyme | Sigma-Aldrich | Z273287 | Working concentration 1% m/v |
| Ascorbic acid (ASAC) | Sigma | A4403 | Dissolve 100 mg into 250 mL of dH2O to get 0.4mg/ml stock. Sterile filter through a 0.22 µm filter, aliquot and freeze at -80 ºC. Dilute at 1:1000 for use. (working concentration 0.4 µg/mL). |
| Axion CytoView MEA 24 plates (M384-tMEA-24W) | Axion | OPT-24 | |
| Axion Edge MEA platform | Axion | Maestro Edge | |
| Basement Membrane matrix – Matrigel | Corning | 354277 | Details in the protocol |
| Benchtop microscope (sterile under cell culture hood) | Zeiss | 415510-1100-000 | Primo Vert |
| Bicuculline | Sigma Aldrich | 14340 | Working concentration10 μM |
| Ciliary neurotrophic factor (CNTF) | Peprotech | 450-13 | Dissolve 100 µg in 1mL of sterile PBS, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL). |
| CO2 tanks and regulator for Axion Edge | AirGas/Harris | 9296NC | |
| Compound E | Abcam | ab142164 | Dissolve 250 µg in 2.0387 mL of DMSO to get a 250 µM stock. Aliquot and freeze at -80 ºC. Dilute 1:2000 for use. (working concentration 125 nM). |
| Cyanquixaline (CNQX) | Sigma Aldrich | C239 | Working concentration 50 μM |
| Dihydrokainic acid (DHK) | Tocris | 111 | Working concentration 50 μM and 300 μM |
| DMEM/F12 | Thermofisher | 113300 | Working concentration 1x |
| Essential 8 Medium + Essential 8 Supplement | Thermofisher | A1517001 | Combine 10 mL of Essential 8 (10x) supplement with 500 mL of Essential 8 Growth Medium (1x) |
| Fetal Bovine Serum (FBS) | Thermofisher | 16140071 | Working concentration 1x |
| Glial cell line-derived neurotrophic (GDNF) | Peprotech | 450-10 | Dissolve 100 µg in 1mL of PBS to 100 µg/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL). |
| Hemocytometer | Election Microscopy Sciences | 63510-20 | |
| Heparin | Millipore-sigma | H3149-100KU | Dissolve 200 mg in 100 mL of PBS to get 2 mg/mL stock solution. Aliquot the stock in 15 mL and 500 µL tubes. Dilute 1:1000 for use. (working concentration 2 µg/mL). |
| Humidity controlled Cell culture incubator | ThermoFisher | 370 | set to 37 ºC, 5 % CO2 |
| Insulin-like growth factor 1 (IGF-1) | R&D systems | 291-G1-200 | Dissolve 200 µg in 2 mL of PBS to 100 µg/mL, then add 18 mL of 0.1%BSA-PBS to make 10 µg/mL stock and store at -80 ºC. Aliquot and freeze at -80 ºC. Dilute 10 µg/mL stock at 1: 1000 for use. (working concentration 10 ng/mL). |
| Kainc acid | Abcam | ab144490 | Working concentration 5 μM |
| Knockout Serum Replacement (KSR) | Thermofisher | 10828 | Working concentration 1x |
| Laminin | Thermofisher | 23017-015 | Stock solution 1 mg/mL, working concentration 10 µg/mL (coating), 1 µg/mL (cell media) |
| LDN193189 | Stemgent | 04-0074 | Dissolve 2 mg of LDN into 500 µL of Chloroform to get 10 mM stock. Aliquot this and freeze at -80 ºC. For using, dilute the stock 1 to 10 into DMSO [to 1 mM] first, then dilute 1:5000 of 1 mM into the desired media to get 0.2 µM working solution |
| L-Glutamine | Thermofisher | 25030 | Working concentration 100x |
| MEA glass plates | MultiChannel Systems | 60MEA200/30iR-Ti-gr | |
| Multichannel Pipet P200 | Gilson | PJ22224 | |
| Neurobasal | Thermofisher | 21103049 | Working concentration 1x |
| Non-Essential Amino Acids (NEAA) | Thermofisher | 11140050 | Working concentration 100x |
| Pencillin/Streptomycin | Thermofisher | 15140122 | Working concentration 100x |
| Polyornithine (PLO) | Sigma-Aldrich | P3655 | Dissolve 100 mg in 1 mL of ddW to get 100 mg/mL stock solution. Aliquot the stock in 100 µL tubes. (working concentration 100 µg/mL) |
| Potassium chloride (KCl) | NA | NA | Working concentration100 mM |
| Purmorphamine (PMN) | Millipore-Sigma | 540223 | Dissolve 5 mg in 9.6 mL of DMSO to get 1 mM solution. Aliquot and freeze at -80 ºC. Dilute 1:1000 for use. (working concentration 1 µM). |
| Recombinant human-brain-derived neurotrophic factor (BDNF) | Peprotech | 450-02 | Centrifuge briefly before reconstitution. Dissolve 100 µg in 1 mL of PBS to 100 µg/mL, and then add 9 mL of sterile 0.1% BSA-PBS to 10 µg/mL. Aliquot and freeze at -80 ºC. Dilute at 1: 1000 for use. (working concentration 10 ng/mL). |
| Retinoic acid (RA) | Sigma | R2625 | Dissolve 100 mg into 3.3 mL of DMSO to get 100 mM stock solution. Aliquot the stock 100 µL/tube and freeze at -80 ºC. Take 200 µL of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 µL/tube and store at -80 ºC. Dilute at 1:10000 for use. (working concentration 2 µM). |
| ROCK-I nhibitor | Peprotech | 1293823 | Dissolve 5 mg in 1480 µL of dH2O to get 10 mM stock, aliquot and freeze at -80 ºC. Dilute at 1: 500 for use. (working concentration 20 µM). |
| SB431542 | Sigma | S4317 | Dissolve 5mg into 1.3 mL of DMSO to get 10 mM stock solution. Aliquot and freeze at -80 ºC. Dilute at 1:1000 for use. (working con 10 µM) |
| Sterile cell culture hoods | Baker Company | SG-600 | |
| Supplement B – B27 Supplement | Thermofisher | 21985023 | Working concentration 50x |
| Supplement N – N2 Supplement | Thermofisher | 17502048 | Working concentration 100x |
| Table top cell culture centrifuge | ThermoFisher | 75004261 | Sorvall Legend X1R |
| Thermoplastic film – Parafilm | PARAFILM | P7793 | |
| Tissue dissociation protease – Dispase | StemCell Technologies | 7923 | Working concentration 1x |
| Trypsin Inhibitor | Sigma | T6522-1G | Dissolve 1g in 100mL ddH2O to get 10 mg/mL stock. Aliquot and store at 4 ºC. Dilute 1:10 of trypsin volume for use. (working concentration 1 mg/mL). |
| Trypsin-EDTA (0.05%) | Thermofisher | 2530054 | Working concentration 1x |
| Waterbath | ThermoFisher | 2332 | Isotemp |
Riferimenti
- Ferraiuolo, L., Maragakis, N. J. Mini-review: Induced pluripotent stem cells and the search for new cell-specific ALS therapeutic targets. Neuroscience Letters. 755, 135911 (2021).
- Taga, A., et al. Role of human-induced pluripotent stem cell-derived spinal cord astrocytes in the functional maturation of motor neurons in a multi-electrode array system. Stem Cells Translational Medicine. 8 (12), 1272-1285 (2019).
- Klapper, S. D., et al. Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks. Glia. 67 (10), 1893-1909 (2019).
- Zhao, C., et al. Mutant C9orf72 human iPSC-derived astrocytes cause non-cell autonomous motor neuron pathophysiology. Glia. 68 (5), 1046-1064 (2020).
- Almad, A. A., et al. Connexin 43 in astrocytes contributes to motor neuron toxicity in amyotrophic lateral sclerosis. Glia. 64 (7), 1154-1169 (2016).
- Wainger, B. J., et al. Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. Cell Reports. 7 (1), 1-11 (2014).
- Tyzack, G., Lakatos, A., Patani, R. Human stem cell-derived astrocytes: Specification and relevance for neurological disorders. Current Stem Cell Reports. 2, 236-247 (2016).
- Imaizumi, K., et al. Controlling the regional identity of hPSC-derived neurons to uncover neuronal subtype specificity of neurological disease phenotypes. Stem Cell Reports. 5 (6), 1010-1022 (2015).
- Odawara, A., Matsuda, N., Ishibashi, Y., Yokoi, R., Suzuki, I. Toxicological evaluation of convulsant and anticonvulsant drugs in human induced pluripotent stem cell-derived cortical neuronal networks using an MEA system. Scientific Reports. 8 (1), 10416 (2018).
- Haidet-Phillips, A. M., et al. Gene profiling of human induced pluripotent stem cell-derived astrocyte progenitors following spinal cord engraftment. Stem Cells Translational Medicine. 3 (5), 575-585 (2014).
- Roybon, L., et al. Human stem cell-derived spinal cord astrocytes with defined mature or reactive phenotypes. Cell Reports. 4 (5), 1035-1048 (2013).
- Shimojo, D., et al. simple motor neuron differentiation from human pluripotent stem cells. Molecular Brain. 8 (1), 79 (2015).
- Chandrasekaran, A., et al. Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells. Stem Cell Reports. 25, 139-151 (2017).
- Krencik, R., Weick, J. P., Liu, Y., Zhang, Z. J., Zhang, S. C. Specification of transplantable astroglial subtypes from human pluripotent stem cells. Nature Biotechnology. 29 (6), 528-534 (2011).
- Li, X. J., et al. Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells. Development. 136 (23), 4055-4063 (2009).
- Liu, H., Zhang, S. C. Specification of neuronal and glial subtypes from human pluripotent stem cells. Cellular and Molecular Life Sciences. 68 (24), 3995-4008 (2011).
- Borghese, L., et al. Inhibition of notch signaling in human embryonic stem cell-derived neural stem cells delays G1/S phase transition and accelerates neuronal differentiation in vitro and in vivo. Stem Cells. 28 (5), 955-964 (2010).
- Shi, Y., Kirwan, P., Smith, J., Robinson, H. P., Livesey, F. J. Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nature Neuroscience. 15 (3), 477-486 (2012).
Citazione di questo articolo
Taga, A., Habela, C. W., Johns, A., Liu, S., O’Brien, M., Maragakis, N. J. Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons. J. Vis. Exp. (174), e62726, doi:10.3791/62726 (2021).