成人皮层的器官型培养作为人类干细胞移植和验证的 离体 模型
Summary
该协议描述了成人皮层的长期器官型培养与诱导多能干细胞衍生皮质祖细胞的 离体 皮质内移植相结合，这提供了一种新方法来进一步测试基于干细胞的治疗人类神经退行性疾病。
Abstract
神经退行性疾病在症状和细胞影响方面是常见且异质的，由于缺乏完全模仿人类疾病的适当动物模型以及死后人类脑组织的可用性差，他们的研究变得复杂。成人神经组织培养为研究神经系统疾病的不同方面提供了可能性。分子，细胞和生化机制可以很容易地在这个系统中解决，以及测试和验证药物或不同的治疗方法，如基于细胞的疗法。该方法结合了从接受切除手术的癫痫患者获得的成人皮层的长期器官型培养物，以及诱导多能干细胞来源的皮质祖细胞的 体外 皮质移植。该方法将允许研究细胞存活，神经元分化，突触输入和输出的形成以及移植到完整的成人皮质组织后人类来源细胞的电生理特性。这种方法是开发3D人类疾病建模平台之前的重要一步，该平台将使基础研究更接近基于干细胞的治疗的临床转化，用于患有不同神经系统疾病的患者，并允许开发用于重建受损神经回路的新工具。
Introduction
神经退行性疾病，如帕金森病、阿尔茨海默病或缺血性中风，是一组具有神经元功能障碍或死亡共同特征的疾病。它们在受影响的大脑区域和神经元群体方面是异质的。不幸的是，由于缺乏模仿人脑中发生的事情的动物模型，这些疾病的治疗方法很少或疗效有限1，2。干细胞疗法是大脑再生最有前途的策略之一3。近年来，从不同来源的干细胞中产生神经元祖细胞已经有了很大的发展4，5。最近的出版物表明，人类诱导多能干（iPS）细胞衍生的长期自我更新神经上皮样干（lt-NES）细胞，遵循皮质分化方案，并在缺血性中风影响躯体感觉皮层的大鼠模型中进行皮质内移植后，产生成熟的皮质神经元。此外，移植物衍生的神经元接收来自宿主神经元的传入和传出突触连接，显示它们整合到大鼠神经元网络中6，7。移植物来源的轴突被髓鞘化，发现在大鼠大脑的不同区域，包括梗死周围区域、胼胝体和对侧躯体感觉皮层。最重要的是，iPS细胞衍生的移植逆转了中风动物的运动缺陷7。
即使动物模型有助于研究移植存活，神经元整合以及移植细胞对运动和认知功能的影响，该系统中也缺少有关人类细胞（移植物 – 宿主）之间相互作用的信息8，9。出于这个原因，这里描述了一种长期人脑器官型培养与人iPS细胞来源神经元祖细胞离体移植的组合方法。从神经外科切除中获得的人脑器官型培养物是与大脑的生理相关的3D模型，使研究人员能够增加对人类中枢神经系统回路的理解以及测试人类脑部疾病治疗的最准确方法。然而，在这种情况下还没有进行足够的研究，在大多数情况下，已经使用了人类海马脑器官型培养物10，11。大脑皮层受到几种神经退行性疾病的影响，例如缺血性中风12 或阿尔茨海默病13，因此拥有一个人类皮质 3D 系统非常重要，该系统使我们能够扩展我们的知识并测试和验证不同的治疗策略。过去几年的几项研究使用成人皮质（hACtx）组织的培养物来模拟人类脑部疾病14，15，16，17，18，19;然而，在干细胞治疗方面可获得的信息有限。两项研究已经证明了此处描述的系统的可行性。2018年，用不同转录因子编程并移植到hACtx组织中的人类胚胎干细胞被证明会产生成熟的皮质神经元，这些神经元可以整合到成人皮质网络中20。2020 年，将 lt-NES 细胞移植到人类器官型系统中揭示了它们分化成具有功能神经元电生理特性的成熟、层特异性皮质神经元的能力。移植神经元与成人脑切片中的人类皮质神经元建立了传入和传出突触接触，狂犬病病毒逆行单突触追踪、全细胞膜片钳记录和免疫电子显微镜证实了这一点21。
Protocol
该协议遵循瑞典隆德区域伦理委员会批准的指南（道德许可证号 2021-07006-01）。从接受颞叶癫痫择期手术的患者获得健康的新皮质组织。获得所有患者的知情同意。 注意：无论其大小如何，所有获得的组织都经过处理。然而，小于 1-1.5 mm3 的组织在技术上具有挑战性，难以处理和用振动切片机切片。 1. 组织收集、维护、切割和电镀 <…
Representative Results
按照所描述的方案，如上所述，收集和处理来自颞叶癫痫患者的hACtx组织。在培养24小时后固定几片以研究宿主组织的起点。对不同神经细胞群的分析，如神经元（表达NeuN和Map2，图1A），少突胶质细胞（Olig2和MBP，图1B）和星形胶质细胞（人类特异性GFAP，也称为STEM123，图1C）显示组织的最佳保存。 下一步是研…
Discussion
获得足够高质量的hACtx切片是该协议中最关键的一步。 皮质组织是从接受切除手术的癫痫患者获得的24。切除组织的质量以及切除和培养之间的组织暴露时间至关重要;组织从手术室转移到实验室并切割的速度越快，器官型培养就越理想。理想情况下，应在收集后的最初几个小时内切割组织并将其转移到细胞培养实验室。在此过程中组织的氧合也提高了切片的质量。?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了瑞典研究委员会，瑞典脑基金会，瑞典中风基金会，斯科讷地区，Thorsten and Elsa Segerfalk基金会以及瑞典政府战略研究领域倡议（StemTherapy）的资助。
Materials
| Tissue Cutting and electrophysiology | |||
| Adenosine 5'-triphosphate magnesium salt | Sigma | A9187 | |
| Bath temperature controller | Luigs & Neumann | TC0511354 | |
| Calcium Chloride dihydrate | Merck | 102382 | |
| Carbogen gas | Air Liquide | NA | |
| Cooler | Julaba FL 300 | 9661012.03 | |
| D-(+)Glucose | Sigma-Aldrich | G7021 | |
| Double Patch-Clamp amplifier | HEKA electronic | EPC10 | |
| Guanosine 5'-Triphosphate disodium salt | Millipore | 371701 | |
| HEPES | AppliChem | A1069 | |
| Magnesium Chloride hexahydrate | Sigma-Aldrich | M2670 | |
| Magnesium Sulfate heptahydrate | Sigma-Aldrich | 230391 | |
| Patchmaster | HEKA electronic | Patchmaster 2×91 | |
| Pipette Puller | Sutter | P-2000 | |
| Plastic Petri dish | Any suitable | ||
| Potassium chloride | Merck | 104936 | |
| Potassium D-gluconate | ThermoFisher | B25135 | |
| Rubber teat + glass pipette | Any suitable | ||
| Sodium Bicarbonate | Sigma-Aldrich | S5761 | |
| Sodium Chloride | Sigma-Aldrich | S7653 | |
| Sodium dihydrogen phosphate monohydrate | Merck | 106346 | |
| Sucrose | Sigma-Aldrich | S7903 | |
| Tissue adhesive: Acryl super glue | Loctite | 2062278 | |
| Upright microscope | Olympus | BX51WI | |
| Vibratome | Leica | VT1200 S | |
| RINSING SOLUTION | |||
| D-(+)Glucose | Sigma-Aldrich | G7021 | |
| HBSS (without Ca, Mg, or PhenolRed) | ThermoFisher Scientific | 14175095 | |
| HEPES | AppliChem | A1069 | |
| Penicillin-Streptomycin (10,000 U/mL) | ThermoFisher Scientific | 15-140-122 | |
| MANTAINANCE AND CULTURE OF HUMAN NEOCORTICAL TISSUE | |||
| 6-well plate | ThermoFisher Scientific | 140675 | |
| Alvetex scaffold 6 well insert | Reinnervate Ltd | AVP004-96 | |
| B27 Supplement (50x) | ThermoFisher Scientific | 17504001 | |
| BrainPhys without Phenol Red | StemCell technologies | #05791 | Referenced as neuronal medium in the text |
| Filter units 250 mL or 500 mL | Corning Sigma | CLS431096/97 | |
| Forceps | Any suitable | ||
| Gentamicin (50 mg/mL) | ThermoFisher Scientific | 15750037 | |
| Glutamax Supplement (100x) | ThermoFisher Scientific | 35050061 | Referenced as L-glutamine in the text |
| Rubber teat + Glass pipette | Any suitable | ||
| GENERATION OF lt-NES cells | |||
| 2-Mercaptoethanol 50 mM | ThermoFisher Scientific | 31350010 | |
| Animal Free Recombinant EGF | Peprotech | AF-100-15 | |
| B27 Suplemment (50x) | Thermo Fisher Scientific | 17504001 | |
| bFGF | Peprotech | AF-100-18B | |
| Bovine Albumin Fraction V (7.5% solution) | ThermoFisher Scientific | 15260037 | |
| Cyclopamine, V. calcifornicum | Calbiochem | # 239803 | |
| D (+) Glucose solution (45%) | Sigma | G8769 | |
| Dimethyl sulfoxide (DMSO) | Sigma Aldrich | D2438-10mL | |
| DMEM/F12 | ThermoFisher Scientific | 11320074 | |
| Dulbecco's Phosphate Buffer Saline (DPBS) | Thermo Fisher Scientific | 14190-144 | Without calcium and magnesium |
| Laminin Mouse Protein, Natural | Thermo Fisher Scientific | 23017015 | |
| MEM Non-essential aminoacids solutions (100x) | ThermoFisher Scientific | 11140050 | |
| N-2 Supplement (100 x) | ThermoFisher Scientific | 17502001 | |
| Poly-L-Ornithine | Merk | P3655 | |
| Recombinant Human BMP-4 Protein | R&D Systems | 314-BP-010 | |
| Recombinant Human Wnt-3a Protein | R&D Systems | 5036-WN | |
| Sodium Pyruvate (100 mM) | ThermoFisher Scientific | 11360070 | |
| Soybean Trypsin Inhibitor, powder | Thermo Fisher Scientific | 17075029 | |
| Sterile deionized water | MilliQ | MilliQ filter system | |
| Trypsin EDTA (0.25%) | Sigma | T4049-500ML | |
| EQUIPMENT FOR CELL CULTURE | |||
| Adjustable volume pipettes 10, 100, 200, 1000 µL | Eppendorf | Various | |
| Basement membrane matrix ESC-qualified (Matrigel) | Corning | CLS354277-1EA | |
| Centrifuge | Hettich Centrifugen | Rotina 420R | 5% CO2, 37 °C |
| Incubator | ThermoForma Steri-Cult CO2 | HEPA Class100 | |
| Stem cell cutting tool 0.190-0.210 mm | Vitrolife | 14601 | |
| Sterile tubes | Sarstedt | Various | |
| Sterile Disposable Glass Pasteur Pipettes 150 mm | VWR | 612-1701 | |
| Sterile pipette tips 0.1-1000 µL | Biotix VWR | Various | |
| Sterile Serological Pipettes 5, 10, 25, 50 mL | Costar | Various | |
| T25 flasks Nunc | ThermoFisher Scientific | 156367 | |
| IMMUNOHISTOCHEMISTRY | |||
| 488-conjugated AffinityPure Donkey anti-mouse IgG | Jackson ImmunoReserach | 715-545-151 | |
| 488-conjugated AffinityPure Donkey anti-rabbit IgG | Jackson ImmunoReserach | 711-545-152 | |
| 488-conjugated AffinityPure Donkey anti-chicken IgG | Jackson ImmunoReserach | 703-545-155 | |
| Alexa fluor 647-conjugated Streptavidin | Jackson ImmunoReserach | 016-600-084 | |
| Bovine Serum Albumin | Jackson ImmunoReserach | 001-000-162 | |
| Chicken anti-GFP | Merk Millipore | AB16901 | |
| Chicken anti-MAP2 | Abcam | ab5392 | |
| Cy3-conjugated AffinityPure Donkey anti-chicken IgG | Jackson ImmunoReserach | 703-165-155 | |
| Cy3-conjugated AffinityPure Donkey anti-goat IgG | Jackson ImmunoReserach | 705-165-147 | |
| Cy3-conjugated AffinityPure Donkey anti-mouse IgG | Jackson ImmunoReserach | 715-165-151 | |
| Diazabicyclooctane (DABCO) | Sigma Aldrich | D27802 | Mounting media |
| Goat anti-AIF1 (C-terminal) | Biorad | AHP2024 | |
| Hoechst 33342 | Molecular Probes | Nuclear staining | |
| Mouse anti-MBP | BioLegend | 808402 | |
| Mouse anti-SC123 | Stem Cells Inc | AB-123-U-050 | |
| Normal Donkey Serum | Merk Millipore | S30-100 | |
| Paint brush | Any suitable | ||
| Paraformaldehyde (PFA) | Sigma Aldrich | 150127 | |
| Potassium Phospate Buffer Saline, KPBS (1x) | |||
| Distilled water | |||
| Potassium dihydrogen Phospate (KH2PO4) | Merk Millipore | 104873 | |
| Potassium phospate dibasic (K2HPO4) | Sigma Aldrich | P3786 | |
| Sodium chloride (NaCl) | Sigma Aldrich | S3014 | |
| Rabbit anti-NeuN | Abcam | ab104225 | |
| Rabbit anti-Olig2 | Abcam | ab109186 | |
| Rabbit anti-TMEM119 | Abcam | ab185333 | |
| Sodium azide | Sigma Aldrich | S2002-5G | |
| Sodium citrate | |||
| Distilled water | |||
| Tri-Sodium Citrate | Sigma Aldrich | S1804-500G | |
| Tween-20 | Sigma Aldrich | P1379 | |
| Triton X-100 | ThermoFisher Scientific | 327371000 | |
| EQUIPMENT FOR IMMUNOHISTOCHEMISTRY | |||
| Confocal microscope | Zeiss | LSM 780 | |
| Microscope Slides 76 mm x 26 mm | VWR | 630-1985 | |
| Microscope Coverslips 24 mm x 60 mm | Marienfeld | 107242 | |
| Microscope Software | Zeiss | ZEN Black edition | |
| Rubber teat + Glass pipette | Any suitable |
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Cite This Article
Palma-Tortosa, S., Martínez-Curiel, R., Aretio-Medina, C., Avaliani, N., Kokaia, Z. Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation. J. Vis. Exp. (190), e64234, doi:10.3791/64234 (2022).