开发HiPSC衍生的无血清胚胎体用于使用生理相关测定的3-D干细胞培养物的询问
Summary
在这里，我们报告了一种由称为无血清胚状体（SFEB）的人诱导多能干细胞（hiPSCs）开发三维（3-D）系统的方案。这种3-D模型可以像器官切片培养物一样用于模拟人类皮质发育和生理学询问发展中的神经回路。
Abstract
虽然已经使用hiPSC开发了许多体外疾病模型，但一个限制是这些二维（2-D）系统可能不代表携带疑似疾病变异的受影响个体的基础细胞结构和功能复杂性。传统的二维模型仍然是体内结构的不完整表现，并没有充分地捕捉大脑的复杂性。因此，现在需要更多的基于3-D hiPSC的模型，其可以更好地概括在体内系统中看到的细胞相互作用和功能。
在这里，我们报告了一种基于无血清胚状体（SFEB）从未分化的hiPSC开发3-D系统的方案。这种3-D模型反映了开发的腹侧新皮层的各个方面，并且允许研究活体神经细胞和完整组织的整合功能，例如迁移，连通性，通讯和垫uration。具体来说，我们证明使用我们的方案的SFEB可以使用生理相关和高含量的基于细胞的测定法进行询问，如钙成像和多电极阵列（MEA）记录，而不进行冷冻切片。在MEA记录的情况下，我们证明SFEB在长期培养期间增加了穗活动和网络爆发活动。该SFEB协议提供了一个强大和可扩展的系统，用于研究三维模型中网络形成的研究，捕捉早期皮质发育的方面。
Introduction
我们以前报告了由患者衍生的人类多能干细胞（hiPSC）产生的3-D模型系统，其概括了早期皮层网络发展的某些方面1 。此3-D模型，无血清胚状体（SFEB），改善了以前的简单聚合的hiPSC模型2,3。工作的一个增长的身体被显露像我们SFEBs 3-D结构，神经发育的近似方面在体内和在比2维（2-D）观察到更早的时间点通常观察到的/单层的hiPSC模型4,5。初步研究集中在3-D体的自组织复杂性，而不表现出其生理复杂性2 。
本文描述的方案已用于源自成纤维细胞的未分化的hiPSC外周血单核细胞（PBMCs）。这些细胞维持在γ照射的小鼠胚胎喂养器（MEFs）上。将这些hiPSC菌落手工清洗自发分化的细胞，酶促收获，并重新悬浮于含Rho-Kinase抑制剂Y-27632（ROCKi）的培养基中。将未分化的hiPSC在转移到96孔低粘附性V底板之前进行离解和离心。电镀后，使用双SMAD抑制（SB431542和LDN193189以及dickkopf 1（DKK-1））启动神经诱导，以驱动前额叶前脑神经元命运谱系6 。 14天后，将SFEB转移到6孔板中的细胞培养插入物中。一旦转移，圆形SFEB开始扩散和稀释，同时维持局部网络连接，如使用类似的细胞培养插入物1的海马器官切片培养物制备中常见的那样，ss =“xref”> 7。
以这种格式使用基于SFEB的3-D平台适用于可以使用基于细胞的生理测定例如钙成像或电生理测定（例如单细胞记录或多电极阵列（MEA））来询问的皮层网络的有效生产） 1 。虽然3-D系统具有早期皮层发育的标志，但其他研究已经表明，这些3-D体可能需要更长的孵育时间，以允许人体组织发育的本质上较慢的速度8 。该SFEB协议成功地从未分化的hiPSC产生3-D SFEB，其捕获皮质早期发育的方面。
SFEBs在神经系统疾病中模拟网络畸变的潜力是该系统的优势。源自患者组织的hiPSC可以生长成受屁股的神经系统的细胞与细胞生物学有关的ays以及伴随的基因表达。人类iPSC被用来确定大群体的个体的遗传谱与不同神经障碍具有复杂的病因，如孤独症谱系障碍（ASD），精神分裂症9，Rett综合症10，和阿尔茨海默氏病11，12。直到最近，iPSC模型通常是单层制剂，虽然精通评估分子相互作用，但在破译体内发现的复杂细胞相互作用方面是不够的。动物模型已经成为重建整个器官平台的默认替代品。这些动物模型受到研究结果差的翻译困扰，复制由大型遗传筛选研究确定的人类遗传图谱的能力有限。因此，iPSC开发的3-D系统增加了人类所需的复杂层次疾病建模13，14。用于3D的hiPSC平台下一个步骤是，以适应使用基于细胞的测定筛选15高通量的大规模要求。
Protocol
神经祖细胞的产生 在补充有小分子的人iPSC培养基中，在γ-照射的小鼠胚胎饲养细胞（MEF）细胞层上，将来自成纤维细胞和PBMC的hiPSC保持在6孔板中（参见材料表）。 注：日常保养是以前报道的程序1，16修改后的版本。 在6孔组织培养级板的每个孔上以600μL/孔推荐的培养基（以下制造商的方案）将6×10 5个 MEF板铺板?…
Representative Results
使用我们的技术生长的SFEB产生具有形态学特征的组织，其类似于具有广泛的Tuj1阳性神经元以及神经祖细胞的早期发展的皮质脑室下区域（ 图3A ）。在SFEB的外层和内层观察到许多发育中的皮质玫瑰花结（ 图3B ）。 SFEB的外缘类似于含有神经元神经元的显影皮层。 Brn2的表达支持这一表达，Brn2是最外侧皮质层中神经祖细胞的标志…
Discussion
这里描述的方案提供了将hiPSC源区分为重现前额叶皮质早期发育阶段的3-D结构的条件。该过程产生可以询问电生理学的结构，同时也适用于显微镜。 SFEB的最终形态类似于器官型脑切片培养物的形态，并允许高质量的详细共聚焦成像。该方案可以成功地从成纤维细胞和外周血单核细胞衍生的hiPSC中产生SFEB。由外周血单个核细胞来源的hiPSCs制成的SFEB表现出与从成纤维细胞衍生的hiPSC产生的形态特征相…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢伊丽莎白Benevides校对文章。感谢Drs。 John Hussman和Gene Blatt进行了有益的讨论和评论。
Materials
| SFEB Neuronal Differentiation Cell culture Media. Reagents. Components | |||
| STEMdiff Neural Induction Medium (hiPSC Media) | STEMCELL Technologies | 0-5835 | 250ml |
| PluriQ ES-DMEM Medium (MEF Media) | GlobalStem | GSM-2001 | |
| Name | Company | Catalog Number | Comments |
| DM1 Media Components | |||
| D-MEM/F-12 (1X), Glutamax liquid, 1:1 | Invitrogen | 10565018 | 385ml |
| Knockout Serum Replacement | Invitrogen | 10828028 | 20% 100ml |
| Pen/Strep | Invitrogen | 15140122 | 5ml |
| Glutamax 200mM | Invitrogen | 35050061 | 5ml |
| MEM Non-Essential Amino Acids Solution 10 mM (100X), liquid | Invitrogen | 11140050 | 5ml |
| 2-Mercaptoethanol (1,000X), liquid | Invitrogen | 21985023 | 900ul |
| Name | Company | Catalog Number | Comments |
| DM2 Media Components | |||
| D-MEM/F-12 (1X), Glutamax liquid, 1:1 | Invitrogen | 10565018 | 500ml |
| Glutamax 200mM | Invitrogen | 35050061 | 5ml |
| Pen/Strep | Invitrogen | 15140122 | 5ml |
| N-2 Supplement (100X), liquid | Invitrogen | 17502048 | 10ml |
| Name | Company | Catalog Number | Comments |
| DM3 Media Components | |||
| NEUROBASAL Medium (1X), liquid | Invitrogen | 21103049 | 500ml |
| B-27 Supplement Minus Vitamin A (50X), liquid | Invitrogen | 12587010 | 10ml |
| Glutamax 200mM | Invitrogen | 35050061 | 5ml |
| Pen/Strep | Invitrogen | 15140122 | 5ml |
| Name | Company | Catalog Number | Comments |
| Small Molecules | |||
| Thiazovivin | Stemgent | 04-0017 | 2uM |
| SB431542 | Stemgent | 04-0010-10 | 1:1000 (10uM) |
| Dorsomorphin | Stemgent | 04-0024 | 1uM |
| LDN-193189 | Stemgent | 04-0074-10 | 250nM |
| Y27632 (ROCKi) | Stemgent | 04-0012-10 | 10uM |
| Name | Company | Catalog Number | Comments |
| Recombinant Protiens | |||
| DKK-1 | Peprotech | 120-30 | 200ng/ml |
| Name | Company | Catalog Number | Comments |
| Components/Materials | |||
| Cell Culture inserts 0.4uM, 30mm Diameter | Millicell | PICM0RG50 | |
| Mouse Embryonic Fibroblasts | GlobalStem | GSC-6301G | |
| 96 well V bottom w/Lids | Evergreen | 222-8031-01V | |
| StemPro Accutase Cell Dissociation Reagent | ThermoFisher | A1110501 | |
| TritonX-100 | ThermoFisher | 85111 | |
| Phosphate Buffered Saline (PBS) | ThermoFisher | 10010023 | 500 mL |
| Normal Donkey Serum | Jackson Labs | 017-000-121 | |
| Leibovitz's L-15 Medium | ThermoFisher | 11415114 | 500 mL |
| DRAQ5 (Nuclear Marker) | ThermoFisher | 65-0880-96 | |
| MEA Plates | Axion Biosystems | M768-GL1-30Pt200 | |
| 6 well flat bottom | Falcon | 353046 | |
| Name | Company | Catalog Number | Comments |
| Antibody | |||
| Nestin | Millipore | MAB5326 | |
| Brn-2 | Protein tech | 14596-1-AP | |
| VGLUT1 | Synaptic Systems | 135 303 | |
| Pax6 | abcam | ab5790 | |
| Calretinin | abcam | ab702 | |
| Calbindin | abcam | ab11426 | |
| CoupTFII | R&D Systems | PPH714700 | |
| Nkx 2.1 | abcam | ab12650 | |
| Tuj1 | abcam | ab41489 | |
| Reelin | Millipore | MAB5364 | |
| Tbr1 | Millipore | MAB2261 | |
| NFH | Dako | M0762 |
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Citazione di questo articolo
Phillips, A. W., Nestor, J. E., Nestor, M. W. Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays. J. Vis. Exp. (125), e55799, doi:10.3791/55799 (2017).