从人类多能干细胞生成神经视网膜
Summary
本方案描述了一种优化的3D神经视网膜诱导系统，该系统以高可重复性和效率减少视网膜类器官的粘附和融合。
Abstract
视网膜病变是全球失明的主要原因之一。研究其发病机制对于视网膜病变的早期诊断和及时治疗至关重要。不幸的是，道德障碍阻碍了从人类那里收集证据。最近，大量研究表明，人类多能干细胞（PSCs）可以使用不同的诱导方案分化为视网膜类器官（RO），这在视网膜病变中具有巨大的潜力，可用于疾病建模、药物筛选和基于干细胞的治疗。本研究描述了一种优化的诱导方案，以产生神经视网膜 （NR），显着降低囊泡和融合的可能性，提高生产成功率，直到第 60 天。基于PSCs解离后自重组的能力，结合某些互补因子，这种新方法可以特异性地驱动NR分化。此外，该方法简单、具有成本效益、具有显着的可重复性和效率，为视网膜疾病的个性化模型提供了令人鼓舞的前景，并为细胞治疗、药物筛选和基因治疗测试等应用提供了丰富的细胞库。
Introduction
眼睛是人类感觉器官的主要信息来源，视网膜是哺乳动物眼睛的主要视觉感觉组织1。视网膜病变是导致眼部疾病的全球主要原因之一，导致失明2.全世界约有 285 万人因视网膜病变而遭受不同程度的视力损害3.因此，研究其发病机制对于早期诊断和及时治疗至关重要。大多数关于人类视网膜病变的研究主要集中在动物模型上4,5,6。然而，人类视网膜是一个复杂的多层组织，由各种细胞类型组成。传统的二维 （2D） 细胞培养和动物模型系统通常无法忠实地概括人类天然视网膜的正常时空发育和药物代谢 7,8。
最近，3D 培养技术已经发展到可以从多能干细胞 （PSC） 生成组织样器官9,10。在3D悬浮培养系统中，由人PSC产生的视网膜类器官（ROs）不仅包含七种视网膜细胞类型，而且还表现出与体内人视网膜相似的独特分层结构11,12,13。人 PSC 衍生的 RO 已获得普及和广泛使用，是目前研究人视网膜发育和疾病的最佳体外模型14,15。在过去的几十年中，许多研究人员已经证明，人类PSC，包括胚胎干细胞（ESC）和诱导多能干细胞（iPSC），可以使用各种诱导方案分化为RO。这些进展在视网膜病变的疾病建模、药物筛选和干细胞疗法方面具有巨大的潜力16,17,18。
然而，从人类多能干细胞 （PSC） 生成神经视网膜 （NR） 是一个复杂、繁琐且耗时的过程。此外，组织类器官的批次间差异可能导致结果的重现性降低19,20。许多内在和外在因素可以影响视网膜类器官（ROs）的产量，例如起始细胞的数量或种类以及转录因子和小分子化合物的使用21,22,23。自从Sasai实验室11产生第一个人类RO以来，多年来已经提出了多种修改，以提高诱导过程的易用性和有效性13,21,24,25。不幸的是，迄今为止，尚未建立在所有实验室中生成RO的金标准协议。事实上，由于不同的诱导方法，以及视网膜标志物的表达及其结构的稳健性存在很大差异，RO存在一定程度的差异22,26。这些问题可能会使样本采集和研究结果的解释严重复杂化。因此，需要一种更巩固和更强大的分化方案，以最大限度地提高效率，同时将RO产生的异质性降至最低。
本研究描述了一种基于 Kuwahara 等人 12 和 Döpper 等人 27 组合的优化诱导方案，并附有详细说明。新方法显著降低了类器官囊泡化和融合的概率，提高了产生NR的成功率。这一发展为视网膜疾病的疾病建模、药物筛选和细胞治疗应用带来了巨大的希望。
Protocol
本研究是根据《赫尔辛基宣言》原则进行的，并经中国人民解放军总医院机构伦理委员会批准。WA09（H9）电调线是从WiCell研究所获得的。 1.培养基和试剂制备 人胚胎干细胞培养基和传代溶液维持培养基 （MM）：无菌制备 500 mL 完全 MM（基础培养基 + 5x 补充剂;参见 材料表）。在室温（RT）下解冻5x补充剂或在2-8°C下过夜。 预热至室温，使?…
Representative Results
修改后的协议的图形概述如 图 1 所示。当细胞生长到70%-80%的密度时，使用H9-ESCs产生RO。H9-ESCs在96个V型底锥形孔中的单细胞悬浮液在第1天聚集，并在第6天形成边界良好的圆形EB。随着培养时间的增加，EB的体积逐渐增加。第30天，神经上皮样结构在长期NR分化过程中明显形成并增厚。 此外，将改进的方法与其他两种方法（Kuwahara 等人 12 …
Discussion
人类RO可以在空间和时间上概括胎儿视网膜的发育，早期RO在同等发育阶段表现出与胎儿视网膜的高度相似性15。在组织形态和分子表达方面，人类RO密切反映了视网膜组织的实际生长状态，为疾病建模、药物筛选和再生医学领域提供了巨大而前所未有的机会。目前，已经建立了几种不同的方法在 体外从人PSCs中产生RO，并且正在进行持续的修改和优化，以进一步提高效率<sup…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
没有。
Materials
| 0.01 M TPBS | Servicebio | G0002 | Washing slices |
| 4% Paraformaldehyde | Servicebio | G1101-500ML | Fix retinal organoids |
| 5 mL Pasteur pipette | NEST Biotechnology | 318516 | Pipette retinal organoids |
| 96 V-bottomed conical wells | Sumitomo Bakelit | MS-9096VZ | |
| Adhesion Microscope Slides | CITOTEST | 188105 | Fix slices |
| AggreWell medium | STEMCELL Technologies | 5893 | Medium |
| Anhydrous ethanol | SINOPHARM | 10009218 | Dehydrate |
| Anti-CHX10 | Santa Cruz | sc-365519 | Primary antibody |
| Antifade Solution | ZSGB-BIO | ZLI-9556 | |
| Anti-KI67 | Abcam | ab16667 | Primary antibody |
| Anti-NESTIN | Sigma | N5413 | Primary antibody |
| Anti-Neuronal Class III β-Tubulin(TUJ1) | Beyotime | AT809 | Primary antibody |
| Anti-PAX6 | Abcam | ab195045 | Primary antibody |
| Cell dissociation solution(CDS) | STEMCELL Technologies | 7922 | Cell dissociation |
| CHIR99021 | Selleckchem | S2924 | GSK-3α/β inhibitor |
| Cholesterol Lipid Concentrate | Gibco | 12531018 | 250× |
| Citrate Antigen Retrieval Solution | Servicebio | G1202-250ML | 20×, pH 6.0 |
| CS10 | STEMCELL Technologies | 1001061 | Cell Freezing Medium |
| DAPI | Roche | 10236276001 | Nuclear counterstain |
| Dimethyl sulfoxide(DMSO) | Sigma | D2650 | |
| DMEM/F12 | Gibco | 11330032 | Medium |
| DMEM/F12-GlutaMAX | Gibco | 10565018 | Medium |
| Donkey anti-Mouse Alexa Fluor Plus 488 | Invitrogen | A32766 | Secondary Antibody |
| Donkey anti-Rabbit Alexa Fluor 568 | Invitrogen | A10042 | Secondary Antibody |
| Ethylene Diamine Tetraacetic Acid (EDTA) | Biosharp | BL518A | 0.5 M, pH 8.0, cell dissociation |
| Extracellular matrix (ECM) | Corning | 354277 | Coating plates |
| F12-Glutamax | Gibco | 31765035 | Medium |
| Fetal Bovine Serum | Gibco | A5669701 | |
| Flow-like tissue cell quantitative analyzer | TissueGnostics | TissueFAXS Plus | Scan sections |
| IMDM-GlutaMAX | Gibco | 31980030 | Medium |
| IWR1-endo | Selleckchem | S7086 | Wnt-inhibitor |
| KnockOut Serum Replacement | Gibco | 10828028 | |
| LDN-193189 2HCl | Selleckchem | S7507 | BMP-inhibitor |
| Low-adhesion 24-well Plates | Corning | 3473 | |
| Low-adhesion 6-well Plates | Corning | 3471 | |
| Maintenance medium (MM) | STEMCELL Technologies | 85850 | Medium |
| N2 supplement | Gibco | 17502048 | |
| Normal Donkey Serum | Solarbio | SL050 | Blocking buffer |
| Paraplast | Leica | 39601006 | Tissue embedding |
| PBS pH 7.4 basic (1x) | Gibco | C10010500BT | Without Ca+,Mg+ |
| Reconbinant human bone morphogenetic protein-4(rhBMP4) | R&D | 314-BP | Key protein factor |
| Retinoic acid | Sigma | R2625 | Powder, keep out of light |
| SB431542 | Selleckchem | S1067 | ALK5-inhibitor |
| SU5402 | Selleckchem | S7667 | Tyrosine kinase inhibitor |
| Super PAP Pen | ZSGB-BIO | ZLI-9305 | |
| Taurine | Sigma | T0625-10G | |
| Thioglycerol | Sigma | M1753 | |
| Triton X-100 | Sigma | X100 | Permeabilization |
| WA09 embryonic stem cell line | WiCell Research Institute | Cell line | |
| Xylene | SINOPHARM | 10023418 | Dewaxing |
| Y-27632 2HCL | Selleckchem | S1049 | ROCK-inhibitor |
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Citazione di questo articolo
Li, W., Li, H., Yan, H., Gao, L., Wang, X., Zhao, L., Yan, Y., Ye, Z., Xi, J., Yue, W., Li, Z. Generating Neural Retina from Human Pluripotent Stem Cells. J. Vis. Exp. (202), e66246, doi:10.3791/66246 (2023).