从健康和视网膜疾病特异性人诱导的多能干细胞中生成视网膜类器官
Summary
该协议描述了一种将hiPSCs分化为眼场簇并使用涉及贴壁和悬浮培养系统的简化培养条件生成神经视网膜类器官的有效方法。其他眼细胞类型，如RPE和角膜上皮，也可以在视网膜培养物中从成熟的眼视野中分离出来。
Abstract
多能干细胞可以产生复杂的组织类器官，可用于体外疾病建模研究和开发再生疗法。该协议描述了一种更简单，稳健和逐步的方法，该方法在视网膜分化的前4周内在由贴壁单层培养物组成的杂交培养系统中生成视网膜类器官，直到出现独特的，自组织的眼场原始簇（EFP）。此外，每个EFP内的甜甜圈形，圆形和半透明神经视网膜岛在视网膜分化培养基中使用非粘附培养皿在悬浮液下手动挑选和培养1-2周以产生多层3D光学杯（OC-1M）。这些未成熟的视网膜类器官含有PAX6+和ChX10+增殖的多能视网膜前体。前体细胞在类器官内线性自组装，表现为明显的径向条纹。悬浮培养后4周，视网膜祖细胞经历有丝分裂后停滞和谱系分化，形成成熟的视网膜类器官（OC-2M）。光感受器谱系承诺的前体在视网膜类器官的最外层发育。这些CRX+和RCVRN+感光细胞形态成熟，显示出内段样延伸。该方法可用于使用人胚胎干细胞（hESCs）和诱导多能干细胞（iPSCs）生成视网膜类器官。所有步骤和程序都得到清晰的解释和演示，以确保可复制性并在基础科学和转化研究中得到更广泛的应用。
Introduction
视网膜是存在于脊椎动物眼睛后部的感光组织，它通过一种称为光转导途径的生化现象将光信号转化为神经冲动。视网膜感光细胞中产生的初始神经冲动被转导到其他视网膜中间神经元和视网膜神经节细胞（RGC）并到达大脑的视觉皮层，这有助于图像感知和视觉反应。
根据世界卫生组织（世卫组织）的数据，估计有150万儿童失明，其中100万在亚洲。遗传性视网膜营养不良症（IRD）是一种主要的致盲性疾病，全球每4，000人中就有1人受到影响1，2，3，而在发展中国家，与年龄相关性黄斑变性（AMD）相关的失明患病率在0.6%-1.1%之间4。IRD是由300多种不同基因的遗传缺陷引起的，这些基因涉及视网膜发育和功能5。这种遗传变化导致视网膜正常功能的破坏和视网膜细胞（即感光细胞和视网膜色素上皮（RPE））的逐渐退化，从而导致严重的视力丧失和失明。在涉及角膜、晶状体等的其他致盲疾病方面取得了巨大进展。然而，视网膜营养不良和视神经萎缩迄今没有任何经过验证的治疗方法。由于成人视网膜没有干细胞6，胚胎干细胞（ESC）和患者来源的诱导多能干细胞（iPSC）等替代来源可以提供无限供应所需的细胞类型，并为开发体外疾病建模研究和开发再生疗法所需的复杂组织类器官7提供了巨大的希望，8，9，10.
几年的视网膜研究使人们对协调早期视网膜发育的分子事件有了更好的理解。大多数从 PSC 生成视网膜细胞和 3D 类器官的方案旨在通过在生长因子和小分子的复杂混合物中培养细胞来逐步调节已知的生物过程，从而在体外概括这些发育事件。由此产生的视网膜类器官由主要的视网膜细胞组成：视网膜神经节细胞（RGC），中间神经元，光感受器和视网膜色素上皮（RPE）11，12，13，14，15，16，17，18，19.尽管成功尝试使用视网膜类器官对IRD进行建模，但在分化过程中对生长因子和小分子的复杂混合物的需求以及视网膜类器官生成效率相对较低，这对大多数方案构成了重大挑战。它们主要包括胚体的形成，然后在体外发育的不同阶段使用复杂的培养条件逐步分化为视网膜谱系20，21，22。
在这里，报道了一种从健康对照和视网膜疾病特异性hiPSCs开发复杂3D神经视网膜类器官的简化且稳健的方法。此处描述的方案利用近乎融合的hiPSC培养物的直接分化，而无需形成拟胚体。此外，培养基的复杂性也得到了简化，使其成为一种经济高效且可重复的技术，新研究人员可以轻松采用。它涉及一个混合培养系统，该系统由贴壁单层培养物组成，在视网膜分化的前 4 周内，直到出现独特的自组织眼野原始簇 （EFP）。此外，手动采摘每个EFP内的圆形神经视网膜岛并在悬浮培养物中生长1-2周，以制备由PAX6 +和CHX10 +增殖神经视网膜前体组成的多层3D视网膜杯或类器官。视网膜类器官在100μM含牛磺酸的培养基中进一步培养4周，导致RCVRN+和CRX+感光器前体的出现以及具有基本内段样延伸的成熟细胞。
Protocol
Representative Results
Discussion
hiPSCs是体外研究器官和组织发育的有力工具。通过将健康与疾病特异性hiPSC与视网膜谱系区分开来概括疾病表型，有助于获得对不同形式遗传性视网膜营养不良的病理生理学的新见解。已经描述并采用了几种方案，用于将PSCs体外分化为视网膜细胞类型。其中大多数涉及使用含有重组生长因子、补充剂、小分子和试剂的复杂混合物的培养基，例如：N1、N2 和 B27 补充剂;BMP 和 TGFβ 信号阻…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢遗传学家Chitra Kannabiran博士的科学和技术支持;Subhadra Jalali博士，视网膜顾问;米林德·奈克博士，眼部整形外科医生;以及海得拉巴LV Prasad眼科研究所的眼部肿瘤学家Swathi Kaliki博士，致力于生成正常和患者特异性iPSC系。作者感谢科学与工程研究委员会，科学和技术部（IM）（SB / SO/HS / 177 / 2013），生物技术系（IM）（BT / PR32404 / MED / 30 / 2136 / 2019）的研发资助，以及来自ICMR（S.M.，D.P.），UGC（T.A.）和CSIR（V.K.P.）印度政府的高级研究奖学金。
Materials
| 0.22 µm Syringe filters | TPP | 99722 | |
| 15 mL centrifuge tube | TPP | 91015 | |
| 50 mL centrifuge tube | TPP | 91050 | |
| 6 well plates | TPP | 92006 | |
| Anti-Chx10 Antibody; Mouse monoclonal | Santa Cruz | SC365519 | 1:50 dilution |
| Anti-CRX antibody; Rabbit monoclonal | Abcam | ab140603 | 1:300 dilution |
| Anti-MiTF antibody, Mouse monoclonal | Abcam | ab3201 | 1:250 dilution |
| Anti-Recoverin Antibody; Rabbit polyclonal | Millipore | AB5585 | 1:300 dilution |
| B-27 Supplement (50x), serum free | Thermo Fisher | 17504044 | |
| Basic Fibroblast growth factor (bFGF) | Sigma Aldrich | F0291 | |
| Centrifuge 5810R | Eppendorf | ||
| Coplin Jar (50 mL) | Tarson | ||
| Corning Matrigel hESC-Qualified Matrix | Corning | 354277 | |
| CryoTubes | Thermo Fisher | V7884 | |
| DMEM/F-12, GlutaMAX supplement (basal medium) | Thermo Fisher | 10565-018 | |
| DreamTaq DNA polymerase | Thermo Fisher | EP0709 | |
| Dulbeco’s Phosphate Buffered Saline | Thermo Fisher | 14190144 | |
| Essential 8 medium kit | Thermo Fisher | A1517001 | |
| Ethylene diamine tetraaceticacid disodium salt dihydrate (EDTA) | Sigma Aldrich | E5134 | |
| Falcon Not TC-treated Treated Petri Dish, 60 mm | Corning | 351007 | |
| Fetal Bovine Serum, qualified, United States | Gibco | 26140079 | |
| GelDocXR+ with Image lab software | BIO-RAD | Agarose Gel documentation system | |
| GlutaMAX Supplement | Thermo Fisher | 35050061 | |
| Goat anti-Mouse IgG (H+L), Alexa Fluor 488 | Invitrogen | A11001 | 1:300 dilution |
| Goat anti-Mouse IgG (H+L), Alexa Fluor 546 | Invitrogen | A11030 | 1:300 dilution |
| Goat anti-Rabbit IgG (H+L), Alexa Fluo 546 | Invitrogen | A11035 | 1:300 dilution |
| Goat anti-Rabbit- IgG (H+L), Alexa Fluor 488 | Invitrogen | A11008 | 1:300 dilution |
| HistoCore MULTICUT | Leica | For sectioning | |
| KnockOut Serum Replacement | Thermo Fisher | 10828028 | |
| L-Acsorbic acid | Sigma Aldrich | A92902 | |
| MEM Non-Essential Amino Acids Solution (100x) | Thermo Fisher | 11140-050 | |
| N2 supplement (100x) | Thermo Fisher | 17502048 | |
| NanoDrop 2000 | Thermo Fisher | To quantify RNA | |
| Paraformaldehyde | Qualigens | 23995 | |
| Pasteur Pipets, 9 inch, Non-Sterile, Unplugged | Corning | 7095D-9 | |
| Penicillin-Streptomycin | Thermo Fisher | 15140-122 | |
| Recombinant Anti-Otx2 antibody , Rabbit monoclonal | Abcam | ab183951 | 1:300 dilution |
| Recombinant Anti-PAX6 antibody; Rabbit Monoclonal | Abcam | ab195045 | 1:300 dilution |
| Recombinant Anti-RPE65 antibody, Rabbit Monoclonal | Abcam | ab231782 | 1:300 dilution |
| Recombinant Human Noggin Protein | R&D Systems | 6057-NG | |
| SeaKem LE Agarose | Lonza | 50004 | |
| Serological pipettes 10 mL | TPP | 94010 | |
| Serological pipettes 5 mL | TPP | 94005 | |
| Sodium Chloride | Sigma Aldrich | S7653 | |
| Sodium Citrate Tribasic dihydrate | Sigma Aldrich | S4641 | |
| Starfrost (silane coated) microscopic slides | Knittel | ||
| SuperScript III First-Strand Synthesis System | Thermo Fisher | 18080051 | |
| SuperScript III First-Strand Synthesis System for RT-PCR | Invitrogen | 18080051 | |
| Triton X-100 | Sigma Aldrich | T8787 | |
| TRIzol Reagent | Invitrogen | 15596026 | |
| UltraPure 0.5 M EDTA, pH 8.0 | Thermo Fisher | 15575020 | |
| VECTASHIELD Antifade Mounting Medium with DAPI | Vector laboratories | H-1200 | |
| Vitronectin | Thermo Fisher | A27940 | |
| Y-27632 dihydrochloride (Rho-kinase inhibitor) | Sigma Aldrich | Y0503 | |
| Zeiss LSM 880 | Zeiss | Confocal microscope |
Referências
- Dandona, R., et al. Moderate visual impairment in India: the Andhra Pradesh Eye Disease Study. British Journal of Ophthalmology. 86 (4), 373-377 (2002).
- Hartong, D. T., Berson, E. L., Dryja, T. P. Retinitis pigmentosa. Lancet. 368 (9549), 1795-1809 (2006).
- Sen, P., et al. Prevalence of retinitis pigmentosa in South Indian population aged above 40 years. Ophthalmic Epidemiology. 15 (4), 279-281 (2008).
- Nazimul, H., Rohit, K., Anjli, H. Trend of retinal diseases in developing countries. Expert Review of Ophthalmology. 3 (1), 43-50 (2008).
- . RetNet – Retinal Information Network Available from: https://sph.uth.edu/retnet/ (2022)
- Cicero, S. A., et al. Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells. Proceedings of the National Academy of Sciences. 106 (16), 6685-6690 (2009).
- Guo, Y., et al. Modeling retinitis pigmentosa: retinal organoids generated from the iPSCs of a patient with the USH2A mutation show early developmental abnormalities. Frontiers in Cellular Neuroscience. 13, 361 (2019).
- Lane, A., et al. Modeling and rescue of RP2 Retinitis pigmentosa using iPSC-derived retinal organoids. Stem Cell Reports. 15 (1), 67-79 (2020).
- Li, Y. P., Deng, W. L., Jin, Z. B. Modeling retinitis pigmentosa through patient-derived retinal organoids. STAR Protocols. 2 (2), 100438 (2021).
- Gonzalez-Cordero, A., et al. Recapitulation of human retinal development from human pluripotent stem cells generates transplantable populations of cone photoreceptors. Stem Cell Reports. 9 (3), 820-837 (2017).
- Meyer, J. S., et al. Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment. Stem Cells. 29 (8), 1206-1218 (2011).
- Zhu, J., Lamba, D. A. Small molecule-based retinal differentiation of human embryonic stem cells and induced pluripotent stem cells. Bio-Protocol. 8 (12), 2882 (2018).
- Nakano, T., et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell. 10 (6), 771-785 (2012).
- Reichman, S., et al. From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium. Proceedings of the National Academy of Sciences. 111 (23), 8518-8523 (2014).
- Zhong, X., et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nature Communications. 5, 4047 (2014).
- Wahlin, K. J., et al. Photoreceptor outer segment-like structures in long-term 3D retinas from human pluripotent stem cells. Scientific Reports. 7 (1), 766 (2017).
- Capowski, E. E., et al. Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines. Development. 146 (1), (2019).
- Chichagova, V., et al. Differentiation of retinal organoids from human pluripotent stem cells. Current Protocols in Stem Cell Biology. 50 (1), 95 (2019).
- Kelley, R. A., Chen, H. Y., Swaroop, A., Li, T. Accelerated development of rod photoreceptors in retinal organoids derived from human pluripotent stem cells by supplementation with 9-cis retinal. STAR Protocols. 1 (1), 100033 (2020).
- Zhou, S., et al. Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFbeta and Wnt signaling. Development. 142 (19), 3294-3306 (2015).
- Chambers, S. M., et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology. 27 (3), 275-280 (2009).
- Mellough, C. B., et al. IGF-1 signaling plays an important role in the formation of three-dimensional laminated neural retina and other ocular structures from human embryonic stem cells. Stem Cells. 33 (8), 2416-2430 (2015).
- Susaimanickam, P. J., et al. Generating minicorneal organoids from human induced pluripotent stem cells. Development. 144 (13), 2338-2351 (2017).
