猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植
Summary
这里介绍的是一种将人多能干细胞（hPSC）衍生的视网膜组织移植到大型动物模型的视网膜下空间的手术技术。
Abstract
与光感受器丧失相关的视网膜退行性 （RD） 疾病，如年龄相关性黄斑变性 （AMD）、色素性视网膜炎 （RP） 和 Leber 先天性黑斑病 （LCA） 会导致进行性和使人衰弱的视力丧失。一旦光感受器丧失，对可以恢复视力的疗法的需求尚未得到满足。将人多能干细胞（hPSC）衍生的视网膜组织（类器官）移植到具有晚期RD的眼睛的视网膜下间隙，使视网膜组织薄片具有数千个健康的无突变光感受器，并且有可能通过一个批准的方案治疗与感光器变性相关的大多数/所有致盲疾病。将胎儿视网膜组织移植到动物模型和晚期RD患者的视网膜下间隙已经开发成功，但由于伦理问题和有限的组织供应，不能用作常规治疗。大眼遗传性视网膜变性（IRD）动物模型对于开发视力恢复疗法很有价值，利用先进的手术方法将视网膜细胞/组织移植到视网膜下空间。眼球大小和光感受器分布（例如，黄斑样区域中心 区域的存在）和IRD模型的可用性的相似性，密切概括了人类IRD，将有助于将有前途的疗法快速转化为临床。这里介绍的是一种将hPSC衍生的视网膜组织移植到大型动物模型的视网膜下空间的手术技术，允许在动物模型中评估这种有前途的方法。
Introduction
全世界有数百万人受到视网膜变性（RD）的影响，导致视力障碍或失明，与光敏光感受器（PRs）的丧失有关。年龄相关性黄斑变性（AMD）是由遗传风险因素和环境/生活方式因素共同导致失明的主要原因。此外，已发现超过200个基因和位点可导致遗传性RD（IRD）1。视网膜色素变性 （RP） 是最常见的 IRD，具有遗传异质性，据报道，大约 70 个基因中有 3,000 多个基因突变 2,3,4。导致儿童失明的Leber先天性黑痢病（LCA）也是遗传异质的5,6。基因增强疗法已经开发出来，并正在临床试验中，用于治疗少量IRDs3,7。然而，必须开发一种单独的疗法来治疗每种不同的遗传形式的IRD，从而只治疗一小部分患者。此外，基因增强依赖于可挽救的光感受器群体的存在，因此不适用于晚期变性。
因此，迫切需要开发治疗晚期RD和严重至终末失明的疗法。在过去的20年中，神经假体植入物已经在人类使用之前在大型动物模型中开发和测试，例如猫8,9,10,11,12,13,14。同样，在过去的20年中，已经开发了利用胚胎甚至成熟的哺乳动物视网膜移植视网膜的视网膜替代疗法15,16,17,18,19,20,21,22，甚至在RD患者中成功测试了23,24,25.这两种方法都利用了将新的传感器（在神经修复装置26,27的情况下为光伏硅光电二极管，在视网膜片植入的情况下，在视网膜片植入的情况下，将健康的无突变光感受器）引入具有退化PR的视网膜的想法。最近的研究调查了基于干细胞的方法的使用，例如移植人多能干细胞（hPSC）衍生的视网膜祖细胞28,29，hPSC光感受器30和hPSC-视网膜类器官31,32,33。视网膜类器官能够在培养皿中形成视网膜组织，并衍生出具有数千个无突变PR的感光器片，类似于发育中的人类胎儿视网膜中的感光层34,35,36,37,38,39,40.将hPSC来源的视网膜组织（类器官）移植到RD患者的视网膜下空间是新的和有前途的研究细胞治疗方法之一，许多团队正在研究31,32,41,42。与移植细胞悬液（年轻感光器或视网膜祖细胞）相比，移植的胎儿光感受器片在临床试验中被证明可以改善视力23,24。
这里介绍的方案详细描述了用于整个视网膜类器官（而不是类器官边缘33,41）的视网膜下递送的移植程序，作为引入带有PR的完整视网膜片的潜在更好方法，以增加移植物存活率并改善片状保存。尽管已经开发了引入一块扁平的人类视网膜和RPE贴片的程序43,44,45，但尚未研究较大的3D移植物的移植。干细胞来源的视网膜类器官为开发视力恢复技术提供了取之不尽的感光片来源，不受伦理限制，被认为是人类视网膜组织的极好来源，用于治疗晚期RD和终末失明46。开发用于精确视网膜下植入视网膜类器官的手术方法，同时对宿主视网膜壁龛（神经视网膜，视网膜色素上皮以及视网膜和脉络膜脉络膜脉管系统）的损伤最小，是将这种疗法推向临床应用的关键步骤之一31,32。猫、狗、猪和猴子等大型动物模型已被证明是研究手术递送方法以及证明植入组织片（视网膜色素上皮 （RPE） 细胞）的安全性和研究类器官使用的良好模型 41,44,45,47,48,49,50.大型动物眼睛具有与人类相似的地球大小以及相似的解剖结构，包括存在高感光器密度的区域，包括视锥细胞（中央区域），类似于人类黄斑6,51,52。
在这篇手稿中，描述了一种将hPSC衍生的视网膜组织（类器官）植入猫科动物大型动物模型（野生型和CrxRdy/+猫）的视网膜下空间的技术，该技术与有希望的疗效结果32,53一起为进一步发展这种研究性疗法以临床应用治疗RD疾病奠定了基础。
Protocol
程序是根据视觉和眼科研究协会（ARVO）关于在眼科和视力研究中使用动物的声明进行的。它们还得到了密歇根州立大学机构动物护理和使用委员会的批准。本研究使用了来自密歇根州立大学饲养的猫群的野生型和 CrxRdy/+ 猫。将动物饲养在12小时：12小时明暗循环下，并喂食商业完整的猫饲料。 1. 植入前程序和手术设置 根据研究设计选择野生型或 …
Representative Results
该程序能够在大型眼动物模型的视网膜下空间成功且可重复地植入hPSC衍生的视网膜类器官（此处使用2个示例进行演示：具有健康光感受器（PR）的野生型猫和具有退化PR和视网膜的 CrxRdy/ + 猫）。使用 图1 所示的步骤制备hPSC衍生的视网膜类器官并将其加载到注射装置的硼硅酸盐玻璃套管中，以使类器官不被损坏。这可以通过类器官上样（步骤2.10）和手?…
Discussion
将hPSC衍生的视网膜组织（视网膜类器官）植入视网膜下间隙是一种有前途的实验方法，用于恢复由PR细胞死亡（严重或终末失明）引起的晚期视网膜退行性疾病的视力。所提出的方法建立在早期开发和成功测试的实验疗法的基础上，该疗法基于一块人胎儿视网膜组织的视网膜下移植23,24,25。它介绍了使用源自 hPSC 的替代、可补?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由NEI Fast-track SBIR拨款R44-EY027654-01A1和SBIR拨款3 R44 EY 027654 – 02 S1（I.O.N.，Lineage Cell Therapeutics; Petersen-Jones博士是联合PI）资助。作者要感谢Janice Querbin女士（MSU RATTS）对本研究中包含的动物的麻醉和一般护理的帮助，以及在手术设置和器械制备/灭菌方面的帮助。作者要感谢Paige Winkler博士在植入前一天接受类器官并将其放入培养基中的帮助，以及在植入当天的帮助。作者还感谢Randy Garchar先生（LCTX）勤奋地运输视网膜类器官，组装托运人，并在每次装运后下载温度和G应力记录。这项工作是在作者伊戈尔·纳松金（Igor Nasonkin）受雇于Biotime（现为Lineage）时完成的。
Materials
| 0.22 µm pore syringe filter with PES membrane | Cameo | NA | can be found by various suppliers |
| 23G subretinal injector with extendable 41 G cannula | DORC | 1270.EXT | |
| 250 µL hamilton gas tight luer lock syringe | Hamilton | NA | can be found by various suppliers |
| 6-0 Silk suture | Ethicon | 707G | |
| 6-0/7-0 polyglactin suture | Ethicon | J570G | |
| Acepromazine maleate 500mg/5mL (Aceproject) | Henry Schein Animal Health | NA | can be found by various suppliers |
| Buprenorphine 0.3 mg/mL | Par Pharmaceutical | NA | can be found by various suppliers |
| cSLO + SD-OCT | Heidelberg Engineering | Spectralis HRA+ OCT | |
| Cyclosporine | Novartis | NA | can be found by various suppliers |
| Dexamethasone 2mg/mL (Azium) | Vetone | NA | can be found by various suppliers |
| Doxycyline 25mg/5mL | Cipla | NA | can be found by various suppliers |
| Fatal Plus solution (pentobarnital solution) | Vortech | NA | can be found by various suppliers |
| Gentamicin 20mg/2mL | Hospira | NA | can be found by various suppliers |
| Glass capillary (Thin-Wall Single-Barrel Standard Borosilicate (Schott Duran) Glass Tubing | World Precision Instruments | TW150-4 | |
| Methylprednisolone actetate 40 mg/mL | Pfizer | NA | can be found by various suppliers |
| Microscope | Zeiss | NA | |
| OCT medium (Tissue-Tek O.C.T. Compound) | Sakura | 4583 | |
| Olympic Vac-Pac Size 23 | Natus | NA | can be found by various suppliers |
| Paraformaldehyde 16% solution | EMS | 15719 | |
| Phenylephrine Hydrochloride 10% Ophthalmic Solution | Akorn | NA | can be found by various suppliers |
| Prednisolone 15mg/5mL | Akorn | NA | can be found by various suppliers |
| Propofol 5000mg/50mL (10 mg/mL) (PropoFlo28) | Zoetis | NA | can be found by various suppliers |
| RetCam II video fundus camera | Clarity Medical Systems | NA | can be found by various suppliers |
| Triamcinolone 400mg/10 mL (Kenalog-40) | Bristol -Myers Squibb Company | NA | can be found by various suppliers |
| Tropicamide 1% ophthalmic solution | Akorn | NA | can be found by various suppliers |
| Vitrectomy 23G port | Alcon | Accurus systems | |
| Vitrectomy machine | Alcon | Accurus systems | |
| Vitreo-retinal vertical 80° scissors with squeeze handle | Frimen | FT170206T |
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Cite This Article
Occelli, L. M., Marinho, F., Singh, R. K., Binette, F., Nasonkin, I. O., Petersen-Jones, S. M. Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model. J. Vis. Exp. (174), e61683, doi:10.3791/61683 (2021).