rd10小鼠中人胚胎干细胞衍生的光感受器祖细胞的视网膜下递送
Summary
我们描述了用于制备冷冻保存后 hESC 衍生的光感受器祖细胞以及这些细胞在 rd10 小鼠中的视网膜下递送的详细方案。
Abstract
使用人类多能干细胞再生感光细胞是一种很有前途的疗法，可用于治疗晚期遗传性和衰老性视网膜疾病。我们已经证明，人类重组视网膜特异性层粘连蛋白亚型基质能够支持人胚胎干细胞（hESC）分化为光感受器祖细胞。此外，这些细胞的视网膜下注射在 rd10 啮齿动物和兔子模型中也显示出部分恢复。众所周知，视网膜下注射是一种既定的方法，由于它靠近目标空间，已被用于将药物化合物输送到眼睛的感光细胞和视网膜色素上皮 （RPE） 层。它还被用于将腺相关病毒载体输送到视网膜下空间以治疗视网膜疾病。由于小鼠眼球大小的限制，小鼠模型中药物化合物和细胞的视网膜下递送具有挑战性。该方案描述了用于注射的hESC衍生的光感受器祖细胞的详细程序以及这些细胞在遗传性视网膜色素变性突变 体rd10 小鼠中的视网膜下递送技术。这种方法允许对目标区域进行细胞治疗，特别是视网膜的外核层，在那里发生导致感光细胞变性的疾病。
Introduction
遗传性视网膜疾病和年龄相关性黄斑变性会导致感光细胞丢失并最终失明。视网膜感光器是视网膜的外段层，由负责光转导（即将光转换为神经元信号）的特殊细胞组成。视杆细胞和视锥细胞感光细胞与视网膜色素沉着层 （RPE） 相邻1。补偿细胞损失的感光细胞替代疗法一直是一种新兴和发展中的治疗方法。胚胎干细胞 （ESC）2、3、4、诱导多能干细胞 （iPSCs） 衍生的 RPE 细胞和视网膜祖细胞 （RPC）4、5、6、7、8 用于恢复受损的感光细胞。视网膜下间隙是视网膜和 RPE 之间的密闭空间，是沉积这些细胞以替换受损的感光细胞、RPE 和 Mueller 细胞的有吸引力的位置，因为它位于附近 9,10,11。
在临床前研究中，基因和细胞疗法一直在利用视网膜下空间进行再生医学治疗各种视网膜疾病。这包括以反义寡核苷酸疗法12,13或CRISPR/Cas9的形式递送基因或基因编辑工具的功能拷贝，或通过基于腺相关病毒（AAV）的策略进行碱基编辑14,15,16，植入材料（例如，RPE片，视网膜假体17,18,19）和分化干细胞衍生的视网膜类器官20，21,22 用于治疗视网膜和 RPE 相关疾病。在视网膜下空间使用 hESC-RPE31 治疗 RPE65 相关 Leber 先天性黑朦 （LCA）23,24、CNGA3 连锁色盲25、MERTK 相关视网膜色素变性26、脉络膜血症27、28、29、30已被证明是一种有效的方法。将细胞直接注射到受损区域附近可大大提高细胞在适当区域沉降、突触整合和最终视力改善的机会。
尽管在人类和大眼睛模型（即猪32、33、34、35、兔36、37、38、39、40 和非人灵长类动物41、42、43）中已经建立了视网膜下注射，但由于眼球大小的限制和巨大的占据鼠标眼睛的晶状体44,45,46。然而，转基因模型仅在小动物中容易获得，而在大型动物（即兔子和非人类灵长类动物）中不容易获得，因此小鼠视网膜下注射引起了人们关注视网膜遗传疾病的新治疗方法。目前有三种主要方法将细胞或AAV输送到视网膜下空间，即经角膜途径、经巩膜途径和平坦途径（见图2）。经角膜和经巩膜途径与白内障形成、粘连、脉络膜出血和注射部位反流有关 11、44、45、47、48、49。我们采用pars plana方法作为注射过程的直接可视化，可以在显微镜下实时获得注射部位。
我们最近描述了一种方法，该方法可以使用重组人视网膜特异性层粘连蛋白亚型 LN523，在无异种、化学定义的条件下将人胚胎干细胞 （hESC） 分化为光感受器祖细胞。由于发现 LN523 存在于视网膜中，我们假设人类视网膜的细胞外基质生态位可以在 体外 重现，从而支持与 hESC 的光感受器分化 36。单细胞转录组学分析显示，32 d后生成共表达锥杆同源盒和recoverin的感光祖细胞。使用模拟常染色体人视网膜色素变性的视网膜变性 10 （rd10） 突变小鼠模型来评估第 32 天 hESC 衍生的光感受祖细胞 在体内的疗效。将 hESC 衍生的感光祖细胞注射到 P20 处 rd10 小鼠的视网膜下空间，其中光感受器功能障碍和变性正在进行36。在这里，我们描述了用于制备冷冻保存后 hESC 衍生的光感受器祖细胞并递送至 rd10 小鼠视网膜下空间的详细方案。该方法还可用于将 AAV、细胞悬液、肽或化学物质施用到小鼠的视网膜下空间中。
Protocol
体内实验是根据新加坡保健集团（SingHealth）机构动物护理和使用委员会（IACUC）和视觉和眼科研究协会（ARVO）批准的关于在眼科和视觉研究中使用动物的声明进行的。幼崽通过喂食含有环孢菌素（260 g/L）的饮用水，对幼崽进行从P17（移植前）到P30（移植后）的免疫抑制。 1. 冷冻保存后第 32 天 hESC 衍生的光感受器祖细胞的制备 在37°C水浴中预热的?…
Representative Results
根据制造商的说明组装 10 μL 玻璃注射器（图 1），用于递送细胞悬液/培养基的钝针如 图 1B 所示。视网膜下注射的不同方法如 图 2 所示。我们描述了该协议中的pars plana方法（图2C）。安装在玻璃注射器上的钝针通过硬化切开术伤口插入，并进入全球的视网膜下空间。如 图3A所示，在?…
Discussion
视网膜下注射已用于细胞悬浮移植，以治疗RPE和视网膜疾病23,25,26,27,28,31,40。这种方法在啮齿动物研究中非常重要，不仅对于细胞移植和基因治疗方法，而且对于评估视网膜疾病的新型治疗化合物也是如此。目前，有三种?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
我们感谢 Wei Sheng Tan、Luanne Chiang Xue Yen、Xinyi Lee 和 Yingying Chung 为冷冻保存后第 32 天 hESC 衍生的光感受器祖细胞的制备提供技术援助。这项工作得到了美国国家医学研究委员会青年研究员研究资助奖（NMRC / OFYIRG / 0042 / 2017）和国家研究基金会第24届 竞争性研究计划资助（CRP24-2020-0083）对HGT的部分支持。
Materials
| 0.3% Tobramycin | Novartis | NDC 0078-0813-01 | Tobrex (3.5 g) |
| 0.3% Tobramycin and 0.1% Dexamethasone | Novartis | NDC 0078-0876-01 | Tobradex (3.5 g) |
| 0.5% Proparacaine hydrochloride | Alcon | NDC 0998-0016-15 | 0.5% Alcaine (15 mL) |
| 1 mL Tuberculin syringe | Turemo | SS01T2713 | |
| 1% Tropicamide | Alcon | NDC 0998-0355-15 | 1% Mydriacyl (15 mL) |
| 2.5% Phenylephrine hydrochloride | Alcon | NDC 0998-0342-05 | 2.5% Mydfrin (5 mL) |
| 24-well tissue culture plate | Costar | 3526 | |
| 30 G Disposable needle | Becton Dickinson (BD) | 305128 | |
| 33 G, 20 mm length blunt needles | Hamilton | 7803-05 | |
| Automated Cell Counter | NanoEnTek | Model: Eve | |
| B27 without Vitamin A | Life Technologies | 12587001 | 2%36 |
| Buprenorphine | Ceva | Vetergesic vet (0.3 mg/mL) | |
| CKI-7 | Sigma | C0742 | 5 µM36 |
| Cyclosporine | Novartis | 260 g/L in drinking water | |
| Day 32 hESC-derived photoreceptor progenitor cells | DUKE-NUS Medical School | Human embryonic stem cells are differentiated for 32 days. See protocol in Ref 36. | |
| Gauze | Winner Industries Co. Ltd. | 1SNW475-4 | |
| Glasgow Minimum Essential Medium | Gibco | 11710–035 | |
| hESC cell line H1 | WiCell Research Institute | WA01 | |
| Human brain-derived neurotrophic factor (BDNF) | Peprotech | 450-02-50 | 10 ng/mL36 |
| Human ciliary neurotrophic factor (CNTF) | Prospec-Tany Technogene | CYT-272 | 10 ng/mL36 |
| Ketamine hydrochloride (100 mg/mL) | Ceva Santé Animale | KETALAB03 | |
| LN-521 | Biolamina | LN521-02 | 1 µg36 |
| mFreSR | STEMCELL Technologies | 5854 | |
| Microlitre glass syringe (10 mL) | Hamilton | 7653-01 | |
| N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) | Selleckchem | S2215 | 10 µM36 |
| N-2 supplement | Life Technologies | A13707-01 | 1%36 |
| Non-essential amino acids (NEAA) | Gibco | 11140–050 | 1x36 |
| NutriStem XF Media | Satorius | 05-100-1A | |
| Operating microscope | Zeiss | OPMI LUMERA 700 | With Built-in iOCT function |
| PRDM (Photoreceptor differentiation medium, 50ml) | DUKE-NUS Medical School | See media composition36. Basal Medium, 10 µM DAPT, 10 ng/mL BDNF, 10 ng/mL CNTF, 0.5 µM Retinoic acid, 2% B27 and 1% N2. Basal Medium: 1x GMEM, 1 mM sodium pyruvate, 0.1 mM B-mercaptoethanol, 1x Non-essential amino acids (NEAA). | |
| Pyruvate | Gibco | 11360–070 | 1 mM36 |
| Rd10 mice | Jackson Laboratory | B6.CXB1-Pde6brd10/J mice | Gender: male/female, Age: P20 (injection), Weight: 3-6 g |
| Retinoic acid | Tocris Bioscience | 0695/50 | 0.5 µM36 |
| Round Cover Slip (12 mm) | Fisher Scientific | 12-545-80 | |
| SB431542 | Sigma | S4317 | 0.5 µM36 |
| Vidisic Gel (10 g) | Dr. Gerhard Mann | ||
| Xylazine hydrochloride (20 mg/mL) | Troy Laboratories | LI0605 | |
| β-mercaptoethanol | Life Technologies | 21985–023 | 0.1 mM36 |
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Diesen Artikel zitieren
Tun, S. B. B., Shepherdson, E., Tay, H. G., Barathi, V. A. Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice. J. Vis. Exp. (200), e65848, doi:10.3791/65848 (2023).