Name: subretinal transplantation of human embryonic stem cell derived-retinal pigment epithelial cells into a large-eyed model of geographic atrophy
Uploaded: 2018-01-22T14:30:00
Description: Watch this Scientific Journal Video about Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy at JoVE.com

这项研究得到了卡研究所、皇冠公主玛加丽塔·瓦尔斯特伦的视力受损基金会、埃德温∙乔丹基金会眼科研究、瑞典眼基金会、国王古斯塔夫五世基金会、ARMECLindeberg 基金会和 Cronqvist 基金会

下面的协议遵循卡 Instituet 的动物保育指南。所有使用新西兰白化兔的动物实验 (材料表) 已得到区域动物伦理委员会 (召开 Norra Djurförsöksetiska Nämnd) 的批准 (许可证: dnr 56/15)。使用干细胞 (dnr 2011/745-31/3) 和干细胞-RPE (dnr 2013/813-31/2) 的转让和操纵也符合瑞典立法和卡学院条例, 并已得到区域人类伦理委员会的批准 (Regionala Etikprövningsnämnden 我斯德哥尔摩)。
1. 视网膜?...

<ol>
	<li>Plaza Reyes, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xeno-Free+and+Defined+Human+Embryonic+Stem+Cell-Derived+Retinal+Pigment+Epithelial+Cells+Functionally+Integrate+in+a+Large-Eyed+Preclinical+Model.">Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model.</a> Stem Cell Rep. 6 (1), 9-17 (2016).</li><li>Bartuma, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Imaging+of+Subretinal+Bleb-Induced+Outer+Retinal+Degeneration+in+the+Rabbit.">In Vivo Imaging of Subretinal Bleb-Induced Outer Retinal Degeneration in the Rabbit.</a> Invest Ophthalmol Vis Sci. 56 (4), 2423-2430 (2015).</li><li>Petrus-Reurer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+Subretinal+Suspension+Transplants+of+Human+Embryonic+Stem+Cell-Derived+Retinal+Pigment+Epithelial+Cells+in+a+Large-Eyed+Model+of+Geographic+Atrophy.">Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy.</a> Invest Ophthalmol Vis Sci. 58 (2), 1314-1322 (2017).</li><li>Carido, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+mouse+model+with+complete+RPE+loss+and+its+use+for+RPE+cell+transplantation.">Characterization of a mouse model with complete RPE loss and its use for RPE cell transplantation.</a> Invest Ophthalmol Vis Sci. 55 (8), 5431-5444 (2014).</li><li>Lund, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+cells+rescue+visual+function+in+dystrophic+RCS+rats.">Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.</a> Cloning Stem Cells. 8 (3), 189-199 (2006).</li><li>Vugler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+the+phenomenon+of+HESC-derived+RPE:+anatomy+of+cell+genesis,+expansion+and+retinal+transplantation.">Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation.</a> Exp Neurol. 214 (2), 347-361 (2008).</li><li>Schwartz, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> Lancet. 379 (9817), 713-720 (2012).</li><li>Hughes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+schematic+eye+for+the+rabbit.">A schematic eye for the rabbit.</a> Vision Res. 12 (1), 123-138 (1972).</li><li>Blanch, R. J., Ahmed, Z., Berry, M., Scott, R. A., Logan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+retinal+injury.">Animal models of retinal injury.</a> Invest Ophthalmol Vis Sci. 53 (6), 2913-2920 (2012).</li><li>Nork, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+anatomic+consequences+of+subretinal+dosing+in+the+cynomolgus+macaque.">Functional and anatomic consequences of subretinal dosing in the cynomolgus macaque.</a> Arch Ophthalmol. 130 (1), 65-75 (2012).</li></ol>

在本协议中, 对遗传算法的大眼模型的生成及其在评价干细胞-RPE 积分中的临床应用进行了描述。
为了将 GA 及相关疾病的再生疗法翻译为临床7, 必须开发和优化方法, 以忠实地捕获移植和影像学的临床方法。兔子在这方面有吸引力: 它有一个相对较大的眼睛, 允许眼内手术和标准成像的使用, 是便宜和容易安置相比, 其他大眼睛动物。
<p class="jove_conten...

该协议描述了一种大眼睛的地理萎缩 (GA) 的临床前模型的产生, 允许评价移植干细胞-RPE 在视网膜空间的整合。本文详细介绍的方法已在3份最近的出版物中使用, 它们展示了干细胞1的视网膜色素上皮细胞的丰富、纯净和功能性种群的产生, 以及产生外视损伤和类似 GA 的表型由视网膜注射的生理盐溶液 (即, BSS 和 PBS) 或 NaIO3在兔子眼中2,3。我们进一步证明, 亚视网膜悬吊移植的干细胞-RPE 形成广泛功能单分子膜与感光剂抢救能力2。
利用兔眼的几个优点来产生一种 GA 模型的疾病。首先, 兔眼的大小, 这是70% 的成人眼睛的体积, 允许临床有意义的移植使用的细胞密度远远低于常规使用的小鼠眼 (1000 细胞/µL vs. 5万细胞/µL)4,5. 其次, 啮齿类动物的手术通常是通过脉络膜巩膜的, 这会损害视网膜屏障, 并可能引发炎症反应和可能的拒绝6。这两个因素一起可能导致移植细胞的多层和结块, 以及移植细胞在一个被破坏的本地视网膜组织中整体的不良整合。然而, 大眼睛的兔子模型允许执行一项与临床设置完全相同的手术技术。第三, 大眼睛模型也允许高分辨率的在体内成像和监测移植细胞和上覆视网膜通过时间1,2,3。因此, 我们描述了一个临床相关和成本效率的临床前模型, 应该是一个有吸引力的替代啮齿目动物的人有兴趣的研究正常和病变的视网膜和亚视网膜空间。

<table >
	<tbody>
		<tr >
			<td >NutriStem hESC XF differentiation medium &#8211;bFGF and &#8211;TGFb</td>
			<td >Biological Industries</td>
			<td >06-5100-01-1A</td>
			<td ></td>
		</tr>
		<tr >
			<td >TrypLE Select 1x</td>
			<td >Gibco, ThermoFisher Scientific Corp</td>
			<td >12563-011</td>
			<td ></td>
		</tr>
		<tr >
			<td >PBS without Ca2+ and Mg2+</td>
			<td >Gibco, ThermoFisher Scientific Corp</td>
			<td >14190-094</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell strainer 40 &#956;m Nylon</td>
			<td >VWR</td>
			<td >732-2757</td>
			<td ></td>
		</tr>
		<tr >
			<td >Needle 30g 0.5&#8217;&#8217;; 0.3 x 13mm</td>
			<td >BD Microlance</td>
			<td >304827</td>
			<td ></td>
		</tr>
		<tr >
			<td >Acrodisc 25mm Syringe Filter</td>
			<td >Acrodisc</td>
			<td >PN4612</td>
			<td></td>
		</tr>
		<tr >
			<td >0.4 % trypan blue</td>
			<td >ThermoFisher Scientific Corp</td>
			<td >15250061</td>
			<td >Use at 0.2%</td>
		</tr>
		<tr >
			<td >NaIO3</td>
			<td >Sigma-Aldrich Corp</td>
			<td>S4007</td>
			<td ></td>
		</tr>
		<tr >
			<td >BSS</td>
			<td >Alcon Nordic A/S</td>
			<td >65079550</td>
			<td ></td>
		</tr>
		<tr >
			<td >70% Ethanol</td>
			<td >Solveco AB</td>
			<td>1047</td>
			<td></td>
		</tr>
		<tr >
			<td >Ketaminol, 100 mg/mL</td>
			<td >Intervet, Boxmeer</td>
			<td >511519</td>
			<td >Use 35 mg/kg ketamine</td>
		</tr>
		<tr >
			<td >Rompun vet, 20 mg/mL</td>
			<td >Bayer Animal Health</td>
			<td >22545</td>
			<td >Use 5 mg/kg xylazine</td>
		</tr>
		<tr >
			<td >Triescence, 40 mg/mL</td>
			<td >Alcon Nordic A/S</td>
			<td >412915</td>
			<td >2 mg intraviterial</td>
		</tr>
		<tr >
			<td >Cyklopentolat-phenylephrine, 0.75% + 2.5%</td>
			<td >APL</td>
			<td >321968</td>
			<td >Use 1 drop in each eye</td>
		</tr>
		<tr >
			<td >Viscotears</td>
			<td>Laboratoires Th&#233;a</td>
			<td >597562</td>
			<td ></td>
		</tr>
		<tr >
			<td >Topical saline</td>
			<td >Apotea AB</td>
			<td>7053249369080</td>
			<td></td>
		</tr>
		<tr >
			<td >Allfatal vet. 100 mg/mL</td>
			<td >Omnidea</td>
			<td>77168</td>
			<td >Use 100 mg/mL pentobarbital</td>
		</tr>
		<tr >
			<td >Extension tube (Hammer)</td>
			<td >MedOne Surgical Inc</td>
			<td >3223</td>
			<td ></td>
		</tr>
		<tr >
			<td >25G/38G polytip subretinal cannula</td>
			<td >MedOne Surgical Inc</td>
			<td >3219</td>
			<td >25G/38G</td>
		</tr>
		<tr >
			<td >Single Use Flat Lens</td>
			<td >Volk</td>
			<td >#VWFD10</td>
			<td ></td>
		</tr>
		<tr >
			<td >Barraquer Colibri lid retractor</td>
			<td >AgnTho&#39;s AB</td>
			<td >42-020-030</td>
			<td></td>
		</tr>
		<tr >
			<td >Non-valved trocars</td>
			<td >Alcon Nordic A/S</td>
			<td>8065751448</td>
			<td></td>
		</tr>
		<tr >
			<td >Clawed forceps</td>
			<td >Bausch &#38; Lomb Nordic AB</td>
			<td >ET1811</td>
			<td></td>
		</tr>
		<tr >
			<td >Alcon Accurus 400VS Vitrectomy machine</td>
			<td >Alcon Nordic A/S</td>
			<td >8065740238</td>
			<td ></td>
		</tr>
		<tr >
			<td >Accurus 25+ Gauge Vitrectomy TotalL Plus Pak</td>
			<td >Alcon Nordic A/S</td>
			<td >8065751493</td>
			<td ></td>
		</tr>
		<tr >
			<td >SD-OCT device</td>
			<td>Heidelberg Engineering</td>
			<td>Spectralis HRA+OCT</td>
			<td>Use Heidelberg Eye Explorer version 1.9.10.0</td>
		</tr>
		<tr >
			<td >24 well plates</td>
			<td >Sarstedt</td>
			<td >83.3922</td>
			<td></td>
		</tr>
		<tr >
			<td >Neubauer hemocytometer</td>
			<td >VWR</td>
			<td >631-0925</td>
			<td></td>
		</tr>
		<tr >
			<td >New Zealand albino rabbits</td>
			<td>Lidk&#246;pings Rabbit Farm, Sweden</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >hESC-RPE cells</td>
			<td ></td>
			<td ></td>
			<td>See reference number 1</td>
		</tr>
	</tbody>
</table>

subretinal transplantation of human embryonic stem cell derived-retinal pigment epithelial cells into a large-eyed model of geographic atrophy

地理萎缩 (GA), 晚期与干年龄相关的黄斑变性的特点是失去视网膜色素上皮 (RPE) 层, 这导致后退化的重要视网膜结构 (如, 光) 导致严重视力损害。类似地, 视网膜色素上皮的丧失和视力减退见于长期随访的晚期与湿性黄斑变性 (AMD) 接受玻璃抗血管内皮生长因子 (VEGF) 治疗的患者。因此, 一方面, 这是至关重要的有效地从一个无限的来源, 可以作为替代治疗的视网膜色素上皮细胞。另一方面, 重要的是要评估的行为和整合的模型中衍生细胞的疾病所涉及的外科和影像学方法尽可能接近于那些应用于人类。在这里, 我们提供了一个详细的协议的基础上, 我们以前的出版物, 描述了使用白化兔眼的 GA 前体细胞的生成, 用于评价人类胚胎干细胞衍生的视网膜色素上皮细胞 (干细胞-RPE) 在临床相关的设置。分化干细胞-RPE 被移植到天真的眼睛或眼睛与 NaIO3诱导的 GA 样视网膜变性使用25克 transvitreal 部扁平技术。采用多模态高分辨率非侵入实时成像技术对退化和移植区进行评价。

在图 2中显示了典型的活体图像, 如生物滤池、IR-cSLO 和正常白化兔视网膜的 SD OCT。注意不同的视网膜层与他们的独特水平的光反射捕获的 SD OCT 仪器。
在数字 1A和图 1B中, 创建子视网膜泡的设置如下所示: 一个盖子牵引器定位, 使眼睛的盖子, 允许插入3?...

Watch this Scientific Journal Video about Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy at JoVE.com

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

视网膜色素上皮细胞可作为晚期相关黄斑变性的细胞替代疗法。该协议描述了大眼睛的地理萎缩模型的产生和人类胚胎干细胞源性视网膜色素上皮细胞视网膜移植到这种疾病模型。

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Research

JoVE Journal

Biology

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

分离和造血干细胞移植（造血干细胞）

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

气管切开术：干细胞移植肺的方法

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Generation of Human CD40-activated B cells

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

人类干细胞异种移植到鸡胚

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...