Name: differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations
Uploaded: 2018-08-24T12:30:00
Description: Watch this Scientific Journal Video about Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations at JoVE.com

该项目由 NIH 赠款 EY025290、GM127652 和罗切斯特大学开办资金资助。

1. hPSC 与视网膜色素的分化
<ol><li>维护和通过 hPSCs 如前所述18。 注: 所有细胞 (包括 hPSCs 和 hPSC-RPEs) 生长在37摄氏度, 在 5% CO2期间, 整个增长和分化协议。</li>
	<li>
在分化之前, 分裂汇合 hPSCs 到预涂覆的6井板。
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<li>要涂板, 解冻基底膜基质在冰上约1小时, 悬浮液化基质在4°c DMEM 培养基1:50 稀释, 添加800µL 涂层混合物的每一个井, 并孵化至少1小时在37摄氏...

<ol>
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E., 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=Missense+mutations+in+a+retinal+pigment+epithelium+protein,+bestrophin-1,+cause+retinitis+pigmentosa.">Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa.</a> Am J Hum Genet. 85 (5), 581-592 (2009).</li><li>Kramer, F., 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=Mutations+in+the+VMD2+gene+are+associated+with+juvenile-onset+vitelliform+macular+dystrophy+(Best+disease)+and+adult+vitelliform+macular+dystrophy+but+not+age-related+macular+degeneration.">Mutations in the VMD2 gene are associated with juvenile-onset vitelliform macular dystrophy (Best disease) and adult vitelliform macular dystrophy but not age-related macular degeneration.</a> Eur J Hum Genet. 8 (4), 286-292 (2000).</li><li>Marquardt, 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=Mutations+in+a+novel+gene,+VMD2,+encoding+a+protein+of+unknown+properties+cause+juvenile-onset+vitelliform+macular+dystrophy+(Best's+disease).">Mutations in a novel gene, VMD2, encoding a protein of unknown properties cause juvenile-onset vitelliform macular dystrophy (Best's disease).</a> Hum Mol Genet. 7 (9), 1517-1525 (1998).</li><li>Petrukhin, K., 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=Identification+of+the+gene+responsible+for+Best+macular+dystrophy.">Identification of the gene responsible for Best macular dystrophy.</a> Nat Genet. 19 (3), 241-247 (1998).</li><li>Yardley, J., 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=Mutations+of+VMD2+splicing+regulators+cause+nanophthalmos+and+autosomal+dominant+vitreoretinochoroidopathy+(ADVIRC).">Mutations of VMD2 splicing regulators cause nanophthalmos and autosomal dominant vitreoretinochoroidopathy (ADVIRC).</a> Invest Ophthalmol Vis Sci. 45 (10), 3683-3689 (2004).</li><li>Yang, T., Justus, S., Li, Y., Tsang, S. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-evoked+modulation+of+basolateral+membrane+Cl-+conductance+in+chick+retinal+pigment+epithelium:+the+light+peak+and+fast+oscillation.">Light-evoked modulation of basolateral membrane Cl- conductance in chick retinal pigment epithelium: the light peak and fast oscillation.</a> J Neurophysiol. 70 (4), 1669-1680 (1993).</li><li>Li, Y., Nguyen, H. V., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+Biopsy+and+Patient-Specific+Stem+Cell+Lines.">Skin Biopsy and Patient-Specific Stem Cell Lines.</a> Methods Mol Biol. 1353, 77-88 (2016).</li><li>Milenkovic, 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=Bestrophin+1+is+indispensable+for+volume+regulation+in+human+retinal+pigment+epithelium+cells.">Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells.</a> Proc Natl Acad Sci U S A. 112 (20), E2630-E2639 (2015).</li><li>Moshfegh, Y., 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=BESTROPHIN1+mutations+cause+defective+chloride+conductance+in+patient+stem+cell-derived+RPE.">BESTROPHIN1 mutations cause defective chloride conductance in patient stem cell-derived RPE.</a> Hum Mol Genet. 25 (13), 2672-2680 (2016).</li><li>Li, Y., 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=Patient-specific+mutations+impair+BESTROPHIN1's+essential+role+in+mediating+Ca2+-dependent+Cl-+currents+in+human+RPE.">Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca2+-dependent Cl- currents in human RPE.</a> Elife. 6, (2017).</li><li>Marmorstein, L. Y., 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=The+light+peak+of+the+electroretinogram+is+dependent+on+voltage-gated+calcium+channels+and+antagonized+by+bestrophin+(best-1).">The light peak of the electroretinogram is dependent on voltage-gated calcium channels and antagonized by bestrophin (best-1).</a> J Gen Physiol. 127 (5), 577-589 (2006).</li><li>Volland, S., Esteve-Rudd, J., Hoo, J., Yee, C., Williams, D. 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L., Chen, M., Fang, K., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-inspired+voltage-dependent+calcium+channel+blockers.">Bio-inspired voltage-dependent calcium channel blockers.</a> Nat Commun. 4, 2540 (2013).</li><li>Yang, T., Hendrickson, W. A., Colecraft, H. 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最重要的程序, 在一个菜式的方法是区分 hPSCs 与疾病引起的突变的正确的细胞谱系, 这是视网膜色素BEST1。因此, 在每次分化实验后, 通过视网膜色素特异的形貌和蛋白质标记物16、17、18, 应仔细验证所产生的 hPSC 视网膜上皮细胞的成熟状态。为了最小化克隆伪影, 应尽可能使用同一患者或多个人类胚胎干细胞克隆具有相同...

据记载, 至少五的视网膜退行性疾病是由基因突变引起的BEST1基因1,2,3,4,5,6,7,8, 与报告的变异的数量已经超过200并且仍然增加。这些BEST1相关的疾病, 也称为 bestrophinopathies, 导致视力逐渐丧失甚至失明, 目前还没有有效的治疗方法。BEST1的蛋白产物, 即 BESTROPHIN1 (BEST1), 是一种 Ca2 +活化的 Cl 通道 (CaCC), 特别表达在视网膜色素上皮 (RPE) 的眼睛5,6, 8,9。重要的是, 临床表型的BEST1相关疾病是减少视觉反应的光刺激, 称为光峰 (LP) 测量的 electrooculogram10,11;LP 被认为是介导的 CaCC 在视网膜色素12,13,14。为了更好地理解BEST1突变的病理机制, 并致力于潜在的治疗, 有必要研究突变 BEST1 通道内在表达的人视网膜色素细胞。
然而, 直接从活体患者获得视网膜色素上皮细胞是非常不切实际的。虽然本机视网膜色素细胞可以从人体尸体和胎儿的活检中获得, 但这些来源的难以获取却极大地限制了科学研究。因此, 除了人眼以外, 有其他的视网膜色素源是至关重要的。这一呼吁得到了最近的进展, 干细胞技术, 因为功能性视网膜色素细胞现在可以区别于人类多潜能干细胞 (hPSCs), 包括胚胎干细胞 (干细胞) 和诱导多潜能干细胞 (hiPSCs), 后者由捐赠者16,17,18的主要皮肤成纤维细胞重新编程产生。重要的是, hPSCs 的自我更新和干细胞确保了产生 RPEs 的可靠来源, 而干细胞的 hiPSCs 和基因组修饰电位的患者特异性 (如CRISPR) 提供了一种多功能的碟形疾病模型, 用于期望的BEST1突变。
hPSC 在小鼠视网膜色素模型上有几个优势: 1) BEST1敲除小鼠不显示任何视网膜异常19, 提高BEST1在视网膜色素瘤中对小鼠和人类的不同遗传需求的可能性;2) 只有3% 的人视网膜色素细胞是双核的, 与35% 在小鼠20;3) hiPSC 增强自体移植在视网膜疾病临床治疗中的应用21。然而, 动物模型仍然是必不可少的研究视网膜色素生理和病理在一个活体系统, 和致癌的潜力 hiPSC 不能忽视。
这里的过程描述了一个有用的和适度简单的 hPSC 的视网膜色素分化协议, 可用于研究和临床目的。该协议使用烟酰胺 (维生素 B3) 增加 hPSCs 分化为神经组织, 进一步诱导分化为 RPE 的治疗与激活。烟酰胺治疗已被证明增加色素细胞的数量 (一种分化为 RPE 的迹象), 可能通过减轻分化细胞的凋亡活动22。结果 hPSC 细胞显示相同的关键标记, 鹅卵石形态学和细胞功能作为本机人类视网膜色素上皮细胞22。因此, 在一项研究中, 所产生的 hPSC 视网膜色素细胞适用于下游功能分析, 包括免疫印迹法、染色和全细胞贴片钳, 并提供了详细的实验程序。临床上, 从干细胞中提取的视网膜色素上皮细胞在动物研究和人类试验中都显示了移植治疗黄斑变性的巨大潜力23。

<table>
	<tbody>
		<tr>
			<td>Knock-Out (KO) DMEM</td>
			<td>ThermoFisher</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>KO serum replacement</td>
			<td>ThermoFisher</td>
			<td>10829028</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>ThermoFisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>ThermoFisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>ThermoFisher</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Human activin-A</td>
			<td>PeproTech</td>
			<td>120-14</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM (a modification)</td>
			<td>Sigma-Aldrich</td>
			<td>M4526</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>N1 supplement</td>
			<td>Sigma-Aldrich</td>
			<td>N6530</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine-penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1146</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M7156</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0386</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodo-thyronin</td>
			<td>Sigma-Aldrich</td>
			<td>T5516</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR-1 medium</td>
			<td>Stemcell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-664</td>
			<td></td>
		</tr>
		<tr>
			<td>M-PER mammalian protein extraction reagent</td>
			<td>Pierce</td>
			<td>78501</td>
			<td></td>
		</tr>
		<tr>
			<td>proteinase inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr>
			<td>RPE65 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB100-355</td>
			<td></td>
		</tr>
		<tr>
			<td>CRALBP antibody</td>
			<td>Abcam</td>
			<td>ab15051</td>
			<td></td>
		</tr>
		<tr>
			<td>BEST1 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB300-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta Actin antibody</td>
			<td>ThermoFisher</td>
			<td>MA5-15739</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated donkey anti-mouse IgG</td>
			<td>ThermoFisher</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG</td>
			<td>ThermoFisher</td>
			<td>SA5-35521</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG</td>
			<td>LI-COR Biosciences</td>
			<td>926-68071</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>HEKA EPC10 patch clamp amplifier</td>
			<td>Warner Instruments</td>
			<td>895000</td>
			<td></td>
		</tr>
		<tr>
			<td>Patchmaster</td>
			<td>Warner Instruments</td>
			<td>895040</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations

虽然人类BEST1基因中有超过200种基因突变被发现并与视网膜退行性疾病相关联, 但病理机制仍然难以捉摸, 主要是由于缺乏良好的体内模型来研究BEST1及其在生理条件下的突变。BEST1编码离子通道, 即 BESTROPHIN1 (BEST1), 其作用于视网膜色素上皮 (RPE);然而, 人类视网膜色素上皮细胞的极有限的可获得性是科学研究的一个重大挑战。该协议描述了如何通过诱导人类多潜能干细胞 (hPSCs) 的分化而产生人类 RPEs 轴承BEST1致病突变。由于 hPSCs 是可自我再生的, 这种方法允许研究人员有一个稳定的来源的 hPSC RPEs 的各种实验分析, 如免疫印迹法, 免疫荧光和补丁钳, 从而提供了一个非常强大的疾病在一碟模型BEST1相关的视网膜条件。值得注意的是, 这一策略可以应用于研究 rpe (病理) 生理学和本机所表达的兴趣的其他基因。

技术上最具挑战性的一步是手工隔离, 目的是实现高纯度的差异 P0 hPSC-RPE 种群。经过成功的隔离, > 90% 细胞在 P0人口将增长和成熟, 以显示签名的 RPE 形态 (图 1C)。在 P0的人群中, 非 rpe 或未成熟的视网膜色素上皮细胞的存在几乎是不可避免的, 但不会干扰下游实验, 只要色素和鹅卵石状 hPSC 细胞的数量是压倒性的大多数。
<p ...

Watch this Scientific Journal Video about Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations at JoVE.com

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

人视网膜色素上皮细胞的分化、维持和分析: 一种BEST1突变的碟形模型

在这里, 我们提出了一个协议, 以区分视网膜色素上皮 (RPE) 细胞的人多潜能干细胞与患者衍生的突变。突变细胞系可用于功能分析, 包括免疫印迹法、免疫荧光和贴片钳。这一碟形疾病的方法绕过了获取本族人类视网膜色素细胞的困难。

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

Research

JoVE Journal

Genetics

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

科研

遗传学

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

crispr 介导的人类真菌病原体白色念珠菌基因组编辑

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

人类胰腺胰岛的单细胞RNA测序与分析

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

通过高分辨率熔融在水稻种群中识别突变

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

通过RNA-Seq分析，人类原主角细胞的体外分化特征

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...