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。
	<ol>
<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摄氏度。</li>
		<li>当涂层完成后, 吸入混合物并立即将1.5–2毫升介质加入水井中。</li>
		<li>从旧的培养皿中抬起 hPSCs, 用 PBS 加0.5 毫米 EDTA 孵化细胞, 室温5分钟, 吸出溶液, 用培养基将小团簇并用重悬细胞。</li>
		<li>种子细胞在20% 融合在新的板材。 注: 贮存基底膜基质在100µL 整除数在-20 摄氏度, 并使用一个整除涂层6井板。涂层的时间可以延长到4小时, 在37摄氏度, 或在室温过夜。重要的是暂停和种子 hPSCs 作为小团簇的3–5细胞, 而不是单个细胞。</li>
	</ol>
</li>
	<li>当细胞长成全融合时, 用分化培养基 (4 毫升/井) 取代培养基 (表 1, 不激活)。培养14天, 改变介质 (4 毫升/井) 3 x/周。 注: hPSC 板密度加倍大约每24小时. 在分化过程开始后, 明显的细胞死亡是预期的。在培养基变化过程中, 肉眼可以看到死细胞和碎片在水井中的聚集。</li>
	<li>从15天到28天, 补充分化培养基与 100 ng/毫升人类激活 (表 1, 与人类激活 a)。改变介质 (4 毫升/井) 3 x/周。</li>
	<li>停止激活-从29天开始补充 (表 1, 没有人激活)。继续细胞培养在分化培养基为8–10星期, 直到色素 hPSC-RPE 簇出现 (图 1A-B)。 注意: 染色细胞簇可以被肉眼观察到细胞培养板上的小黑点 (图 1A-B, 顶部)。在20X 显微镜下 (图 1A-B, 底部) 可以看到具有特征鹅卵石形状的个体色素细胞。</li>
</ol>2. 分化的视网膜色素上皮细胞的分离和培养
<ol><li>移除分化介质, 用 PBS 冲洗一次, 在每个井中加入1毫升0.05% 胰蛋白酶加 1 U/µL 胶原酶, 并在37摄氏度内孵育 20–30. 除去胰蛋白酶/胶原酶, 轻轻地将2毫升预热的 RPE 介质加入6井板的每一井 (c0>Table 2)。 注: 使用倒置显微镜监测 hPSC 视网膜色素上皮细胞的形态, 在胰蛋白酶/胶原酶治疗前是多边形, 并在充分消化后变成圆形。</li>
	<li>
使用本生燃烧器拉玻璃巴斯德吸管。
	<ol>
<li>用双手, 保持长 (9 英寸) 巴斯德吸管在两端, 所以它是水平的, 上面的本生燃烧器约2/3 的长度的薄部分的吸管。</li>
		<li>一旦玻璃从火焰中变软, 迅速把两端分开, 这样在吸管的新端形成一个微型刮刀状尖端 (图 2)。</li>
		<li>重复数次以创建多个 "单元格刮刀"。喷雾的拉吸管与70% 乙醇灭菌和空气干燥他们在细胞培养罩。</li>
	</ol>
</li>
	<li>
在显微镜下, 用微 "细胞刮刀" 轻轻地将色素 hPSC-RPE 簇从盘子中分离出来。
	<ol>
<li>轻轻地轻敲 (不要刮) 直接对色素细胞, 它将漂浮到培养基中, 一旦它们从井底离解。</li>
		<li>立即使用20µL 微轻轻吸入分离的 hPSC 视网膜色素细胞, 并将其转移到预涂6井板与视网膜色素中等24。</li>
		<li>收集 ~ 5 x 103细胞 (~ 10% 融合, 观察下20X 显微镜下) 在每一个12井板的预涂层井通过重复步骤 2.3. 1–2.3. 2 多次, 根据需要。把最后的音量从中到2毫升。 注: 为了防止游离 hPSC 视网膜色素上皮细胞漂浮, 避免在离解过程中板块的移动, 使离解细胞漂浮在培养基中, 但仍保持局部集中。</li>
	</ol>
</li>
	<li>培养在视网膜色素培养基上的12井板上新隔离的 hPSC 细胞 (P0), 使其形成染色单层 (图 1C)。</li>
	<li>更改中等 3 x/周。当改变培养基时, 在每个井中留出大约1/3 体积的旧介质, 并添加2/3 体积的新鲜、预热的视网膜色素培养基。在这个阶段, 使用2毫升的 RPE 介质为每个井在一个12井板 (因此, 交换1.3 毫升的媒介每次)。 注: P0 hPSC-RPE 细胞的单层可能形成圆顶, 表明细胞正积极输送液体, 并由紧密连接25锚定。因此, 圆顶的形成是一个成熟的视网膜色素状态的指标。</li>
</ol>3. 视网膜色素免疫印迹法的命运验证
<ol><li>利用哺乳动物蛋白提取缓冲剂, 用蛋白酶抑制剂鸡尾酒, 制作 hPSC 细胞裂解物。在30分钟的冰上, 在 1.3万 x g的离心前孵育细胞裂解物, 以在4摄氏度处丢弃难溶细胞碎片。测定裂解液的总蛋白浓度。</li>
	<li>变性40µg 的总蛋白在 Laemmli SDS 样品缓冲区75摄氏度10分钟. 将10% 三甘氨酸 SDS 页凝胶上的蛋白质样品分离为90伏的恒定电压, 然后 15 v, 直到染料前端达到凝胶的底部。</li>
	<li>在预冷的三甘氨酸缓冲液中将蛋白质转移到硝化膜上, 再辅以10% 甲醇100伏, 1 小时。</li>
	<li>在室温下, 用0.1% 非离子洗涤剂 (TBST) 和5% 非脂肪干牛奶在三层缓冲盐水中阻断膜。</li>
	<li>在4摄氏度的阻塞缓冲液中, 用最初的抗体将细胞膜孵化出来。稀释针对 RPE 特异标记蛋白的抗体如下: RPE65, 1:1, 000;CRALBP, 1:1, 000;BEST1, 1:1, 000。稀释抗体对β肌动蛋白在 1:10,000, 以检测β肌动蛋白作为负荷控制。</li>
	<li>用 TBST 洗3次膜, 每次洗5分钟。</li>
	<li>用荧光共轭山羊抗小鼠 IgG 二次抗体在室温下以1小时的阻塞缓冲液稀释1:10,000。</li>
	<li>用 TBST 洗4次膜, 每次洗10分钟。</li>
	<li>使用红外成像系统检测蛋白质 (图 3)。</li>
</ol>4. 染色检测 BEST1 亚细胞定位
<ol><li>用2毫升的 PBS 清洗 hPSC 视网膜色素细胞, 并在室温下固定2毫升4% 多聚甲醛45分钟。</li>
	<li>用2毫升的 pbs 冲洗两次, 并在2毫升的 pbs 中孵化出0.1% 个非离子表面活性剂和2% 驴血清以45分钟来阻断非特定的结合部位。</li>
	<li>室温下用 BEST1 抗体 (1:200 稀释) 孵化细胞, 2 小时。</li>
	<li>用2毫升 PBS 冲洗细胞3次。用荧光共轭二级 IgG (1:1, 000) 在室温下孵育1小时。</li>
	<li>用2毫升 PBS 冲洗3次以去除未绑定的二级抗体。</li>
	<li>用共焦显微镜观察染色细胞 (图 4)。</li>
</ol>5. 用全细胞膜片钳记录 hPSC -RPE 中 Ca2 +相关 Cl 电流
<ol><li>24–72 h 在膜片钳之前, 分裂一个完全汇合的井 (在一个12井板) 成熟 P0 hPSC-RPE 细胞 (鹅卵石形状和着色)。吸入培养基, 用 PBS 冲洗一次, 并在井中加入1毫升0.05% 胰蛋白酶加 1 U/µL 胶原酶。 注: 预热的胰蛋白酶/胶原酶到37摄氏度的权利使用前。</li>
	<li>孵育37°c 8 分钟。 注意: 在这一步之后, hPSC 细胞仍然黏附和连接。</li>
	<li>用1毫升的微轻轻地将细胞从盘子里冲洗出来, 将消化混合物中的细胞转移到15毫升锥形管上。用1毫升预热的胰蛋白酶/胶原酶洗井, 收集残余细胞, 并将其组合成15毫升锥形管。 注意: 在这一点上, hPSC 视网膜上皮细胞仍在大团簇中。不要试图通过剧烈的吹打打破细胞团簇。有些团簇可能粘在1毫升尖端的内壁上。</li>
	<li>在37°c 干浴中孵化锥形管8分钟。</li>
	<li>用1毫升的微轻轻地吸收10–15倍的消化混合。 注意: 细胞团簇变得小得多, 但仍然可见。避免剧烈的吹打。</li>
	<li>重复前面的两个步骤。 注: 大部分细胞应在吹打后单细胞悬浮。可能还有一些微小的细胞团簇, 这是可以接受的。可选: 在37摄氏度孵育锥形管, 再加5分钟, 然后用温和的吹打。胰蛋白酶/胶原酶在37°c 的总时间不应超过30分钟 (8 + 8 + 8 + 5 = 29)。</li>
	<li>将5毫升预热的 RPE 培养基添加到含有所消化细胞的15毫升圆锥管中。旋转 200 x g 5 分钟收集细胞。在10–20% 融合上预涂上 35 mm 的菜肴上重新悬浮和种子细胞 (例如, 在12井板上的100% 汇合井, 35 融合的五10% 毫米菜肴)。 注意: 在任何时候, 避免剧烈的吹打, 这可能导致显着的细胞死亡, 由明显的黑细胞碎片在上清后, 离心。良好分离的单细胞播种是修补钳记录的关键。</li>
	<li>按照前面描述的18、26、27、28、29、30、31, 执行全细胞贴片钳。从一个阶梯电位族 (-100 到 +100 mv, 从持有电位 0 mv) 获取当前痕迹 (图 5)。 注: 内部和外部解决方案的食谱分别在表 3和表 4中进行了说明。</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+some+organizational+characteristics+of+the+mouse+central+retina+and+the+human+macula.">A comparison of some organizational characteristics of the mouse central retina and the human macula.</a> PLoS One. 10 (4), e0125631 (2015).</li><li>Kamao, 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=Characterization+of+human+induced+pluripotent+stem+cell-derived+retinal+pigment+epithelium+cell+sheets+aiming+for+clinical+application.">Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application.</a> Stem Cell Reports. 2 (2), 205-218 (2014).</li><li>Idelson, 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=Directed+differentiation+of+human+embryonic+stem+cells+into+functional+retinal+pigment+epithelium+cells.">Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.</a> Cell Stem Cell. 5 (4), 396-408 (2009).</li><li>Mandai, 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=Autologous+Induced+Stem-Cell-Derived+Retinal+Cells+for+Macular+Degeneration.">Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration.</a> N Engl J Med. 376 (11), 1038-1046 (2017).</li><li>Maminishkis, 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=Confluent+monolayers+of+cultured+human+fetal+retinal+pigment+epithelium+exhibit+morphology+and+physiology+of+native+tissue.">Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.</a> Invest Ophthalmol Vis Sci. 47 (8), 3612-3624 (2006).</li><li>Maruotti, 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=A+simple+and+scalable+process+for+the+differentiation+of+retinal+pigment+epithelium+from+human+pluripotent+stem+cells.">A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells.</a> Stem Cells Transl Med. 2 (5), 341-354 (2013).</li><li>Yang, T., He, L. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preassociated+apocalmodulin+mediates+Ca2+-dependent+sensitization+of+activation+and+inactivation+of+TMEM16A/16B+Ca2+-gated+Cl-+channels.">Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl- channels.</a> Proc Natl Acad Sci U S A. 111 (51), 18213-18218 (2014).</li><li>Yang, T., 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=Structure+and+selectivity+in+bestrophin+ion+channels.">Structure and selectivity in bestrophin ion channels.</a> Science. 346 (6207), 355-359 (2014).</li><li>Yang, T., Puckerin, A., 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=Distinct+RGK+GTPases+differentially+use+alpha1-+and+auxiliary+beta-binding-dependent+mechanisms+to+inhibit+CaV1.2/CaV2.2+channels.">Distinct RGK GTPases differentially use alpha1- and auxiliary beta-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels.</a> PLoS One. 7 (5), e37079 (2012).</li><li>Yang, T., Suhail, Y., Dalton, S., Kernan, T., 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=Genetically+encoded+molecules+for+inducibly+inactivating+CaV+channels.">Genetically encoded molecules for inducibly inactivating CaV channels.</a> Nat Chem Biol. 3 (12), 795-804 (2007).</li><li>Yang, T., Xu, X., Kernan, T., Wu, V., 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=Rem,+a+member+of+the+RGK+GTPases,+inhibits+recombinant+CaV1.2+channels+using+multiple+mechanisms+that+require+distinct+conformations+of+the+GTPase.">Rem, a member of the RGK GTPases, inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase.</a> J Physiol. 588 (Pt 10), 1665-1681 (2010).</li><li>Gong, 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=Differentiation+of+Human+Protein-Induced+Pluripotent+Stem+Cells+toward+a+Retinal+Pigment+Epithelial+Cell+Fate.">Differentiation of Human Protein-Induced Pluripotent Stem Cells toward a Retinal Pigment Epithelial Cell Fate.</a> PLoS One. 10 (11), e0143272 (2015).</li><li>Zhang, 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=ATP+activates+bestrophin+ion+channels+through+direct+interaction.">ATP activates bestrophin ion channels through direct interaction.</a> Nat Commun. 9 (1), 3126 (2018).</li></ol>

作者没有什么可透露的。

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 ...

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

6.7K 次观看.  University of Rochester, School of Medicine and Dentistry. 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。

视频: 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

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

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

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

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

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

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

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

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

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

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

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

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

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

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

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

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 ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

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

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.

In this article, we present the protocol that is described as high-resolution melting analysis (HRM)-based Target Induced Local Lesions In Genomes (TILLING). This method utilizes fluorescence changes during the melting of the DNA duplex and is suitable for high-throughput screening of both insertion/deletion (Indel) and single base substition (SBS).

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 ...