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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
该项目由 NIH 赠款 EY025290、GM127652 和罗切斯特大学开办资金资助。
Materials
| Knock-Out (KO) DMEM | ThermoFisher | 10829018 | |
| KO serum replacement | ThermoFisher | 10829028 | |
| Nonessential amino acids | ThermoFisher | 11140050 | |
| Glutamine | ThermoFisher | 35050061 | |
| Penicillin-streptomycin | ThermoFisher | 10378016 | |
| Nicotinamide | Sigma-Aldrich | N0636 | |
| Human activin-A | PeproTech | 120-14 | |
| MEM (a modification) | Sigma-Aldrich | M4526 | |
| Fetal Bovine Serum | VWR | 97068-085 | |
| N1 supplement | Sigma-Aldrich | N6530 | |
| Glutamine-penicillin-streptomycin | Sigma-Aldrich | G1146 | |
| Nonessential amino acids | Sigma-Aldrich | M7156 | |
| Taurine | Sigma-Aldrich | T0625 | |
| Hydrocortisone | Sigma-Aldrich | H0386 | |
| Triiodo-thyronin | Sigma-Aldrich | T5516 | |
| mTeSR-1 medium | Stemcell Technologies | 5850 | |
| Matrigel | Corning | 356230 | |
| Collagenase | Gibco | 17104019 | |
| Trypsin | VWR | 45000-664 | |
| M-PER mammalian protein extraction reagent | Pierce | 78501 | |
| proteinase inhibitor cocktail | Sigma-Aldrich | 4693159001 | |
| RPE65 antibody | Novus Biologicals | NB100-355 | |
| CRALBP antibody | Abcam | ab15051 | |
| BEST1 antibody | Novus Biologicals | NB300-164 | |
| Beta Actin antibody | ThermoFisher | MA5-15739 | |
| Alexa Fluor 488-conjugated donkey anti-mouse IgG | ThermoFisher | A-21202 | |
| Goat anti-mouse IgG | ThermoFisher | SA5-35521 | |
| Goat anti-Rabbit IgG | LI-COR Biosciences | 926-68071 | |
| Hoechst 33342 | ThermoFisher | 62249 | |
| HEKA EPC10 patch clamp amplifier | Warner Instruments | 895000 | |
| Patchmaster | Warner Instruments | 895040 |
Riferimenti
- Allikmets, R., et al. Evaluation of the Best disease gene in patients with age-related macular degeneration and other maculopathies. Hum Genet. 104 (6), 449-453 (1999).
- Burgess, R., et al. Biallelic mutation of BEST1 causes a distinct retinopathy in humans. Am J Hum Genet. 82 (1), 19-31 (2008).
- Davidson, A. E., et al. Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa. Am J Hum Genet. 85 (5), 581-592 (2009).
- Kramer, F., et al. 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. Eur J Hum Genet. 8 (4), 286-292 (2000).
- Marquardt, A., et al. Mutations in a novel gene, VMD2, encoding a protein of unknown properties cause juvenile-onset vitelliform macular dystrophy (Best’s disease). Hum Mol Genet. 7 (9), 1517-1525 (1998).
- Petrukhin, K., et al. Identification of the gene responsible for Best macular dystrophy. Nat Genet. 19 (3), 241-247 (1998).
- Yardley, J., et al. Mutations of VMD2 splicing regulators cause nanophthalmos and autosomal dominant vitreoretinochoroidopathy (ADVIRC). Invest Ophthalmol Vis Sci. 45 (10), 3683-3689 (2004).
- Yang, T., Justus, S., Li, Y., Tsang, S. H. BEST1: the Best Target for Gene and Cell Therapies. Mol Ther. 23 (12), 1805-1809 (2015).
- Marmorstein, A. D., et al. Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium. Proc Natl Acad Sci U S A. 97 (23), 12758-12763 (2000).
- Boon, C. J., et al. The spectrum of ocular phenotypes caused by mutations in the BEST1 gene. Prog Retin Eye Res. 28 (3), 187-205 (2009).
- Marmorstein, A. D., Cross, H. E., Peachey, N. S. Functional roles of bestrophins in ocular epithelia. Prog Retin Eye Res. 28 (3), 206-226 (2009).
- Fujii, S., Gallemore, R. P., Hughes, B. A., Steinberg, R. H. Direct evidence for a basolateral membrane Cl- conductance in toad retinal pigment epithelium. Am J Physiol. 262, C374-C383 (1992).
- Gallemore, R. P., Steinberg, R. H. Effects of DIDS on the chick retinal pigment epithelium. II. Mechanism of the light peak and other responses originating at the basal membrane. J Neurosci. 9 (6), 1977-1984 (1989).
- Gallemore, R. P., Steinberg, R. H. Light-evoked modulation of basolateral membrane Cl- conductance in chick retinal pigment epithelium: the light peak and fast oscillation. J Neurophysiol. 70 (4), 1669-1680 (1993).
- Li, Y., Nguyen, H. V., Tsang, S. H. Skin Biopsy and Patient-Specific Stem Cell Lines. Methods Mol Biol. 1353, 77-88 (2016).
- Milenkovic, A., et al. Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells. Proc Natl Acad Sci U S A. 112 (20), E2630-E2639 (2015).
- Moshfegh, Y., et al. BESTROPHIN1 mutations cause defective chloride conductance in patient stem cell-derived RPE. Hum Mol Genet. 25 (13), 2672-2680 (2016).
- Li, Y., et al. Patient-specific mutations impair BESTROPHIN1’s essential role in mediating Ca2+-dependent Cl- currents in human RPE. Elife. 6, (2017).
- Marmorstein, L. Y., et al. The light peak of the electroretinogram is dependent on voltage-gated calcium channels and antagonized by bestrophin (best-1). J Gen Physiol. 127 (5), 577-589 (2006).
- Volland, S., Esteve-Rudd, J., Hoo, J., Yee, C., Williams, D. S. A comparison of some organizational characteristics of the mouse central retina and the human macula. PLoS One. 10 (4), e0125631 (2015).
- Kamao, H., et al. Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application. Stem Cell Reports. 2 (2), 205-218 (2014).
- Idelson, M., et al. Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell. 5 (4), 396-408 (2009).
- Mandai, M., et al. Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration. N Engl J Med. 376 (11), 1038-1046 (2017).
- Maminishkis, A., et al. Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest Ophthalmol Vis Sci. 47 (8), 3612-3624 (2006).
- Maruotti, J., et al. A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells. Stem Cells Transl Med. 2 (5), 341-354 (2013).
- Yang, T., He, L. L., Chen, M., Fang, K., Colecraft, H. M. Bio-inspired voltage-dependent calcium channel blockers. Nat Commun. 4, 2540 (2013).
- Yang, T., Hendrickson, W. A., Colecraft, H. M. Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl- channels. Proc Natl Acad Sci U S A. 111 (51), 18213-18218 (2014).
- Yang, T., et al. Structure and selectivity in bestrophin ion channels. Science. 346 (6207), 355-359 (2014).
- Yang, T., Puckerin, A., Colecraft, H. M. Distinct RGK GTPases differentially use alpha1- and auxiliary beta-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels. PLoS One. 7 (5), e37079 (2012).
- Yang, T., Suhail, Y., Dalton, S., Kernan, T., Colecraft, H. M. Genetically encoded molecules for inducibly inactivating CaV channels. Nat Chem Biol. 3 (12), 795-804 (2007).
- Yang, T., Xu, X., Kernan, T., Wu, V., Colecraft, H. M. Rem, a member of the RGK GTPases, inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase. J Physiol. 588 (Pt 10), 1665-1681 (2010).
- Gong, J., et al. Differentiation of Human Protein-Induced Pluripotent Stem Cells toward a Retinal Pigment Epithelial Cell Fate. PLoS One. 10 (11), e0143272 (2015).
- Zhang, Y., et al. ATP activates bestrophin ion channels through direct interaction. Nat Commun. 9 (1), 3126 (2018).
