Differentiation of Human Embryonic Stem Cells into Cone Photoreceptors
人胚胎干细胞向视锥细胞的分化
发布: 2016年07月20日第6卷第14期 DOI: 10.21769/BioProtoc.1870 浏览次数: 10122
评审: Agnieszka PastulaNicoletta CordaniAnonymous reviewer(s)
下载 PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Oct 2015
Abstract
Photoreceptors are specialized retinal neurons able to respond to light in order to generate visual information. Among photoreceptors, cones are involved in colors discrimination and high-resolution central vision and are selectively depleted in macular degenerations and cone dystrophies. A possible therapeutic solution for these disorders is to replace degenerating cells with functional cones. Here, we describe a simple protocol for the rapid production of large amount of cone photoreceptors from human pluripotent stem cells. The differentiation protocol is based on the “default pathway” of neural induction using the BMP, TGFβ and WNT antagonist COCO.
Keywords: Cone photoreceptor (视锥细胞)
Human (人类)
Embryonic stem cell (胚胎干细胞)
Default pathway (默认路径)
Coco (可可)
Materials and Reagents
- Tissue culture 6-well plates (Thermo Fischer Scientific, catalog number: 130184 )
- Pipets (5 ml, 10 ml)
- 50 ml conical tubes (Thermo Fischer Scientific, catalog number: 339653CS )
- Micro cover glass (VWR International, catalog number: 89015-724 )
- Cell scraper (Thermo Fischer Scientific, catalog number: 179693 )
- 0.22 μm sterilize filter Filtropur (Sarstedt, catalog number: 83.1826.001 )
- Human embryonic stem (hES) cell line HUES9 (Harvard stem cell institute) or H9 cells (WiCell, catalog number: WA09 )
- hES cell media (Peprotech, catalog number: BM-HESC-500 )
- N2 supplement (Gibco, catalog number: 17502-048 )
- B27 supplement (Gibco, catalog number: 17504-044 )
- DMEM/F12 (Gibco, catalog number: 11320-033 )
- NEAA (Gibco, catalog number: 11140-050 )
- Human FGF-basic (Peprotech, catalog number: 100-18B-50UG )
- Heparin (Sigma-Aldrich, catalog number: H3149-10KU )
- Human IGF-I (Peprotech, catalog number: 100-11 )
- Human recombinant COCO (R&D Systems, catalog number: 3047-CC )
- Accutase solution (100 ml) (Sigma-Aldrich, catalog number: A6964 )
- Rock inhibitor Y-27632 (hydrochloride) (Cayman chemical, catalog number: 10005583 )
- Reduced growth factor matrigel (Corning, catalog number: 354230 )
- hES cell qualified matrigel (Corning, catalog number: 354277 )
- Trizol reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596026 )
- Protease inhibitor cocktail (Sigma-Aldrich, catalog number: 04693116001 )
- Na2HPO4·7H2O (FisherBiotech, catalog number: BP332-500 )
- NaCl (Amresco, catalog number: 0241 )
- KCl (EMD Millipore, catalog number: PX1405-1 )
- KH2PO4 (FisherBiotech, catalog number: BP362-1 )
- Crystalline PFA (Sigma-Aldrich, catalog number: P6148 )
- 1x PBS
- Antibodies against CRX, S-opsin, ABCA4 and cone arrestin
- CI media (see Recipes)
- 10x phosphate-buffer saline (PBS) (see Recipes)
- 4% paraformaldehyde (PFA) (see Recipes)
- hES-qualified matrigel plate coating (see Recipes)
Equipment
- 37 °C, 5% CO2 water jacketed incubator (Thermo Fisher Scientific, catalog number: 3110 )
- P1000, P200 pipets
- Microscope (Leica, model: DM IL )
- Refrigerated Tabletop Centrifuge for 50 ml conical tubes (Thermo Fisher Scientific, Centra GP8R, 031220F )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Flamier, A., Barabino, A. and Bernier, G. (2016). Differentiation of Human Embryonic Stem Cells into Cone Photoreceptors. Bio-protocol 6(14): e1870. DOI: 10.21769/BioProtoc.1870.
Download Citation in RIS Format
分类
发育生物学 > 细胞生长和命运决定 > 神经元
干细胞 > 胚胎干细胞 > 维持和分化
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
