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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.

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Differentiation of Human Embryonic Stem Cells into Cone Photoreceptors
人胚胎干细胞向视锥细胞的分化

发育生物学 > 细胞生长和命运决定 > 神经元
作者: Anthony Flamier
Anthony FlamierAffiliation 1: Stem Cell and Developmental Biology Laboratory, Montreal, Canada
Affiliation 2: Department of Neurosciences, Montreal, Canada
Affiliation 3: Department of Ophthalmology, University of Montreal, Montreal, Canada
Bio-protocol author page: a3320
Andrea Barabino
Andrea BarabinoAffiliation 1: Stem Cell and Developmental Biology Laboratory, Montreal, Canada
Affiliation 2: Department of Neurosciences, Montreal, Canada
Affiliation 3: Department of Ophthalmology, University of Montreal, Montreal, Canada
Bio-protocol author page: a3321
 and Gilbert Bernier
Gilbert BernierAffiliation 1: Stem Cell and Developmental Biology Laboratory, Montreal, Canada
Affiliation 2: Department of Neurosciences, Montreal, Canada
Affiliation 3: Department of Ophthalmology, University of Montreal, Montreal, Canada
For correspondence: gbernier.hmr@ssss.gouv.qc.ca
Bio-protocol author page: a3322
Vol 6, Iss 14, 7/20/2016, 1357 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1870

[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)

[Abstract]

Materials and Reagents

  1. Tissue culture 6-well plates (Thermo Fischer Scientific, catalog number: 130184 )
  2. Pipets (5 ml, 10 ml)
  3. 50 ml conical tubes (Thermo Fischer Scientific, catalog number: 339653CS )
  4. Micro cover glass (VWR International, catalog number: 89015-724 )
  5. Cell scraper (Thermo Fischer Scientific, catalog number: 179693 )
  6. 0.22 μm sterilize filter Filtropur (Sarstedt, catalog number: 83.1826.001 )
  7. Human embryonic stem (hES) cell line HUES9 (Harvard stem cell institute) or H9 cells (WiCell, catalog number: WA09 )
  8. hES cell media (Peprotech, catalog number: BM-HESC-500 )
  9. N2 supplement (Gibco, catalog number: 17502-048 )
  10. B27 supplement (Gibco, catalog number: 17504-044 )
  11. DMEM/F12 (Gibco, catalog number: 11320-033 )
  12. NEAA (Gibco, catalog number: 11140-050 )
  13. Human FGF-basic (Peprotech, catalog number: 100-18B-50UG )
  14. Heparin (Sigma-Aldrich, catalog number: H3149-10KU )
  15. Human IGF-I (Peprotech, catalog number: 100-11 )
  16. Human recombinant COCO (R&D Systems, catalog number: 3047-CC )
  17. Accutase solution (100 ml) (Sigma-Aldrich, catalog number: A6964 )
  18. Rock inhibitor Y-27632 (hydrochloride) (Cayman chemical, catalog number: 10005583 )
  19. Reduced growth factor matrigel (Corning, catalog number: 354230 )
  20. hES cell qualified matrigel (Corning, catalog number: 354277 )
  21. Trizol reagent (Thermo Fisher Scientifc, AmbionTM, catalog number: 15596026 )
  22. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: 0 4693116001 )
  23. Na2HPO4·7H2O (FisherBiotech, catalog number: BP332-500 )
  24. NaCl (Amresco, catalog number: 0 241 )
  25. KCl (EMD Millipore, catalog number: PX1405-1 )
  26. KH2PO4 (FisherBiotech, catalog number: BP362-1 )
  27. Crystalline PFA (Sigma-Aldrich, catalog number: P6148 )
  28. 1x PBS
  29. Antibodies against CRX, S-opsin, ABCA4 and cone arrestin
  30. CI media (see Recipes)
  31. 10x phosphate-buffer saline (PBS) (see Recipes)
  32. 4% paraformaldehyde (PFA) (see Recipes)
  33. hES-qualified matrigel plate coating (see Recipes)

Equipment

  1. 37 °C, 5% CO2 water jacketed incubator (Thermo Fisher Scientific, catalog number: 3110 )
  2. P1000, P200 pipets
  3. Microscope (Leica, model: DM IL )
  4. Refrigerated Tabletop Centrifuge for 50 ml conical tubes (Thermo Fisher Scientific, Centra GP8R, 031220F )

Procedure

  1. Preparation and plating of human Embryonic Stem (hES) cells-Time: day -6 to day 0
    1. Culture hES cell line HUES9 on matrigel-coated plates (see hES qualified Matrigel in Recipes) until they form large colonies (~1 x 106 cells) using hES cell media (see Materials and Reagents) (Figure 1A).
    2. Dilute reduced growth factor matrigel in ice cold DMEM/F12 using pre-chilled 5 ml pipets and 200 μl pipet tips: mix 100 μl of matrigel on ice and 6 ml of ice cold DMEM/F12.
    3. Add 1 ml of freshly prepared matrigel per well of a 6-well plate.
      Note: Place a sterile coverslip before adding the matrigel for immunofluorescence analysis.
    4. Evenly distribute the matrigel and incubate for 30 min at 37 °C into a water jacketed incubator.
    5. During matrigel incubation, discard hES cell media and wash with 1 ml of 1x PBS pre-warmed at 37 °C.
      Note: This protocol is adapted to the use of 3 well of a 6-well plate with large hES cell colonies as starting material.
    6. Discard PBS and add 1 ml of Accutase pre-warmed at room temperature.
    7. Incubate the hES cell plate until the cells start to detach (round shaped cells) at 37 °C (1-2 min).
    8. Stop the reaction with 1 ml of hES cell media.
    9. Collect the cells into a 50 ml conical tube with a P1000 pipet tip by pipetting up and down.
    10. Add 6 ml of warmed hES cell media.
    11. Add ROCK inhibitor Y27632 to reach the final concentration of 10 μM.
    12. Centrifuge at 1,300 rpm (400 x g) for 5 min at room temperature.
    13. Discard the supernatant and resuspend the cells into 12 ml of warm hES cell media.
    14. Add ROCK inhibitor Y27632 to reach the final concentration of 10 μM.
    15. Take the matrigel-coated plate from the incubator and discard the DMEM/F12 remaining on each well.
    16. Distribute 2 ml of hES cells suspension in each well of the matrigel-coated plate.
    17. Swirl the plate and incubate into an incubator at 37 °C.
    18. Change culture media every day until the cells reach full confluency (100%).

  2. Differentiation-Time: day 0 to day 21
    1. Prepare and sterile filter CI media needed for two weeks.
    2. Upon full confluency, remove hES cell media from the plate and add 2 ml of freshly prepared CI media (Figure 1B).
    3. Change media every day by slowly dispensing warmed CI media on the side of the well for at least 21 days (Figure 1C).

  3. Cell collection and analysis
    1. For gene expression analysis
      1. Discard the media, wash with 1 ml of 1x PBS and add 1 ml of Trizol reagent.
      2. Pipet up and down with a P1000 pipet tip and collect all the cells.
      3. Store at -80 °C or proceed to RNA extraction.
      4. Differentiation efficiency was confirmed by real-time PCR using primers against SIX6, CRX, S-opsin (OPN1SW), cone arrestin [also known as arrestin 3 (ARR3)], cone transducing (GNAT2), phosphodiesterase 6H (PDE6H) and phosphodiesterase 6C (PDE6C). Primers sequences are: SIX6 (F; CCTGCAGGATCCATACCCTA and R; ACCTGCTGCTGGAGTCTGTT), CRX (F; CCTTCTGACAGCTCGGTGTT and R; CCACTTTCTGAAGCCTGGAG), S-opsin (F; TGTGCCTCTCTCCCTCATCT and R; GGCACGTAGCAGACACAGAA), Cone Arrestin (F; CCCAGAGCTTTGCAGTAACC and R; CACAGGACACCATCAGGTTG), PdE6h (F; TACTCTGCCTGCTCCAGCTT and R; GAGAGTGGCAGAACCTCTGG), PdE6c (F; TTGGGAACAAGGAGATCTGG and R; GGCTCCTCCTTCTTGCTTTT) (Zhou et al., 2015).
    2. For protein analysis
      Detach the cells in the culture media using a cell scraper. Centrifuge at 1,300 rpm for 5 min and discard the supernatant. Resuspend the pellet in 200 μl of protease inhibitor and store at -20 °C or proceed to protein extraction. Protein expression can be analyzed by western blotting using antibodies against CRX, S-opsin, cone arrestin and cone transducing (Zhou et al., 2015).
    3. For immunofluorescence analysis
      Discard the media, wash with 1 ml of 1x PBS and add 1 ml of fresh PFA at 4%. Incubate 10 min at room temperature. Discard PFA and proceed to immunofluorescence using antibodies against CRX, S-opsin, ABCA4 and cone arrestin. Coverslips can be mounted on slides after secondary antibody incubation (Figure 1D) (Zhou et al., 2015).

Representative data



Figure 1: Representative images of hES cells at different time points of the protocol. A. Large hES cell colony ready for passaging. Scale bar: 500 μm B. Confluent layer of hES cells ready to start the differentiation (100% confluency). Scale bar: 100 μm C. Cells obtained after 21 days of differentiation. Scale bar: 100 μm D. Differentiated cells after 21 days of differentiation labelled with the photoreceptor markers CRX and S-Opsin. Scale bar: 100 μm.

Notes

The quality of the starting hES cells is essential to obtain a good efficiency of differentiation. Therefore, it is recommended to assess pluripotency of the cells by detecting hES cell markers such as Tra1-60, Oct3/4, SSEA4 and Nanog. This protocol has been tested using either HUES9 cell line or H9 cells and differentiation efficiency was confirmed by the detection of CRX, S-Opsin, Recoverin and ABCA4.
The use of Accutase is essential to obtain a good cell viability after re-plating at the opposite of Trypsin.

Recipes

  1. 10x Phosphate-Buffer Saline (PBS)
    Mix 25.6 g Na2HPO4·7H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4
    Bring to 1 liter with H2O.
    Autoclave for 40 min at 121 °C.
  2. CI media
    Add all of these components:
    48 ml DMEM/F12
    0.5 ml N2 supplement (100x)
    1 ml B27 supplement (100x)
    0.5 ml NEAA (100x)
    12.5 μl human FGF-basic (10 ng/μl)
    5 μl Heparin (10 μg/μl)
    5 μl human IGF-I (100 ng/μl)
    7.5 μl human recombinant COCO (200 μg/ml)
    Filter sterilize (0.22 μm) and store at 4 °C.
    Use within two weeks.
  3. 4% PFA
    Weigh 16 g of crystalline PFA in a beaker and add 400 ml of 1x PBS. Cover and heat the solution until the powder is fully dissolved (50 °C). Aliquot and store at -20 °C.
  4. hES-qualified matrigel plate coating
    Dilute hES-qualified matrigel in ice cold DMEM/F12 using pre-chilled 10 ml pipets and P1000 pipet tips: mix a volume of matrigel on ice corresponding to the dilution factor (see certificate) and 25 ml of ice cold DMEM/F12. Dispend 1 ml of diluted matrigel per well of a 6-well plate and incubate for 30 min at 37 °C into a water jacketed incubator. Discard the excess of DMEM/F12 before use.

Acknowledgements

We are grateful to D. Melton and Harvard Stem Cell Institute for the HUES9 cell line. This work was supported by grants from the Foundation Fighting Blindness Canada, Turmel Family Foundation for Macular Degeneration Research, Canadian Stem Cell Network, C. Durand Foundation, the GO Foundation, and Natural Science and Engineering Research Council of Canada (Grant #250970-2012). A.F. is supported by fellowships from the Réseau Vision du Québec and from University of Montreal Molecular Biology Program. G.B is supported by the Fonds de Recherche en Santé du Québec.

References

  1. Zhou, S., Flamier, A., Abdouh, M., Tetreault, N., Barabino, A., Wadhwa, S. and Bernier, G. (2015). Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFbeta and Wnt signaling. Development 142(19): 3294-3306.

材料和试剂

  1. 组织培养6孔板(Thermo Fischer Scientific,目录号:130184)
  2. 吸移管(5ml,10ml)
  3. 50ml锥形管(Thermo Fischer Scientific,目录号:339653CS)
  4. 微覆盖玻璃(VWR International,目录号:89015-724)
  5. 细胞刮刀(Thermo Fischer Scientific,目录号:179693)
  6. 0.22μm消毒过滤器Filtropur(Sarstedt,目录号:83.1826.001)
  7. 人胚胎干(hES)细胞系HUES9(Harvard干细胞研究所)或H9细胞(WiCell,目录号:WA09)
  8. hES细胞培养基(Peprotech,目录号:BM-HESC-500)
  9. N2补充剂(Gibco,目录号:17502-048)
  10. B27补充剂(Gibco,目录号:17504-044)
  11. DMEM/F12(Gibco,目录号:11320-033)
  12. NEAA(Gibco,目录号:11140-050)
  13. 人类FGF碱性(Peprotech,目录号:100-18B-50UG)
  14. 肝素(Sigma-Aldrich,目录号:H3149-10KU)
  15. 人IGF-I(Peprotech,目录号:100-11)
  16. 人重组COCO(R& D Systems,目录号:3047-CC)
  17. Accutase溶液(100ml)(Sigma-Aldrich,目录号:A6964)
  18. 岩石抑制剂Y-27632(盐酸盐)(Cayman chemical,目录号:10005583)
  19. 减少生长因子基质胶(Corning,目录号:354230)
  20. hES细胞基质胶(Corning,目录号:354277)
  21. Trizol试剂(Thermo Fisher Scientifc,Ambion TM,目录号:15596026)
  22. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:04693116001)
  23. (FisherBiotech,目录号:BP332-500)。
  24. NaCl(Amresco,目录号:0241)
  25. KCl(EMD Millipore,目录号:PX1405-1)

  26. (FisherBiotech,目录号:BP362-1)
  27. 结晶PFA(Sigma-Aldrich,目录号:P6148)
  28. 1x PBS
  29. 针对CRX,S-视蛋白,ABCA4和锥细胞抑制素的抗体
  30. CI介质(参见配方)
  31. 10x磷酸盐缓冲盐水(PBS)(参见配方)
  32. 4%多聚甲醛(PFA)(参见配方)
  33. hES合格的基质胶板涂层(见配方)

设备

  1. 37℃,5%CO 2水夹套培养箱(Thermo Fisher Scientific,目录号:3110)
  2. P1000,P200移液器
  3. 显微镜(Leica,型号:DM IL)
  4. 用于50ml锥形管(Thermo Fisher Scientific,Centra GP8R,031220F)的冷冻台式离心机

程序

  1. 人胚胎干细胞(hES)细胞的制备和电镀 - 时间:第-6天至第0天
    1. 使用hES细胞培养基(参见材料和试剂)(图1),在基质胶包被的平板上培养hES细胞系HUES9(参见hES限定的Matrigel in Recipes)直到它们形成大的集落(?1×10 6个细胞) 1A)。
    2. 使用预冷的5ml移液管和200μl移液管吸头在冰冷的DMEM/F12中稀释还原的生长因子基质胶:将100μl基质胶在冰上和6ml冰冷的DMEM/F12混合。
    3. 向6孔板的每孔中加入1ml新鲜制备的基质胶。
      注意:在添加基质胶进行免疫荧光分析之前,请放置无菌盖玻片。
    4. 均匀地分布基质胶并在37℃下孵育30分钟到水夹套培养箱中
    5. 在基质胶孵育期间,丢弃hES细胞培养基并用1ml在37℃预热的1×PBS洗涤。
      注意:该方案适用于使用具有大hES细胞集落的6孔板的3孔作为起始材料。
    6. 弃去PBS,加入1ml在室温下预热的Accutase
    7. 孵育hES细胞平板,直到细胞在37℃(1-2分钟)开始分离(圆形细胞)。
    8. 停止与1毫升hES细胞培养基的反应。
    9. 通过上下吹吸,用P1000移液管吸头将细胞收集到50 ml锥形管中
    10. 加入6ml温热的hES细胞培养基
    11. 加入ROCK抑制剂Y27632以达到10μM的终浓度
    12. 在室温下以1,300rpm(400xg)离心5分钟
    13. 弃去上清液并将细胞重悬于12ml温热的hES细胞培养基中
    14. 加入ROCK抑制剂Y27632至终浓度为10μM
    15. 从培养箱中取出基质胶包被的板,弃去每个孔上剩余的DMEM/F12。
    16. 在基质胶包被的板的每个孔中分配2ml hES细胞悬浮液
    17. 旋转板并孵育在37℃的培养箱中
    18. 每天更换培养基,直到细胞达到完全融合(100%)
  2. 区分时间:第0天到第21天
    1. 准备和无菌过滤器CI介质需要两周。
    2. 完全融合后,从板中取出hES细胞培养基,加入2ml新鲜制备的CI培养基(图1B)。
    3. 通过在孔侧面缓慢分配温暖的CI培养基至少21天来更换培养基(图1C)。

  3. 细胞收集和分析
    1. 用于基因表达分析
      1. 弃去培养基,用1ml 1×PBS洗涤并加入1ml Trizol试剂。
      2. 用P1000移液管吸头上下移动并收集所有细胞。
      3. 储存于-80℃或进行RNA提取。
      4. 通过实时PCR,使用针对SIX6 ,CRX ,S-视蛋白( OPN1SW ),视锥抑制素[也称为抑制蛋白3( ARR3 )],锥体转导()。引物序列是:SIX6(F; CCTGCAGGATCCATACCCTA和R; ACCTGCTGCTGGAGTCTGTT),CRX(F; CCTTCTGACAGCTCGGTGTT和R; CCACTTTCTGAAGCCTGGAG),S-视蛋白(F; TGTGCCTCTCTCCCTCATCT和R; GGCACGTAGCAGACACAGAA),圆锥抑制蛋白(F; CCCAGAGCTTTGCAGTAACC和R; CACAGGACACCATCAGGTTG) ,PdE6h(F; TACTCTGCCTGCTCCAGCTT和R; GAGAGTGGCAGAACCTCTGG),PdE6c(F; TTGGGAACAAGGAGATCTGG和R; GGCTCCTCCTTCTTGCTTTT)(Zhou等人,2015)。
    2. 用于蛋白质分析
      使用细胞刮刀分离培养基中的细胞。以1,300rpm离心5分钟,弃去上清液。将沉淀重悬于200μl蛋白酶抑制剂中,-20℃保存或进行蛋白提取。可以通过使用针对CRX,S-视蛋白,锥细胞抑制蛋白和锥体转导的抗体的蛋白质印迹分析蛋白质表达(Zhou等人,2015)。
    3. 用于免疫荧光分析 弃去培养基,用1ml 1×PBS洗涤并加入1ml 4%的新鲜PFA。在室温下孵育10分钟。丢弃PFA,并使用针对CRX,S-视蛋白,ABCA4和锥细胞抑制蛋白的抗体进行免疫荧光。盖玻片可以在二次抗体孵育后安装在载玻片上(图1D)(Zhou等人,2015)。

代表数据



图1:在方案的不同时间点的hES细胞的代表性图像。 A.大的hES细胞集落准备用于传代。比例尺:500μmB.准备开始分化的hES细胞的铺满层(100%融合)。比例尺:100μm。C.分化21天后获得的细胞。比例尺:100μm。分化21天后的分化细胞用光感受器标记物CRX和S-Opsin标记。比例尺:100μm。

笔记

起始hES细胞的质量对于获得良好的分化效率是必需的。因此,建议通过检测hES细胞标志物如Tra1-60,Oct3/4,SSEA4和Nanog来评估细胞的多能性。该协议已使用HUES9细胞系或H9细胞进行测试,并通过检测CRX,S-Opsin,Recoverin和ABCA4来确认分化效率。
使用Accutase对于在与胰蛋白酶相反的再接种后获得良好的细胞活力是必需的。

食谱

  1. 10x磷酸盐缓冲液(PBS)
    将25.6g Na 2 HPO 4 7H 2 O 2,80g NaCl,2g KCl,2g KH 2 PO 4, sub> PO 4
    用H 2 O调至1升。
    在121°C高压灭菌40分钟。
  2. CI媒体
    添加所有这些组件:
    48 ml DMEM/F12
    0.5ml N2补充液(100x)
    1 ml B27补充剂(100x)
    0.5ml NEAA(100x)
    12.5μl人FGF碱性(10ng /μl)
    5微升肝素(10微克/微升)
    5μl人IGF-I(100ng /μl)
    7.5μl人重组COCO(200μg/ml)
    过滤灭菌(0.22μm)并储存在4℃。
    在两周内使用。
  3. 4%PFA
    在烧杯中称取16g结晶PFA,加入400ml 1×PBS。盖上并加热溶液直到粉末完全溶解(50℃)。等分并储存在-20°C
  4. hES合格的基质胶板涂层
    使用预冷的10ml移液管和P1000移液管吸头在冰冷的DMEM/F12中稀释hES-限定的基质胶:将对应于稀释因子(见证书)的一定体积的基质胶在冰上混合,并与25ml冰冷的DMEM/F12混合。分配1毫升稀释基质胶每孔的6孔板和孵育30分钟,在37℃下,在水夹套孵化器。在使用前丢弃多余的DMEM/F12

致谢

我们感谢D.梅尔顿和哈佛大学干细胞研究所为HUES9细胞系。这项工作得到了基金会战斗盲人加拿大,图尔特家庭基金会黄斑变性研究,加拿大干细胞网络,C.杜兰德基金会,GO基金会和加拿大自然科学和工程研究理事会(批准#250970-2012 )。 A.F.得到魁北克地区RéseauVision大学和蒙特利尔大学分子生物学计划的奖学金支持。 G.B得到魁北克省魁北克省政府的支持。

参考文献

  1. Zhou,S.,Flamier,A.,Abdouh,M.,Tetreault,N.,Barabino,A.,Wadhwa,S. and Bernier,G.(2015)。  通过同时抑制BMP,TGFbeta和Wnt信号分化将人胚胎干细胞分化成锥形感受器。 142(19):3294-3306。
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How to cite this protocol: 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; Full Text



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