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Retinal Differentiation of Mouse Embryonic Stem Cells
小鼠胚胎干细胞的视网膜分化   

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Abstract

Groundbreaking studies from Dr. Yoshiki Sasai’s laboratory have recently introduced novel methods to differentiate mouse and human Embryonic Stem Cells (mESCs and hESCs) into organ-like 3D structures aimed to recapitulate developmental organogenesis programs (Eiraku et al., 2011; Eiraku and Sasai, 2012; Nakano et al., 2012; Kamiya et al., 2011). We took advantage of this method to optimize a 3D protocol to efficiently generate retinal progenitor cells and subsequently retinal neurons in vitro. This culture system provides an invaluable platform both to study early developmental processes and to obtain retinal neurons for transplantation approaches. The protocol described here has been successfully applied to several mouse ESC (including the R1, WD44 and G4 cell lines) and mouse induced-Pluripotent Stem Cell (iPSCs) lines.

Keywords: Retina(视网膜), Mouse Embryonic Stem Cells(小鼠胚胎干细胞), Embryoid Bodies(胚状体), Developmental Biology(发育生物学), Organogenesis(器官发生)

Materials and Reagents

  1. 96-well ultra low-cell-adhesion plate, Lipidure-Coat (Amsbio LLC, catalog number: AMS.51011610 ) or PrimeSurface 96U plate (Sumitomo Bakelite, catalog number: MS-9096U )
  2. 6-well ultra low-cell-adhesion plate (Thermo Fisher Scientific, CorningTM, catalog number: 07-200-601 )
  3. 100 mm cell culture dish (Corning, BD Falcon®, catalog number: 353003 )
  4. Glass Pasteur pipette
  5. Mouse Embryonic Stem Cells (mESCs) or mouse Induced-Pluripotent Stem Cells (miPSCs)
  6. Phosphate Buffer Saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
  7. Water, cell culture grade (Thermo Fisher Scientific, catalog number: 15230-162 )
  8. Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 10564-011 )
  9. Glasgow’s MEM (GMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11710-035 )
  10. Dulbecco’s modified Eagle’s medium/nutrient F-12 Ham (DMEM/F12 Ham) (Thermo Fisher Scientific, GibcoTM, catalog number: 10565-018 )
  11. Fetal Bovine Serum (FBS), ESC Qualified (Thermo Fisher Scientific, catalog number: 10439-024 )
  12. Knock-Out Serum Replacement (KSR) (Thermo Fisher Scientific, GibcoTM, catalog number: 10828-028 )
  13. Mixture of penicillin and streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  14. Leukemia Inhibitory Factor (LIF) (Merck Millipore Corporation, catalog number: ESG1107 )
  15. GSK3b inhibitor (Stemolecule CHIR99021) (Stemgent, catalog number: 04-0004-02 )
  16. MAPK/ERK inhibitor (Stemolecule PD0325901) (Stemgent, catalog number: 04-0006-02 )
  17. TrypLE Trypsin Replacement (Thermo Fisher Scientific, catalog number: 12605-028 )
  18. 10 mM non-essential amino acids (NEAA) solution (Thermo Fisher Scientific, GibcoTM, catalog number: 11140-050 )
  19. Sodium pyruvate solution (Thermo Fisher Scientific, GibcoTM, catalog number: 11360-070 )
  20. 2-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M7522 )
  21. Growth factor reduced Matrigel® (GFR Matrigel) (Corining®, catalog number: 356230 )
  22. B27 supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  23. N2 supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17502-048 )
  24. All-trans retinoic acid (Sigma-Aldrich, catalog number: R2625-50MG )
  25. Taurine (Sigma-Aldrich, catalog number: T-8691 )
  26. MEF medium (see Recipes)
  27. LIF + 2i medium (see Recipes)
  28. Retinal Differentiation medium (RD medium) (see Recipes)
  29. Tom’s medium (see Recipes)

Equipment

  1. Incubator (37 °C and 5% CO2) (Thermo Scientific, model: HERACell 150i )
  2. Centrifuge (Eppendorf, model: Conical centrifuge 5702 )
  3. Tissue culture hood, Type B2 Biological Safety Cabinet (Thermo Scientific, model: 1300 Series Class II )
  4. Hemocytometer (Neubauer chamber) or equivalent method
  5. Inverted microscope (Leica, model: DMi8 )

Procedure

  1. Coating plates
    1. Thaw an aliquot of Growth Factor Reduced-Matrigel by submerging the tube in ice for 1 h.
    2. Dilute the Matrigel 1:50 in cold MEF medium and transfer the media and the Matrigel to a TC dish. For 6-well plates, we use 1 ml/well and 7 ml for 10 cm plates.
    3. Incubate at 37 ºC for at least 1 h.
    Note: Because Matrigel solidifies at warm temperatures, it should be kept on ice at all times and we recommend using pre-cooled pipet tips.

  2. Maintenance of mESCs
    1. mESCs are plated on a 10 cm dish pre-coated with Growth Factor Reduced-Matrigel, in LIF + 2i medium.
    2. mESCs are next incubated in a 5% CO2 incubator at 37 ºC until passage or retinal differentiation.
    3. Medium is changed every other day.
      Note: Special attention should be taken to avoid overconfluency of mESCs in maintenance cultures as it might result in detrimental differentiation of the cells.

  3. Generation of Retinal Cells from undifferentiated mESCs
    1. Remove the media from a 50% confluent plate of undifferentiated mESC (at least four passages off feeder layers, Figure1) and add TrypLE Trypsin replacement to the cells. Place the cells back in the incubator for 3-5 min until the mESCs colonies start to lift. Since TrypLE Trypsin replacement is not inhibited by the serum in the media, this method does not require a pre-wash with PBS.
    2. Add 5 ml of MEF media to the plate and gently lift off the cells using a glass Pasteur pipette. Observe that the cells are removed from the plate and pipette up and down to achieve a single cell suspension being careful to avoid bubbles.


      Figure 1. Typical morphologies of undifferentiated mESCs (Day 0). Feeder-free undifferentiated R1 mESCs (originally generated by Andras Nagy) are shown. Scale bars: 500 μm (A) and 250 μm (B).

    3. mESC cells are pelleted at 190 x g for 5 min.
    4. Carefully disperse the pellet using 5 ml of Retinal Differentiation medium (RD medium).
    5. Count the cell density using a hemocytometer (Neubauer chamber) or equivalent method.
    6. Plate 3,000 cells per 100 μl in a 96-well low attachment plate (100 μl/well) in RD medium. Cell aggregates (Embryoid Bodies) should form in less than 12 h. The day on which the culture is started is designated as Day 0.
    7. 24 h later (Day 1), add Growth Factor Reduced-Matrigel to a final concentration of 2% (vol/vol). The lot-to-lot variability in commercial Matrigel products can change the efficiency of retinal induction; we preferentially use one of relatively high concentration (> 9.5 mg/ml). We recommend diluting the Matrigel 1:10 in cold RD medium and add 20 μl of the dilution/well.
    8. On Day 3 a clear layer of neuroepithelial cells is apparent (Figure 2).
      Note: Different cell lines might grow at different rates and consequently the initial number of cells per well might need to be adjusted. We found that for most of the lines tested 3,000-5,000 cells/well was the optimal cell density.


      Figure 2. Typical morphologies of embryoid bodies at the different differentiation stages. R1 mESCs were differentiated using the method described here. Around each EB (embryoid body) a clear neuroepithelial layer develops from Day 3 (D-F), and from Day 5 this layer is conspicuous (G-K), optic vesicle and optic cup-like structures are apparent from day 7-8 (L-O). Scale bars: 150 μm.
      Note: Retinal Pigmented Epithelium (RPE) cells are observed after Day 13 (red arrows in O and P).

  4. Retinal maturation 
    1. On Day 7, the embryoid bodies (EBs) are transferred to 6-well low-attachment plates in Tom’s medium supplemented with B27 and N2 to promote neuronal differentiation. From Day 7 medium should be changed every other day.
    2. From Day 10 to Day 15, in addition to B27 and N2, Tom’s medium is also supplemented with 0.5 μM retinoic acid and 1 mM taurine. RPE cells are apparent from Day 13 (Figure 2)
    3. From Day 15 onwards, EBs are maintained in Tom’s medium. Rhodopsin+ Rod photoreceptors are detected from Day 24 and after Day 35, the EBs exhibit all the layers of the mature retina.

Recipes

  1. MEF medium (100 ml)
    89 ml DMEM
    10 ml FBS
    1 ml Pen/Strep
  2. LIF + 2i medium (100 ml)
    90 ml of MEF medium
    10 ml FBS
    1 ml sodium pyruvate
    100 μl 2-ME (Stock 0.1 M)
    100 μl LIF (Stock 10 millions units/Ml)
    On the day of use, supplement with 3 µM of GSK3β inhibitor Stemolecule CHIR99021 and 0.4 µM of MEK inhibitor Stemolecule PD0325901
  3. 2-ME (monthly preparation)
    35 µl 2-ME
    5 ml of distilled water
  4. Retinal Differentiation (RD) medium (100 ml)
    96.4 ml Glasgow minimum essential medium (GMEM)
    1.5 ml KSR
    1 ml non-essential amino acids (stock 100x)
    1 ml sodium pyruvate (stock 100x)
    100 µl 2-ME (stock 0.1 M)
  5. Tom’s medium (100 ml)
    98 ml DMEM: F12
    1 ml non-essential amino acids (stock 100x)
    1 ml sodium pyruvate (stock 100x)
    - From Day 7 to Day 15:
    1 ml N2 (stock 100x)
    2 ml B27 (stock 50x)
    - From Day 10 to Day 15:
    0.5 μM retinoic acid (stock 1 mM)
    1 mM taurine (stock 1 M)

Acknowledgments

This protocol was originally published as part of: La Torre et al. (2015). The author wishes to thank all present and past members of the Reh and Bermingham-McDonogh laboratories for many helpful discussions. Special thanks to Tom Reh and Akina Hoshino for invaluable help and advice, and NIH 1 PO1 GM081619 and the imaging core of the Vision Core Grant to the University of Washington, P30EY01730 (PI: Reh).

References

  1. Eiraku, M., Takata, N., Ishibashi, H., Kawada, M., Sakakura, E., Okuda, S., Sekiguchi, K., Adachi, T. and Sasai, Y. (2011). Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472(7341): 51-56.
  2. Eiraku, M. and Sasai, Y. (2012). Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues. Nat Protoc 7(1): 69-79.
  3. Kamiya, D., Banno, S., Sasai, N., Ohgushi, M., Inomata, H., Watanabe, K., Kawada, M., Yakura, R., Kiyonari, H., Nakao, K., Jakt, L. M., Nishikawa, S. and Sasai, Y. (2011). Intrinsic transition of embryonic stem-cell differentiation into neural progenitors. Nature 470(7335): 503-509.
  4. La Torre, A., Hoshino, A., Cavanaugh, C., Ware, C. B. and Reh, T. A. (2015). The GIPC1-Akt1 pathway is required for the specification of the eye field in mouse embryonic stem cells. Stem Cells 33(9): 2674-2685.
  5. Nakano, T., Ando, S., Takata, N., Kawada, M., Muguruma, K., Sekiguchi, K., Saito, K., Yonemura, S., Eiraku, M. and Sasai, Y. (2012). Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10(6): 771-785.  

简介

来自Yoshiki Sasai博士的实验室的开创性研究最近已经引入了将小鼠和人胚胎干细胞(mESC和hESC)区分为器官样3D结构的新方法,其旨在重现发育器官发生程序(Eiraku等人, ,2011; Eiraku和Sasai,2012; Nakano等人,2012; Kamiya等人,2011)。 我们利用这种方法优化3D协议以有效地生成视网膜祖细胞和随后视网膜神经元体外。 这种文化系统提供了一个宝贵的平台,既研究早期发展过程,并获得视网膜神经元的移植方法。 这里描述的协议已成功应用于几个鼠标ESC(包括R1,WD44和G4细胞系)和小鼠诱导多能干细胞(iPSCs)线。

关键字:视网膜, 小鼠胚胎干细胞, 胚状体, 发育生物学, 器官发生

材料和试剂

  1. 96孔超低细胞粘附板,Lipidure-Coat(Amsbio LLC,目录号:AMS.51011610)或PrimeSurface 96U板(Sumitomo Bakelite,目录号:MS-9096U)
  2. 6孔超低细胞粘附板(Thermo Fisher Scientific,Corning TM ,目录号:07-200-601)。
  3. 100mm细胞培养皿(Corning,BD Falcon ,目录号:353003)
  4. 玻璃巴斯德吸液管
  5. 小鼠胚胎干细胞(mESC)或小鼠诱导多能干细胞(miPSC)
  6. 磷酸盐缓冲液(PBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10010-023)
  7. 水,细胞培养级(Thermo Fisher Scientific,目录号:15230-162)
  8. Dulbecco改良的Eagle培养基(DMEM)高葡萄糖(Thermo Fisher Scientific,Gibco TM ,目录号:10564-011)
  9. 格拉斯哥MEM(GMEM)(Thermo Fisher Scientific,Gibco TM ,目录号:11710-035)
  10. Dulbecco改良的Eagle's培养基/营养F-12 Ham(DMEM/F12 Ham)(Thermo Fisher Scientific,Gibco TM,目录号:10565-018)
  11. 胎牛血清(FBS),ESC Qualified(Thermo Fisher Scientific,目录号:10439-024)
  12. 敲除血清置换(KSR)(Thermo Fisher Scientific,Gibco TM ,目录号:10828-028)
  13. 青霉素和链霉素(Pen/Strep)(Thermo Fisher Scientific,Gibco< sup>,目录号:15140-122)的混合物
  14. 白血病抑制因子(LIF)(Merck Millipore Corporation,目录号:ESG1107)
  15. GSK3b抑制剂(Stemolecule CHIR99021)(Stemgent,目录号:04-0004-02)
  16. MAPK/ERK抑制剂(Stemolecule PD0325901)(Stemgent,目录号:04-0006-02)
  17. TrypLE Trypsin Replacement(Thermo Fisher Scientific,目录号:12605-028)
  18. 10mM非必需氨基酸(NEAA)溶液(Thermo Fisher Scientific,Gibco TM,目录号:11140-050)
  19. 丙酮酸钠溶液(Thermo Fisher Scientific,Gibco TM ,目录号:11360-070)
  20. 2-巯基乙醇(2-ME)(Sigma-Aldrich,目录号:M7522)
  21. 生长因子减少Matrigel (GFR Matrigel)(Corining ,目录号:356230)
  22. B27补充物(Thermo Fisher Scientific,Gibco TM ,目录号:17504044)
  23. N2补充(Thermo Fisher Scientific,Gibco TM ,目录号:17502-048)
  24. 全反式视黄酸(Sigma-Aldrich,目录号:R2625-50MG)
  25. 牛磺酸(Sigma-Aldrich,目录号:T-8691)
  26. MEF介质(参见配方)
  27. LIF + 2i介质(见配方)
  28. 视网膜分化培养基(RD培养基)(参见配方)
  29. 汤姆的媒介(见食谱)

设备

  1. 孵育器(37℃和5%CO 2)(Thermo Scientific,型号:HERACell 150i)
  2. 离心机(Eppendorf,型号:Conical centrifuge 5702)
  3. 组织培养罩,B2型生物安全柜(Thermo Scientific,型号:1300系列II类)
  4. 血细胞计数器(Neubauer室)或等效方法
  5. 倒置显微镜(Leica,型号:DMi8)

程序

  1. 涂层板
    1. 通过将管浸没在冰中1小时来解冻生长因子减少基质胶的等分试样。
    2. 在冷MEF培养基中稀释基质胶1:50,将培养基和Matrigel转移到TC培养皿中。对于6孔板,我们使用1ml /孔和7ml用于10cm板。
    3. 在37℃孵育至少1小时。
    注意:因为Matrigel在温暖的温度下固化,所以应始终保持在冰上,我们建议使用预冷却的吸头。

  2. mESCs的维护
    1. 将mESC接种在用LIF + 2i培养基预涂覆有生长因子减少基质胶的10cm培养皿上。
    2. 然后将mESCs在37℃下在5%CO 2培养箱中孵育,直到传代或视网膜分化。
    3. 每隔一天更换一次 注意:应该特别注意避免mESCs在维持培养中的过度融合,因为它可能导致细胞的有害分化。

  3. 来自未分化mESCs的视网膜细胞的生成
    1. 从未分化的mESC的50%汇合板(至少四个通道离开饲养层,图1)除去培养基,并添加TrypLE胰蛋白酶替换到细胞。将细胞放回培养箱中3-5分钟,直到mESCs菌落开始提起。由于TrypLE胰蛋白酶替换不被培养基中的血清抑制,该方法不需要用PBS预洗涤。
    2. 加入5毫升MEF媒体板和轻轻地脱离细胞用玻璃巴斯德吸管。观察细胞从培养板中取出并用移液管上下移动以获得单细胞悬浮液,小心避免气泡。


      图1.显示了未分化的mESCs(第0天)的典型形态。显示了无饲料的未分化的R1 mESC(最初由Andras Nagy产生)。比例尺:500μm(A)和250μm(B)
    3. mESC细胞在190×g下沉淀5分钟
    4. 使用5ml视网膜分化培养基(RD培养基)小心地分散沉淀
    5. 使用血细胞计数器(Neubauer chamber)或等效方法计数细胞密度。
    6. 将3000个细胞/100μl在96孔低附着板(100μl/孔)的RD培养基中。细胞聚集体(胚胎体)应在不到12小时内形成。培养开始的日期被指定为第0天。
    7. 24小时后(第1天),加入生长因子减少的基质胶至终浓度为2%(vol/vol)。商业Matrigel产品的批次间变异性可以改变视网膜诱导的效率;我们优选使用相对高浓度(> 9.5mg/ml)之一。我们建议在冷RD培养基中稀释Matrigel 1:10,加入20μl稀释液/孔。
    8. 在第3天,清楚的神经上皮细胞层是明显的(图2) 注意:不同的细胞系可能以不同的速率生长,因此每孔的初始细胞数可能需要调整。我们发现,对于大多数测试的线,3,000-5,000个细胞/孔是最佳的细胞密度。


      图2.在不同分化阶段的类胚体的典型形态。使用本文所述的方法分化R1 mESC。在每个EB(胚状体)周围,从第3天(D-F)开始形成清晰的神经上皮层,并且从第5天开始,该层显着(G-K),视觉囊泡和视杯样结构从第7-8天(L-O)比例尺:150μm 注意:在第13天(O和P中的红色箭头)后观察到视网膜色素上皮(RPE)细胞。

  4. 视网膜成熟
    1. 在第7天,将胚状体(EB)转移到补充有B27和N2的Tom's培养基中的6孔低附着板以促进神经元分化。从第7天起,每隔一天更换一次培养基
    2. 从第10天到第15天,除了B27和N2之外,Tom's培养基还补充有0.5μM类视黄酸和1mM牛磺酸。 RPE细胞从第13天(图2)显而易见
    3. 从第15天开始,将EB保持在汤姆氏培养基中。从第24天和第35天后检测到视紫红质+杆感光细胞,EB显示成熟视网膜的所有层。

食谱

  1. MEF培养基(100ml) 89 ml DMEM
    10ml FBS
    1 ml Pen/Strep
  2. LIF + 2i培养基(100ml) 90ml MEF培养基
    10ml FBS
    1ml丙酮酸钠 100μl2-ME(储备0.1M)
    100μlLIF(库存1000万单位/ml)
    在使用当天,补充3μMGSK3β抑制剂干细胞CHIR99021和0.4μMMEK抑制剂干细胞PD0325901
  3. 2-ME(每月准备)
    35μl2-ME
    5ml蒸馏水
  4. 视网膜分化(RD)培养基(100ml)
    96.4ml格拉斯哥最低必需培养基(GMEM)
    1.5 ml KSR
    1 ml非必需氨基酸(原液100x)
    1 ml丙酮酸钠(储备液100x)
    100μl2-ME(原液0.1M)
  5. 汤姆的中等(100毫升)
    98 ml DMEM:F12
    1 ml非必需氨基酸(原液100x)
    1 ml丙酮酸钠(储备液100x)
    - 从第7天到第15天:
    1 ml N2(储备100x)
    2 ml B27(原液50x)
    - 从第10天到第15天:
    0.5μM类视黄酸(1mM储备液) 1mM牛磺酸(原液1M)

致谢

该协议最初是作为:La Torre 等人的一部分发布的。 (2015)。作者要感谢Reh和Bermingham-McDonogh实验室的所有现任和前任成员进行了许多有益的讨论。特别感谢Tom Reh和Akina Hoshino提供宝贵的帮助和建议,以及NIH 1 PO1 GM081619和华盛顿大学P30EY01730(PI:Reh)的Vision Core Grant的成像核心。

参考文献

  1. Eiraku,M.,Takata,N.,Ishibashi,H.,Kawada,M.,Sakakura,E.,Okuda,S.,Sekiguchi,K.,Adachi,T.and Sasai,Y。 自组织光杯在三维文化中的形态发生。/a> Nature 472(7341):51-56。
  2. Eiraku,M。和Sasai,Y。(2012)。  小鼠胚胎干细胞培养物用于产生三维视网膜和皮质组织。 Nat Protoc 7(1):69-79。
  3. Kamiya,D.,Banno,S.,Sasai,N.,Ohgushi,M.,Inomata,H.,Watanabe,K.,Kawada,M.,Yakura,R.,Kiyonari,H.,Nakao, Jakt,LM,Nishikawa,S。和Sasai,Y。(2011)。  胚胎干细胞分化成神经祖细胞的内在转换。

    470(7335):503-509。
  4. La Torre,A.,Hoshino,A.,Cavanaugh,C.,Ware,CB和Reh,TA(2015)。  GIPC1-Akt1通路是小鼠胚胎干细胞中的眼球规格所需的。干细胞 33 9):2674-2685。
  5. Nakano,T.,Ando,S.,Takata,N.,Kawada,M.,Muguruma,K.,Sekiguchi,K.,Saito,K.,Yonemura,S.,Eiraku,M.and Sasai, 2012)。  视杯自我形成和可存储的分层来自人类ESC的神经视网膜。细胞干细胞 10(6):771-785。  


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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:La Torre, A. (2016). Retinal Differentiation of Mouse Embryonic Stem Cells. Bio-protocol 6(13): e1851. DOI: 10.21769/BioProtoc.1851.
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