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A Co-culture Model for Determining the Target Specificity of the de novo Generated Retinal Ganglion Cells
使用共培养模型测定新生成的视网膜神经节细胞的靶点特异性   

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Abstract

In glaucoma, the output neurons of the retina, the retinal ganglion cells (RGCs), progressively degenerate, leading to irreversible blindness (Ahram et al., 2015). The ex vivo stem cell method to replace degenerated RGCs remains a potentially viable approach (Levin et al., 2004). However, the success of the approach depends upon the ability of the de novo generated RGCs to connect over the long distance with specific targets in the central visual pathway. Here, we describe a protocol to examine the target specificity of the de novo generated RGCs using a co-culture approach where the RGCs neurites are allowed to choose between specific (superior colliculus; SC) and non-specific (inferior colliculus; IC) tectal targets.

Keywords: Glaucoma( 青光眼), Human induced pluripotent stem cells(人诱导多能干细胞), Retinal ganglion cells(视网膜神经节细胞), Superior colliculus(上丘), Target specificity(靶点特异性)

Background

Glaucoma is one of the most prevalent causes of irreversible blindness worldwide (Tham et al., 2014). It is characterized by a progressive degeneration of RGCs, the main output neurons of the retina, which connects with the brain for visual perception. Unfortunately, there is no treatment currently available to address RGCs degeneration. The management approaches, whether surgical, pharmacological or neuro-protective do not reverse the degenerative changes (Danesh-Meyer, 2011). Given this intractable situation, stem cell therapy has emerged as a potentially viable approach to replace dead RGCs. The success of this approach requires, 1) directed differentiation of functional and non-tumorigenic RGCs from pluripotent stem cells and 2) target specificity of the de novo generated RGCs. Our lab has recently demonstrated a chemically defined method that allows directed differentiation of RGCs from embryonic stem (ES)/induced pluripotent stem (iPS) cells by recapitulating developmental mechanism (Teotia et al., 2016). The resulting RGCs are stable, functional, and non-tumorigenic. However, the success of the de novo generated cells in the ex vivo stem cell approach to glaucomatous RGC degeneration depends upon their axons ability to find proper targets in the central visual pathways. When transplanted, axons of RGCs must navigate within the retina to exit as optic nerve, decide to cross or not to cross at the optic chiasm, and reach specific targets for establishing retinotopic connections. We have demonstrated that ES/iPS cell-derived RGCs possess target specificity. Here, we describe in detail a co-culture experimental paradigm to test the target specificity of the de novo generated RGCs.

Materials and Reagents

  1. 100-mm Petri dishes (SARSTEDT, catalog number: 83.1802 )
  2. Kimtech science Kim-wipes (KCWW, Kimberly-Clark, catalog number: 34120 )
  3. Double-edge stainless steel razor blades (Personna, catalog number: MPPB-100 )
  4. Whatman filter paper, round (Sigma-Aldrich, catalog number: WHA10539028 )
  5. Plastic pipette tips
    200 μl tips (Fisher Scientific, FisherbrandTM, catalog number: 02-707-452 )
    1 ml tips (Molecular Bio products, catalog number: 3580 )
  6. Tape
  7. 12 mm round coverslips (Fisher Scientific, FisherbrandTM, catalog number: 12-545-80 )
  8. Tissue culture plates, 24-well (Corning, Falcon®, catalog number: 353047 )
  9. 12-well plate
  10. 27 G1/4 gauge needles (BD, catalog number: 305136 )
  11. Sprague-Dawley rats at postnatal day 1/3 (Charles river laboratories)
  12. Ice and ice bucket
  13. Hank’s balanced salt solution (HBSS), Ca2+/Mg2+ free (Mediatech, catalog number: 21-021-CV )
  14. 70% ethanol (Sigma-Aldrich, catalog number: 459844 )
  15. 4% to 7% low melting point agarose (45 to 50 °C) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16520-100 )
  16. Poly D-lysine (Sigma-Aldrich, catalog number: P7886 )
  17. Ultrapure Laminin, mouse (Corning, catalog number: 354239 )
  18. DMEM/F12 media (Thermo Fisher Scientific, GibcoTM, catalog number: 11320033 )
  19. CFDA SE (carboxyfluorescein diacetate succinimidyl ester) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: V12883 )
  20. Vacuum grease
  21. Ames’ Medium (Sigma-Aldrich, catalog number: A1420 )
  22. Toxin tetrodotoxin (TTX) (Tocris Bioscience, catalog number: 1078 )
  23. Lucifer yellow CH dipotassium salt (2 mg/ml) (Sigma-Aldrich, catalog number: L0144 )
  24. Alexa Fluor 568 Hydrazide (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: A10437 )
  25. Sodium chloride (NaCl)
  26. Potassium chloride (KCl)
  27. Sodium phosphate dibasic (Na2HPO4)
  28. Potassium dihydrogen phosphate (KH2PO4)
  29. KCH3SO4
  30. Magnesium chloride (MgCl2)
  31. Calcium chloride (CaCl2)
  32. HEPES
  33. Glucose
  34. EGTA
  35. MgATP
  36. Na2GTP
  37. hiPSC-RGCs specific media as described previously (Teotia et al., 2016)
  38. Phosphate buffer saline (PBS), Ca2+/Mg2+ free (see Recipes)
  39. Intracellular solution (see Recipes)

Equipment

  1. Stereo zoom microscope (Leica Microsystems, model: MZ6 )
  2. Stoelting tissue slicer/chopper (Stoelting)
  3. Sterile biosafety hood (Thermo Fisher Scientific, Forma Scientific, model: Class IIA/B3 Biological safety cabinet)
  4. Dissection hood (Thermo Fisher Scientific, Forma Scientific, Horizontal laminar flow hood)
  5. Micro-dissecting scissors (Roboz, catalog number: RS-5611 )
  6. Tissue forceps (Teeth less) (Harvard apparatus, catalog number: 72-6685 )
  7. Bone rongeur (Roboz, catalog number: RS-8340 )
  8. Beakers (100 ml) (Corning, PYTEX®, catalog number: 1000-100 )
  9. Metal spatula
  10. Vernier micrometer
  11. Water bath set at 37 °C (Thermo Fisher Scientific, Fisher Scientific)
  12. Fine and soft paintbrushes (Colour Shaper, catalog number: 11901 )
  13. -80 °C freezer
  14. Tissue culture hood
  15. Tissue culture incubator set at 37 °C, 5% CO2 (Sanyo)
  16. Standard fluorescein isothiocyanate (FITC) filter sets
  17. Cell scraper
  18. Centrifuge (IEC, model: Centra GP8R )
  19. Fluorescent microscope (Olympus, model: 1X70 )
  20. Recording chamber
  21. Micromanipulator
  22. Patch pipettes with tips of ~1 micron, fabricated out of thin-walled borosilicate glass capillaries (World Precision Instruments, catalog number: TW120F-4 ) using a pipette puller (i.e., NARISHIGE, model: PC-10 or Sutter Instrument, model: P-97 ) and with resistances of 5-10 MΩ
  23. Patch clamp amplifier (Axon Multiclamp, Molecular Devices)
  24. Autoclave

Software

  1. Axiovision 4.8 software
  2. ImageJ (NIH)
  3. GraphPad Prism (Graphpad, La Jolla CA)
  4. Windows Excel (Microsoft, Redmond, USA)

Procedure

  1. Dissection of Superior (SC)/Inferior Colliculus (IC) from PN1/3 rat brain
    Ethical statement: Experimental protocols and the use of animals were approved by the Institutional Animal Care and Use Committee, at the University of Nebraska Medical Center (UNMC) and conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.
    1. Sprague-Dawley rats of both sexes at postnatal day 1/3 are sacrificed by decapitation.
    2. The brains are removed and transferred to 100-mm Petri dish containing HBSS solution and placed on ice (Figures 1A-1C).
    3. Dissections of SC/IC are done under a stereomicroscope.
    4. The tectum was exposed by carefully removing the overlying cortex. A pair of rostral and caudal bumps, superior and inferior colliculi, respectfully, can be observed. Anterior and posterior incisions are made to the rostral bumps to collect SC. Similarly another incision posterior to the caudal bump reveals IC (Figures 1D-1E).
    5. Both sides of SC/IC are carefully separated from each other and kept in ice cold HBSS for further processing (Figures 1F-1G).


      Figure 1. Brain dissection. Decapitate a PN2 rat pup and gently remove the brain from the skull (A-C). Remove the cortex overlying the midbrain, which is removed by horizontal cut to anterior-posterior axis (D-G).

  2. Preparation of slices from SC/IC tissues
    Stoelting tissue slicer is used to slice SC/IC tissues. Section as thin as 100 μm is readily prepared.
    1. Clean the Stoelting tissue slicer with 70% ethanol, wipe completely with Kim-wipe and place inside the sterile dissection hood.
    2. Wipe a new razor blade with Kim-wipe moistened in 70% ethanol and mount on blade holder located on the right side of slicer arm by unscrewing two nuts from the left side of slicer arm to release the blade holder.
    3. Mount the razor with cleaned side of the blade facing towards the side of vernier micrometer (Figures 2A and 2B).


      Figure 2. The stoelting tissue slicer apparatus with double-edge stainless steel blades for slice cutting (A, B)

    4. Move the sample stage (base pad) to the left or to right by turning the vernier micrometer clockwise or anticlockwise to ensure proper functioning of the instrument.
    5. Cut the filter paper into 2.5 x 8 cm and moisten with HBSS.
    6. Transfer the tissue to wet area of filter paper.
    7. Meanwhile, prepare freshly made 4% low melting point agarose.
    8. Weigh 4 mg of agarose in glass beaker and add 100 ml of HBSS. Microwave the beaker for 3-4 min until agarose is completely melted and starts to boil.
    9. Keep the melted agarose in a water bath set at 50 °C to avoid solidification. Keep agarose well mixed throughout the procedure by shaking the beaker intermittently.
    10. Add the melted agarose drop by drop over the tissue with the help of plastic pipette tip, completely covering the SC/IC tissues until a mound of agarose is formed. Remove air bubble, if any (Figure 3).


      Figure 3. Schematic representation of agarose covered tissue

    11. Place the agarose-covered tissue on ice for 5-10 min to allow the agarose to solidify.
    12. Once agarose solidifies completely, place the filter paper on sample stage and secure the paper with tape (Figure 4A).
    13. Position the tissue for its first cut by moving the sample stage either to the left or to right.
    14. Set the slice thickness to 100 µm (range: 100-300 µm).
    15. Bring the slicer arm down, release slowly and let it fall by gravity.
    16. Lift the slicer arm gently once first slice is done, turn the vernier counter clockwise forward by 100 µm and then subsequently cut another slice (Figure 4B).
    17. When the whole tissue is sliced, lift the slicer arm and gently remove the slices off the surface with the help of wet soft paintbrush.
    18. Transfer the slices in to culture Petri dish containing HBSS.
    19. Transfer the slices using plastic pipette tip, the end broadened by a cut, with cut end to PDL and Laminin coated glass coverslips.


      Figure 4. Filter paper mounted on sample stage (A) and tissue slices after slicing (B)

  3. Poly-D-lysine (PDL) and laminin coating and plating of SC/IC tissue slices
    Note: Poly-D-lysine and laminin are stored in a -80 °C freezer as 100 µl aliquots as stock solution of 10 mg/ml and 1 mg/ml, respectively.
    SC/IC slices on to PDL and Laminin coated sterile glass coverslips are transferred to 24-well culture plates.
    1. Briefly, one day prior to SC/IC tissue slices, coverslips/culture plates are coated with PDL and laminin.
    2. Prepare working PDL solution (0.25 mg/ml) in sterile water. Add enough solution to cover surface of sterile glass coverslips.
    3. Incubate overnight at room temperature in a tissue culture hood.
    4. Next day, aspirate PDL solution and wash coverslips with sterile water twice at room temperature and allow to dry at room temperature.
    5. Prepare laminin solution (5 µg/ml) in sterile DMEM/F12 media. Coverslips are then coated with 250-300 µl of laminin/per coverslip/well.
    6. Incubate for 2 h at room temperature.
    7. Aspirate to remove laminin and wash coverslips with DMEM/F12 media twice at room temperature.
    8. Carefully plate SC/IC slices on the coated glass coverslips as described above. Do not allow coating to dry.
    9. Add 50 µl media drop on tissue slices in order to prevent drying and facilitate proper attachment. Incubate at 37 °C/5% CO2 overnight.
    10. Next day, gently add 300 µl of hiPSC-RGCs media per coverslip/well as described previously (Teotia et al., 2016).

  4. Carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) labeling of hiPSC-RGCs
    hiPSC-RGCs are labeled with CFDA cell tracing agent for in vitro tracing of neurite outgrowth. CFDA SE passively diffuses into cells. The well-retained fluorescent conjugates are formed when the succinimidyl ester group reacts with intracellular amines. Preparations of the dye and labeling of cells should be protected from light.
    1. Prepare CFDA solution (5 μM) immediately prior to use by diluting in PBS.
    2. Grow hiPSC-RGCs in a 12-well plate, remove the media from the plate and add CFDA solution to hiPSC-RGCs in a 12-well culture plate.
    3. Incubate cells at 37 °C/5% CO2 for 15 min.
    4. Replace the diluted CFDA solution with fresh medium and incubate cells for another 30 min at 37 °C/5% CO2.
    5. CFDA labeled cells are visualized by fluorescence microscopy using standard fluorescein isothiocyanate (FITC) filter sets. Cells are now ready to use for co-culture experiment. CFDA labeled cells can be cultured for 1-2 weeks post staining.

  5. Co-culture of hiPSC-RGCs with SC/IC tissue slices in vitro
    Setting up the co-culture of hiPSC-RGCs in the same plate as described in Procedure C.
    1. Remove the PDL and laminin coated plate containing SC/IC tissue slices as described in Procedure C from 37 °C/5% CO2 incubator, aspirate the medium and keep in tissue culture hood.
    2. Meanwhile, detach hiPSC-RGCs from 12-well plate using cell scraper.
    3. Centrifuge the cells at 220 x g for 5 min to obtain cell pellet and aspirate the supernatant.
    4. Resuspend the cells in 1 ml of fresh medium, gently mix and add 50 μl of media containing hiPSC-RGCs (~3-6/coverslip) away from the SC/IC slices on the above PDL and laminin coated coverslips.
    5. Return the plate to the incubator and incubate at 37 °C/5% CO2 overnight. Allow the cells to adhere, next day, add 300 µl hiPSC-RGCs medium.
    6. Change the medium every 2-4 days. hiPSC-RGCs can be co-cultured with SC/IC slices up to 8 days in vitro.
    7. Once neurite outgrowth reaches considerable length, mount the coverslips on glass slides and image under the fluorescent microscope (Figure 5).


      Figure 5. CFDA-tagged hiPSC-RGCs elaborated long processes toward SC cells aggregate (A), compared to the one co-cultured with IC cells aggregate (B). *Represents RGCs extending processes. Scale bars = 50 μm

    8. Compare the length and complexity of neurite outgrowth of hiPSC-RGCs extending towards both SC and IC slices. hiPSC-RGCs can be discriminated from clear-cut boundaries of SC/IC slices by CFDA staining of cell soma.

  6. Electrophysiology of hiPSC-RGCs co-cultured with SC tissue slices
    Whole-cell patch clamp electrophysiology allows for recording membrane voltage changes such as action potentials and excitatory and inhibitory synaptic potentials from hiPSC-RGCs co-cultured with SC tissue slices as well as the ionic currents that give rise to those voltage changes. The former is accomplished using the current clamp-recording mode while the latter is accomplished in voltage clamp recordings (Molleman, 2003; Marty and Neher, 2009; Teotia et al., 2016).
    1. Mount a coverslip containing hiPSC-RGCs co-cultured with SC slice on a recording chamber using several dots of vacuum grease.
    2. Position the recording chamber on the stage of an upright, fixed-stage microscope equipped with electrophysiology hardware and superfuse at ~1-4 ml/min with Ames’ Medium bubbled with a gas mixture of 5% CO2 and 95% O2.
    3. To establish a whole-cell recording, provide gentle positive pressure through the pipette and position it on the membrane of the target cell using a micromanipulator while viewing the cell and pipette under the microscope.
    4. Use the patch clamp amplifier to provide a 5 mV depolarizing step to measure resistance. The resistance will climb slightly as the pipette contacts the cell membrane.
    5. Release the positive pressure and provide gentle suction while monitoring resistance to form a GΩ seal (seal resistance > 1 GΩ).
    6. Once the GΩ seal is formed, apply a holding potential (-70 mV) and rupture the patch using either strong pulses of suction, gradual gentle suction with a syringe, or the ‘zap’ feature of the amplifier. The appearance of whole-cell capacitance transients indicates successful rupture of the patch and establishment of whole-cell recording.
    7. In voltage-clamp, to record voltage-gated sodium and potassium currents (INa and IK), design a protocol in which the amplifier depolarizes the cell in increments of 10 mV (up to +50 mV). Use a P/8 leak subtraction protocol to subtract membrane leak and whole-cell capacitance transients from the data.
    8. INa will be a fast inward current that rapidly inactivates, while IK is typically a slowly-developing and non-inactivating outward current (Figure 6).
    9. Monitor the quality of the whole-cell recording by noting access resistance (Ra) using a 5 mV depolarizing pulse. Ra should be as low as possible (i.e., < 40 MΩ) and should remain relatively stable throughout the recording. Large Ra will introduce substantial voltage errors, distorting measures of current voltage-dependence and amplitude.
    10. If INa is sufficiently large, the cell might generate action potentials in response to depolarizing current injections (+10 to +300 pA) in current-clamp mode (Figure 1). INa is typically blocked by the pufferfish toxin tetrodotoxin, which can be added to the extracellular solution and superfused onto the cells at a concentration of 0.5-2 μM.


      Figure 6. Example recording of hiPSC-RGCs co-cultured with SC slices. A. Whole-cell voltage clamp recording of INa and IK in response to step depolarizations (150 msec, -66 mV to +24 mV from a holding potential of -76 mV). INa is blocked by 1 mM TTX. B. Expanded time axis of current responses to a depolarizing step to -26 mV in control conditions and in the presence of 1 mM TTX. C. Current-clamp recording showing voltage responses to hyperpolarizing and depolarizing current injections (-20 to +50 pA). The cell fires action potentials in response to depolarizing current injections. 

Data analysis

Imaging, quantification and analysis of dendritic branching

  1. Mount coverslips on glass slides from 3-6 independent co culture experiments and capture images using fluorescent microscope.
  2. For neurite tracing and sholl analysis collect images using Axiovision 4.8 software at 20x objective on a Zeiss upright fluorescent microscope. Conduct image analysis using ImageJ (NIH). Measure the length and complexity of dendritic branches independently for each group using the plugin Sholl Analysis (v1.50) in ImageJ by placing 20 μm concentric ring from the edge of neural rosettes as described previously (Teotia et al., 2016).
  3. Analyze and plot data using GraphPad Prism (GraphPad, La Jolla CA) and Windows Excel (Microsoft, Redmond, USA). Calculate statistical significance by either a paired Student’s t-test (two-tailed) or by one-way analysis of variance (ANOVA) for multiple groups.

Notes

  1. When imaging cells, it is important to avoid areas where CFDA labeled cells are overlapping on the SC/IC tissue, and try to take into account only those areas where cells are juxtaposed to SC/IC explants.
  2. Cells upon CFDA labeling can be used immediately. Non-toxicity and long-term retention of CFDA allows the fluorescence to be well retained in cells several days post staining. However, in case of cytotoxicity change culture media every day.
  3. When reporting voltages, it is important to account for the liquid junction potential arising at the interface of the pipette solution and the extracellular solution. Although various software programs can calculate this value, it should ideally be empirically measured for each combination of solutions used. The LJP for our intracellular solution and Ames’ Medium was measured as 6 mV.
  4. The intracellular can also contain Lucifer yellow CH dipotassium salt (2 mg/ml) in order to visualize cell morphology. Lucifer yellow CH is a fixable dye with an excitation spectrum peaking around 430 nm and emission peaking around 540 nm that, if desired, can be visualized in combination with immunocytochemistry after whole-cell recording. For different wavelength ranges, other dyes such as Alexa Fluor 568 Hydrazide can be used at 100-200 μM.
  5. TTX solutions should be handled with care and properly disposed of according to institutional guidelines.

Recipes

  1. Phosphate buffer saline (PBS)
    137 mM NaCl
    2.7 mM KCl
    4.3 mM Na2HPO4
    1.47 mM KH2PO4
    Adjust to a final pH of 7.4
    Autoclave at 121 °C for 30 min
  2. Intracellular solution
    98 mM KCH3SO4
    44 mM KCl
    3 mM NaCl
    3 mM MgCl2
    1 mM CaCl2
    5 mM HEPES
    2 mM glucose
    3 mM EGTA
    1 mM MgATP
    1 mM Na2GTP
    Adjusted pH to 7.3
    Psmolality adjusted to ~280 mOsm

Acknowledgments

This work was supported by NIH/NEI: R01-EY022051 (IA).

References

  1. Ahram, D. F., Alward, W. L. and Kuehn, M. H. (2015). The genetic mechanisms of primary angle closure glaucoma. Eye 29(10): 1251-1259.
  2. Danesh-Meyer, H. V. (2011). Neuroprotection in glaucoma: recent and future directions. Curr Opin Ophthalmol 22(2): 78-86.
  3. Levin, L. A., Ritch, R., Richards, J. E. and Borrás, T. (2004). Stem cell therapy for ocular disorders. Arch Ophthalmol 122(4): 621-627.
  4. Marty, A. and Neher, E. (2009). Tight-seal whole-cell recording. In: Sakmann, B. and Neher, E. (Eds.). Single-Channel Recording. Springer, pp 31-52.
  5. Molleman, A. (2003). Patch clamping: an introductory guide to patch clamp electrophysiology. John Wiley & Sons.
  6. Teotia, P., Chopra, D. A., Dravid, S. M., Van Hook, M. J., Qiu, F., Morrison, J., Rizzino, A. and Ahmad, I. (2016). Generation of functional human retinal ganglion cells with target specificity from pluripotent stem cells by chemically defined recapitulation of developmental mechanism. Stem Cells 35(3): 572-585.
  7. Tham, Y. C., Li, X., Wong, T. Y., Quigley, H. A., Aung, T. and Cheng, C. Y. (2014). Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthalmology 121(11): 2081-2090.
  8. Van Hook, M. J. and Thoreson, W. B. (2014). Whole-cell patch-clamp recording. In: Xiong, H. and Gendelman, H. E. (Eds.). Current Laboratory Methods in Neuroscience Research. Springer, pp 353-367.

简介

在青光眼中,视网膜的输出神经元,视网膜神经节细胞(RGC)逐渐退化,导致不可逆的失明(Ahram等人,2015)。 替代退化RGCs的离体干细胞方法仍然是潜在可行的方法(Levin等人,2004)。 然而,该方法的成功取决于生成RGC的远程连接与中心视觉通路中特定目标的能力。 在这里,我们描述了一种协议,用于使用共培养方法来检查产生RG的产生RGCs的靶特异性,其中RGCs神经突被允许在特异性(上丘(SC))和非特异性 (下丘,IC)构造目标。

青光眼是全球不可逆失明的最常见原因之一(Tham等人,2014)。其特征在于RGC的进行性退化,视网膜的主要输出神经元,其与大脑连接用于视觉感知。不幸的是,目前尚无治疗RGCs变性的治疗方法。无论是外科手术,药理学还是神经保护,管理方法都不能扭转退行性变化(Danesh-Meyer,2011)。鉴于这种棘手的情况,干细胞治疗已经成为替代死亡RGCs的潜在可行方法。这种方法的成功需要:1)功能性和非致瘤性RGC与多能干细胞的定向分化,以及2)产生RGC的新生靶标特异性。我们的实验室最近展示了一种化学定义的方法,通过重述发育机制(Teotia等人,2016),允许RGCs从胚胎干(ES)/诱导的多能干细胞(iPS)细胞中的定向分化。所得的RGC是稳定的,功能性的和非致瘤性的。然而,远离干细胞在青光眼RGC变性中的生物细胞的成功取决于它们的轴突在中心视觉途径中找到适当靶标的能力。移植后,RGC的轴突必须在视网膜内导航,作为视神经退出,决定在视交叉处交叉或不交叉,并达到建立视网膜连接的具体目标。我们已经证明ES / iPS细胞衍生的RGC具有靶特异性。在这里,我们详细描述了一个共同培养实验范例,以测试新生RGC的目标特异性。

关键字:青光眼, 人诱导多能干细胞, 视网膜神经节细胞, 上丘, 靶点特异性

材料和试剂

  1. 100毫米培养皿(SARSTEDT,目录号:83.1802)
  2. Kimtech科学Kim-wipes(KCWW,Kimberly-Clark,目录号:34120)
  3. 双刃不锈钢刀片(Personna,目录号:MPPB-100)
  4. Whatman滤纸,圆形(Sigma-Aldrich,目录号:WHA10539028)
  5. 塑料移液器吸头
    200μl提示(Fisher Scientific,Fisherbrand TM ,目录号:02-707-452)
    1 ml提示(Molecular Bio产品,目录号:3580)
  6. 磁带
  7. 12毫米圆盖玻片(Fisher Scientific,Fisherbrand TM ,目录号:12-545-80)
  8. 组织培养板,24孔(Corning,Falcon ®,目录号:353047)
  9. 12孔板
  10. 27 G 1/4 计量针(BD,目录号:305136)
  11. Sprague-Dawley大鼠出生后1/3(查尔斯河实验室)
  12. 冰和冰桶
  13. Hank的平衡盐溶液(HBSS),Ca 2 + /Mg 2 + free(Mediatech,目录号:21-021-CV)
  14. 70%乙醇(Sigma-Aldrich,目录号:459844)
  15. 4%至7%的低熔点琼脂糖(45至50℃)(Thermo Fisher Scientific,Invitrogen TM,目录号:16520-100)
  16. 聚D-赖氨酸(Sigma-Aldrich,目录号:P7886)
  17. 超纯化层粘连蛋白,小鼠(康宁,目录号:354239)
  18. DMEM/F12介质(Thermo Fisher Scientific,Gibco TM,目录号:11320033)
  19. CFDA SE(羧基荧光素二乙酸酯琥珀酰亚胺酯)(Thermo Fisher Scientific,Molecular Probes TM,目录号:V12883)
  20. 真空润滑脂
  21. Ames'Medium(Sigma-Aldrich,目录号:A1420)
  22. 毒素河豚毒素(TTX)(Tocris Bioscience,目录号:1078)
  23. 萤光黄黄色CH 2钾盐(2mg/ml)(Sigma-Aldrich,目录号:L0144)
  24. Alexa Fluor 568酰肼(Thermo Fisher Scientific,Molecular Probes TM,目录号:A10437)
  25. 氯化钠(NaCl)
  26. 氯化钾(KCl)
  27. 磷酸氢二钠(Na 2 HPO 4)
  28. 磷酸二氢钾(KH 2 PO 4)<>>
  29. KCH 3 SO 4
  30. 氯化镁(MgCl 2)
  31. 氯化钙(CaCl 2)
  32. HEPES
  33. 葡萄糖
  34. EGTA
  35. MgATP
  36. Na 2 GTP
  37. 如前所述的hiPSC-RGC特异性培养基(Teotia等人,2016)
  38. 磷酸盐缓冲盐水(PBS),Ca 2+ +/sup>/Mg 2+不含(见配方)
  39. 细胞内溶液(见食谱)

设备

  1. 立体变焦显微镜(Leica Microsystems,型号:MZ6)
  2. 烘烤切片机/切碎机(Stoelting)
  3. 无菌生物安全罩(Thermo Fisher Scientific,Forma Scientific,型号:Class IIA/B3生物安全柜)
  4. 解剖罩(Thermo Fisher Scientific,Forma Scientific,卧式层流罩)
  5. 微解剖剪刀(Roboz,目录号:RS-5611)
  6. 组织镊子(牙齿较少)(哈佛仪器,目录号:72-6685)
  7. 骨骨肉(Roboz,目录号:RS-8340)
  8. 烧杯(100毫升)(康宁,PYTEX ®,目录号:1000-100)
  9. 金属铲子
  10. 游标千分尺
  11. 水浴设置在37°C(Thermo Fisher Scientific,Fisher Scientific)
  12. 精美的油漆刷(彩色成型机,目录号:11901)
  13. -80°C冰箱
  14. 组织文化罩
  15. 组织培养箱设在37℃,5%CO 2(三洋)
  16. 标准荧光素异硫氰酸酯(FITC)过滤器套件
  17. 细胞刮刀
  18. 离心机(IEC,型号:Centra GP8R)
  19. 荧光显微镜(Olympus,型号:1X70)
  20. 录音室
  21. 微操纵器
  22. 使用移液管拉拔器(NARISHIGE,型号:PC)从薄壁硼硅玻璃毛细管(World Precision Instruments,目录号:TW120F-4)制造的具有〜1微米尖端的补片移液管-10或Sutter仪器,型号:P-97),电阻为5-10MΩ
  23. 贴片钳放大器(Axon Multiclamp,Molecular Devices)
  24. 高压灭菌器

软件

  1. Axiovision 4.8软件
  2. ImageJ(NIH)
  3. GraphPad Prism(Graphpad,La Jolla CA)
  4. Windows Excel(Microsoft,Redmond,USA)

程序

  1. 从PN1/3大鼠脑中分离出优良(SC)/下丘(IC) 道德声明:实验方案和动物的使用由内布拉斯加大学医学中心(UNMC)的机构动物护理和使用委员会批准,并根据视力与眼科研究协会(ARVO)进行。使用声明 o f眼睛和视力研究中的动物。
    1. 在出生后第1/3天的Sprague-Dawley两性大鼠被断头处死。
    2. 将脑取出并转移到含有HBSS溶液的100-mm培养皿中并置于冰上(图1A-1C)。
    3. SC/IC的解剖是在立体显微镜下完成的。
    4. 通过小心地去除上覆的皮层,暴露出结构。尊重地可以观察到一对传播和尾部的颠簸,上下颠簸。将前后切口制成脊柱突起以收集SC。类似于尾部凸起后面的另一个切口显示IC(图1D-1E)
    5. SC/IC的两侧仔细分离,保存在冰冷的HBSS中进一步处理(图1F-1G)。


      图1.脑解剖。蜕皮一只PN2大鼠,轻轻地从头骨中取出大脑(A-C)。移除覆盖中脑的皮层,通过水平切割去除前后轴(D-G)。

  2. 从SC/IC组织制备切片
    切片机用于切片SC/IC组织。易于制备薄至100μm的部分。
    1. 用70%乙醇清洗Stoelting组织切片机,用Kim-wipe完全擦拭,放入无菌解剖罩内。
    2. 用Kim-wipe擦拭一个新的剃刀刀片,用70%乙醇润湿,并通过从切片机臂的左侧旋下两个螺母,将其安装在切片机右侧的刀片架上,以释放刀片架。
    3. 将剃须刀的清洁面朝向游标千分尺侧面安装剃刀(图2A和2B)。


      图2.具有用于切片切割的双边不锈钢刀片(A,B)的加热薄纸切片机设备

    4. 通过顺时针或逆时针转动游标测微计将样品台(底座垫)向左或向右移动,以确保仪器正常工作。
    5. 将滤纸切成2.5×8厘米,并用HBSS润湿。
    6. 将纸巾转移到滤纸的湿润区域。
    7. 同时,准备新鲜制作的4%低熔点琼脂糖。
    8. 称取4毫克琼脂糖在玻璃烧杯中并加入100毫升HBSS。微波烧杯3-4分钟,直到琼脂糖完全融化并开始沸腾。
    9. 将融化的琼脂糖保持在50°C的水浴中,以避免凝固。间歇地摇动烧杯,使琼脂糖在整个操作过程中保持良好的混合
    10. 在塑料移液器吸头的帮助下,将熔融的琼脂糖逐滴滴加到组织上,完全覆盖SC/IC组织,直到形成一堆琼脂糖。去除气泡(如果有的话)(图3)

      图3.琼脂糖覆盖组织的示意图

    11. 将琼脂糖覆盖的组织放在冰上5-10分钟,以使琼脂糖凝固
    12. 一旦琼脂糖完全固化,将滤纸放在样品台上并用胶带固定纸张(图4A)
    13. 通过将样品台移动到左侧或右侧来定位其第一次切割的组织。
    14. 将切片厚度设置为100μm(范围:100-300μm)。
    15. 将切片机手臂向下放下,释放缓慢,让其重力下降。
    16. 一旦第一次切片完成,请轻轻拿起切片机臂,将游标逆时针向前转100μm,然后再切割另一个切片(图4B)。
    17. 当整个组织切片时,提起切片机臂并借助湿软刷轻轻取出表面的切屑。
    18. 将切片转移到含有HBSS的培养培养皿中。
    19. 使用塑料移液器吸头将切片转移到切片的末端,切割到PDL和层合蛋白涂覆的玻璃盖玻片上。


      图4.切片后安装在样品台(A)和组织切片上的滤纸(B)

  3. 聚-D-赖氨酸(PDL)和层粘连蛋白涂层和SC/IC组织切片的铺板 注意:聚-D-赖氨酸和层粘连蛋白分别以10μg/ml和1mg/ml的储备溶液的100μl等分试样储存在-80℃冰箱中。
    将SC/IC切片到PDL和层合蛋白涂覆的无菌玻璃盖玻片转移到24孔培养板中。
    1. 简言之,在SC/IC组织切片前一天,用PDL和层粘连蛋白包被盖玻片/培养板。
    2. 在无菌水中制备PDL溶液(0.25 mg/ml)。添加足够的溶液以覆盖无菌玻璃盖玻片的表面。
    3. 在室温下在组织培养罩中孵育过夜。
    4. 第二天,抽取PDL溶液,室温下用灭菌水清洗盖玻片两次,并使其在室温下干燥。
    5. 在无菌DMEM/F12培养基中制备层粘连蛋白溶液(5μg/ml)。然后,盖玻片用250-300μl层粘连蛋白/每盖玻片/孔包被。
    6. 在室温下孵育2小时。
    7. 吸出去除层粘连蛋白,并在室温下用DMEM/F12培养基洗两次盖玻片。
    8. 如上所述,在涂覆的玻璃盖玻片上小心地将SC/IC切片放置。不要让涂层干燥。
    9. 在组织切片上加入50μl介质滴,以防止干燥并促进适当的附着。在37℃/5%CO 2孵育过夜。
    10. 第二天,如前所述(Teotia等人,2016),每盖玻片/孔轻轻加入300μl的hiPSC-RGC培养基。

  4. 半胱氨酸荧光素二乙酸酯,琥珀酰亚胺酯(CFDA SE)标记hiPSC-RGCs
    hiPSC-RGC用CFDA细胞追踪剂标记,用于体外追踪神经突生长。 CFDA SE被动地扩散到细胞中。当琥珀酰亚胺酯基团与细胞内胺反应时,形成良好保留的荧光共轭物。染色剂的制备和细胞标记应避免光照。
    1. 使用前立即用PBS稀释CFDA溶液(5μM)
    2. 在12孔板中生长hiPSC-RGC,从板中除去培养基,并将CFDA溶液加入12孔培养板中的hiPSC-RGC。
    3. 在37℃/5%CO 2孵育细胞15分钟。
    4. 用新鲜培养基替换稀释的CFDA溶液,并在37℃/5%CO 2下孵育细胞另外30分钟。
    5. 使用标准的异硫氰酸荧光素(FITC)过滤器,通过荧光显微镜观察CFDA标记的细胞。细胞现在可以用于共培养实验。染色后可以将CFDA标记的细胞培养1-2周







  5. 在程序C中所述的同一板中建立hiPSC-RGC的共培养
    1. 从37℃/5%CO 2培养箱中取出方法C所述的含有SC/IC组织切片的PDL和层粘连蛋白包被的板,吸出培养基并保持在组织培养罩中。
    2. 同时,使用细胞刮刀从12孔板分离hiPSC-RGC
    3. 以220×g离心细胞5分钟以获得细胞沉淀并抽出上清液。
    4. 将细胞悬浮于1ml新鲜培养基中,轻轻混合并加入50μl含有HiPSC-RGCs(〜3-6 /盖玻片)的培养基,远离上述PDL和层粘连蛋白包被的盖玻片上的SC/IC切片。
    5. 将板返回培养箱,并在37℃/5%CO 2下孵育过夜。允许细胞粘附,第二天,加入300μlhiPSC-RGCs培养基。
    6. 每2-4天更换培养基。 hiPSC-RGC可以与SC/IC切片在体外共培养8天。
    7. 一旦神经突生长达到相当长的长度,将盖玻片安装在玻璃片和荧光显微镜下的图像上(图5)。


      图5.与IC细胞聚集(B)共培养的CFDA标签的hiPSC-RGC,详细阐述了SC细胞聚集的长期过程(A)。比例尺= 50μm

    8. 比较延伸到SC和IC切片的hiPSC-RGCs的神经突生长的长度和复杂性。 hiPSC-RGC可以通过细胞胞浆的CFDA染色来区分SC/IC切片的清晰边界。

  6. 与SC组织切片共同培养的hiPSC-RGCs的电生理学 全细胞膜片钳电生理学允许记录与SC组织切片共同培养的hiPSC-RGCs的膜电压变化,例如动作电位和兴奋性和抑制性突触电位以及引起这些电压变化的离子电流。前者使用电流钳位记录模式完成,而后者在电压钳记录中完成(Molleman,2003; Marty和Neher,2009; Teotia等人,2016)。
    1. 在几个真空润滑脂点上,在记录室上装载含有SC切片共同培养的hiPSC-RGCs的盖玻片。
    2. 将记录室放置在配备电生理学硬件和超级清洁剂的直立式固定级显微镜的台面上,以约1-4ml/min的速度,用Ames's Medium鼓泡5%CO 2气体混合物和95%O 2。
    3. 为了建立全细胞记录,通过移液管提供温和的正压,并使用显微操纵器将其置于靶细胞的膜上,同时在显微镜下观察细胞和移液管。
    4. 使用膜片钳放大器提供5 mV去极化步骤来测量电阻。移液管接触细胞膜时,阻力会稍微上升。
    5. 释放正压力并提供温和的吸力,同时监测阻力以形成GΩ密封(密封电阻> 1GΩ)。
    6. 一旦形成GΩ密封,施加保持电位(-70 mV),并使用强力吸力脉冲,使用注射器逐渐温和抽吸或放大器的"zap"功能来破坏贴片。全细胞电容瞬变的出现表明贴片的成功破裂和全细胞记录的建立。
    7. 在电压钳中,要记录电压门控钠和钾电流(INa和IK),设计一个协议,其中放大器以10 mV(高达+50 mV)的增量去极化电池。使用P/8泄漏减法协议从数据中减去膜泄漏和全细胞电容瞬变
    8. INa将是快速内向电流,其快速灭活,而IK通常是缓慢发展和非钝化的向外电流(图6)。
    9. 通过使用5 mV去极化脉冲注意访问电阻(Ra)来监控全单元记录的质量。 Ra应尽可能低(,即,<40MΩ),并且在整个记录期间应保持相对稳定。大的Ra会引起相当大的电压误差,扭曲当前电压依赖性和振幅的测量。
    10. 如果INa足够大,电池可能会产生动作电位,以响应电流钳模式下的去极化电流注入(+ 10至+300 pA)(图1)。 INa通常被河豚毒素河豚毒素阻断,其可以加入到细胞外溶液中,并以0.5-2μM的浓度超过细胞。


      图6.与SC切片共培养的hiPSC-RGCs的实施例记录。A.全细胞电压钳记录I和N >响应于步进去极化(150msec,从保持电位为-76mV至-66mV至+ 24mV)。 I 被1mM TTX阻断。 B.在对照条件下和存在1mM TTX时,将去极化步骤的电流响应的扩展时间轴扩展至-26mV。 C.电流钳记录显示对超极化和去极化电流注入的电压响应(-20至+50pA)。该细胞响应于去极化电流注射而触发动作电位。 

数据分析

树突状分枝的成像,定量和分析

  1. 使用荧光显微镜从3-6个独立的共同培养实验将玻片上的盖玻片放在玻片上并捕获图像。
  2. 对于神经突追踪和sholl分析,使用Axiovision 4.8软件在Zeiss立式荧光显微镜上以20倍物镜收集图像。使用ImageJ(NIH)进行图像分析。使用ImageJ中的插件Sholl Analysis(v1.50),如前所述,从神经花环的边缘放置20微米的同心圆(Teotia等人),独立地测量树突状枝的长度和复杂性。,2016)。
  3. 使用GraphPad Prism(Graphpad,La Jolla CA)和Windows Excel(Microsoft,Redmond,USA)分析和绘制数据。通过配对的Student's 测试(双尾)或通过单因素方差分析(ANOVA)计算统计学显着性。

笔记

  1. 当成像细胞时,重要的是避免CFDA标记细胞在SC/IC组织上重叠的区域,并尝试仅考虑细胞与SC/IC外植体并置的区域。
  2. CFDA标记后的细胞可立即使用。 CFDA的无毒和长期保留使荧光在染色后数天保持良好。但是,如果细胞毒性每天都会改变培养基。
  3. 当报告电压时,重要的是考虑到在移液管溶液和细胞外溶液的界面处产生的液体接合电位。尽管各种软件程序可以计算出这个值,但理想情况下,对于所使用的解决方案的每种组合都应以经验为基础。将我们的细胞内溶液和Ames培养基的LJP测量为6mV。
  4. 细胞内也可以含有萤光黄黄色二钾盐(2mg/ml),以观察细胞形态。萤光黄黄色CH是具有峰值约430nm的激发光谱的固定染料,并且发射峰值在540nm附近,如果需要,可以在全细胞记录后与免疫细胞化学结合显现。对于不同的波长范围,其他染料如Alexa Fluor 568 Hydrazide可以以100-200μM使用。
  5. TTX解决方案应小心处理,并根据制度准则妥善处理。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    137 mM NaCl
    2.7 mM KCl
    4.3mM Na 2 HPO 4
    1.47mM KH 2 PO 4
    调整到7.4的最终pH值 在121℃高压灭菌30分钟
  2. 细胞内溶液
    98mM KCH 3< 3< 4>< 4>
    44 mM KCl
    3 mM NaCl
    3mM MgCl 2
    1mM CaCl 2
    5 mM HEPES
    2 mM葡萄糖
    3 mM EGTA
    1 mM MgATP
    1mM Na 2 GTP
    调整pH至7.3
    精神压力调整至〜280 mOsm

致谢

这项工作得到NIH/NEI R01-EY022051(IA)的支持。

参考文献

  1. Ahram,DF,Alward,WL和Kuehn,MH(2015)。< a class ="ke-insertfile"href ="http://www.nature.com/eye/journal/v29/n10/abs/eye2015124a .html"target ="_ blank">原发性闭角型青光眼的遗传机制。眼睛 29(10):1251-1259。
  2. Danesh-Meyer,HV(2011)。青光眼神经保护:近期和未来的发展方向。 Curr Opin Ophthalmol 22(2):78-86。
  3. Levin,LA,Ritch,R.,Richards,JE andBorrás,T。(2004)。  眼部疾病的干细胞治疗。 122(4):621-627。
  4. Marty,A.和Neher,E.(2009)。密封全细胞记录。在:Sakmann,B.and Neher,E.(Eds。)。单通道录音。 Springer ,第31-52页。
  5. Molleman,A.(2003)。  贴片夹钳:贴片钳电生理学的介绍指南。 John Wiley&儿子。
  6. Teotia,P.,Chopra,DA,Dravid,SM,Van Hook,MJ,Qiu,F.,Morrison,J.,Rizzino,A。和Ahmad,I.(2016)。< a class =插入文件"href ="http://www.ncbi.nlm.nih.gov/pubmed/27709736"target ="_ blank">通过化学定义的发育机制概述,从多能干细胞产生具有靶特异性的功能性人视网膜神经节细胞。 干细胞 35(3):572-585。
  7. Tham,YC,Li,X.,Wong,TY,Quigley,HA,Aung,T. and Cheng,CY(2014)。  2040年青光眼全球患病率和青光眼负担预测:系统评价和荟萃分析。眼科 121 (11):2081-2090。
  8. Van Hook,MJ和Thoreson,WB(2014)。  全细胞膜片钳记录。在...中:Xiong,H。和Gendelman,HE(Eds。)。目前实验室方法在神经科学研究。 Springer ,第353-367页。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Teotia, P., Van Hook, M. J. and Ahmad, I. (2017). A Co-culture Model for Determining the Target Specificity of the de novo Generated Retinal Ganglion Cells. Bio-protocol 7(7): e2212. DOI: 10.21769/BioProtoc.2212.
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