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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

PT Pooja Teotia
MH Matthew J. Van Hook
IA Iqbal Ahmad

发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2212 浏览次数: 10088

评审: Letizia De ChiaraAnonymous reviewer(s)

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

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

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

干细胞 > 成体干细胞 > 神经干细胞

细胞生物学 > 细胞分离和培养 > 细胞分化

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