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Mar 2019

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A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery
高通量病理生理相关星形胶质细胞运动神经元共培养技术在肌萎缩侧索硬化治疗中的应用   

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Abstract

Amyotrophic lateral sclerosis (ALS) is an adult onset neurological disorder characterized by loss of motor neurons leading to progressive muscle wasting and eventually death. Astrocytes play a key role in disease pathogenesis. However, the ability to study astrocytic support towards motor neurons in ALS has been limited by a lack of sustainable high-throughput human cell models. Moreover, the ability to assess how astrocytic support of motor neurons is influenced by drug treatment or nutritional supplementation has been hampered by the lack of robust methodology. We have developed a high-throughput astrocyte motor neuron co-culture assay, which, by using Hb9-GFP+ motor neurons enables researchers to assess how ALS affects the ability of astrocytes to support motor neurons in 384-well plates. Moreover, astrocyte function can be manipulated by nutritional supplementation or drug treatment to identify possible therapeutic targets.

Keywords: ALS (ALS), Astrocytes (星形胶质细胞), Motor neurons (运动神经元), Drug discovery (药物开发), High-throughput drug screening (高通量药物筛选)

Background

Amyotrophic lateral sclerosis (ALS) is a neurological disorder resulting in degeneration of both upper and lower motor neurons, resulting in the progressive failure of the neuromuscular system. Death typically occurs 2-3 years post-symptom onset, due to a lack of effective therapies. Although death of motor neurons is a key feature of ALS, the disease is non-cell autonomous with neighboring cells such as astrocytes, microglia and oligodendrocytes playing a key role (Ferraiuolo et al., 2011b; Ferraiuolo et al., 2016; Frakes et al., 2017; Vandoorne et al., 2018). Astrocytes play a crucial metabolic role in the CNS as they are the major source of brain glycogen, and astrocyte lactate can be taken up by motor neurons and used as a source of energy (Pellerin and Magistretti, 1994). Several mechanisms have been implicated in astrocyte-mediated motor neuron death, including release of nitric oxide and prostaglandin E2, altered glutamate transporter expression, reduced lactate release and reduced extracellular vesicle mediated release of miRNA (Lin et al., 1998; Ferraiuolo et al., 2011a; Ferraiuolo et al., 2011b; Allen et al., 2019; Varcianna et al., 2019).

Until recently, the ability to study how patient derived astrocytes affect motor neuronal function has been hampered by technological limitations. Initial pioneering studies (Haidet-Phillips et al., 2011, Re et al., 2014) demonstrated that post-mortem human sporadic and familial patient-derived astrocytes could induce motor neuron death in vitro. Although elegant, this methodology presents with limitations in terms of limited availability, scalability and represents end stage of disease. The development of techniques to reprogram fibroblasts into pluripotent stem cells (iPSCs) by Yamanaka and colleagues (Takahashi and Yamanaka, 2006) has radically improved our ability to study human CNS disorders in vitro, for a recent review see (Myszczynska and Ferraiuolo, 2016). This technological advancement was followed up by methodologies to directly reprogram mouse fibroblasts into induced neuronal progenitor cells (iNPCs) and concomitantly by Meyer and Ferraiuolo who developed novel methodologies to reprogram adult human fibroblasts into iNPCs from control and ALS subjects (Kim et al., 2011; Meyer et al., 2014). This advancement in direct reprogramming, has overcome the challenge of phenotypic inconsistency caused by clonal variation in iPSCs and has significantly shortened the reprogramming timescales, reducing costs and increasing throughput (Myszczynska and Ferraiuolo, 2016). Furthermore, an additional beneficial feature of the direct reprogramming approach is that the epigenetic state of the cell is not reset, so any aging phenotype inherent in the donor fibroblasts should be retained (Mertens et al., 2015). This subsequent development has enabled ALS researchers to assess the effect of disease on patient derived cells in vitro, reducing the reliance on post-mortem tissue and animal models of disease that may lack translational efficacy.

In the co-culture approach described in this paper, iNPCs were derived from ALS patient fibroblasts and healthy donor control fibroblasts as described previously (Meyer et al., 2014). iNPCs were differentiated into iAstrocytes by culturing in iAstrocyte media for at least 5 days (Figure 1). Mouse motor neurons expressing the green fluorescent protein (GFP) under the Hb9 motor neuron-specific promoter (called from now on Hb9-GFP+ MN) were differentiated from murine embryonic stem cells (mESCs) via embryonic bodies (mEBs), as previously described (Wichterle et al., 2002; Haidet-Phillips et al., 2011). The advantage of this approach over existing co-culture methods using post-mortem material or iPSCs, is that iAstrocyte support of motor neurons can be measured in a high-throughput, cost-effective fashion, under a number of conditions including drug treatment or nutritional supplementation. Furthermore, traditional cell-based high throughput screening methodologies that have been utilized in the ALS field have focused on animal or human cell models of the disease in monoculture, for a recent review see (McGown and Stopford, 2018). In the methodological approach adopted here, iAstrocytes can be reproducibly differentiated within a week from iNPCs and their effect on motor neuron survival can be measured in co-culture. Survival can be assessed by monitoring fluorescence of the motor neurons over time and counting the number of viable motor neurons (Meyer et al., 2014; Hautbergue et al., 2017; Allen et al., 2019). Moreover, the effect of treated or untreated iAstrocyte conditioned media on motor neuron survival can also be assessed (Varcianna et al., 2019). Therefore, this approach has the advantage over traditional high throughput monoculture methodologies of being more physiologically representative of the in vivo state and therefore has greater translational potential.



Figure 1. Timeline of the high-throughput human iAstrocyte–Hb9-GFP+ mouse motor neuron co-culture assay

Materials and Reagents

  1. Materials
    1. 10 cm tissue culture plates (Thermo Scientific, catalog number: 150350)
    2. 9 cm Petri dish (Scientific Laboratory Supplies, catalog number: PET2000)
    3. 384-well plates (Greiner-Bio, catalog number: 781091)
    4. 384-well PP source plates (Labcyte, catalog number: PP-0200)
    5. 5 ml Disposable Pipets (Fisher Scientific, catalog number: 13-676-10H)
    6. 10 ml Disposable Pipets (Fisher Scientific, catalog number: 13-676-10J)
    7. 25 ml Disposable Pipets (Fisher Scientific, catalog number: 13-678-11)
    8. 10 µl Pipet Tips (Fisher Scientific, catalog number: 02-707-441)
    9. 200 µl Pipet Tips (Fisher Scientific, catalog number: 02-707-422)
    10. 1,000 µl Pipet Tips (Fisher Scientific, catalog number: 02-707-402)
    11. 15 ml Falcon Tube (Greiner-Bio, catalog number: 188271)
    12. 50 ml Falcon Tube (Greiner-Bio, catalog number: 227261)
    13. 0.2 µm Syringe Filter (Sartorius, catalog number: 16534K)
    14. DMSO resistant foil lids (Brooks life sciences, catalog number: 4ti-0512)
    15. Steritop filter (Millipore, catalog number: S2GPT05RE)
    16. Sterile cell strainer, 40 µm mesh (Fisher Scientific, catalog number: 11587522)
    17. Sterile cell strainer, 70 µm mesh (Corning, catalog number: 431751)

  2. Reagents
    1. Primary Mouse Embryonic Fibroblasts (Sigma-Aldrich, catalog number: PMEF-CF)
    2. Pen-Strep (Lonza, catalog number: DE17-603E)
    3. 10x Trypsin (Lonza, catalog number: BE02-007E)
    4. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148)
    5. Accutase (Gibco, catalog number: 11599686)
    6. B-27 (Gibco, catalog number: 11530536)
    7. BDNF (Peprotec, catalog number: 450-02)
    8. CaCl (Sigma-Aldrich, catalog number: C1016)
    9. CNTF (Peprotec, catalog number: 450-13)
    10. D-(+)-Glucose (Sigma-Aldrich, catalog number: G7021)
    11. DMEM (Lonza, catalog number: 12-741F)
    12. DMEM/F-12, GlutaMAX (Gibco, catalog number: 11524436)
    13. DMSO, anhydrous (Sigma-Aldrich, catalog number: 276855)
    14. DNase I (Applichem Lifescience, catalog number: A3778)
    15. EDTA (Thermo Scientific, catalog number: 17892)
    16. ESC FBS (Gibco, catalog number: 11500526)
    17. FBS (Biosera, catalog number: FB-1090)
    18. FGF (Peprotec, catalogue number: 100-18B)
    19. GDNF (Peprotec, catalog number: 450-10)
    20. Ham’s F-12 Nutrient Mix (Gibco, catalog number: 15172529)
    21. Human Fibronectin (Sigma-Aldrich, catalog number: FC010-10MG)
    22. KCl (Sigma-Aldrich, catalog number: P5405)
    23. Knockout DMEM (Gibco, catalog number: 10389172)
    24. KnockOut Serum Replacement (Gibco, catalog number: 11520366)
    25. L-Cysteine (Sigma-Aldrich, catalog number: C7352)
    26. L-glutamine (Lonza, catalog number: BE-17-605E)
    27. MgSO4 (Sigma-Aldrich, catalog number: M2643)
    28. Mouse LIF (Sigma-Aldrich, catalog number: ESG1106)
    29. N-2 (Gibco, catalog number: 11520536)
    30. NaCl (Fisher Scientific, catalog number: 11904061)
    31. NaH2PO4 (Sigma-Aldrich, catalog number: S3139)
    32. NaHCO3 (Sigma-Aldrich, catalog number: S5761)
    33. Nitrogen (oxygen free) (BOC, catalog number: 44-Y)
    34. Non-Essential Amino Acids (Gibco, catalog number: 12084947)
    35. Papain (Sigma-Aldrich, catalog number: P4762)
    36. PBS Tablets (Thermo Scientific, catalog number: BR0014G)
    37. Retinoic Acid (Sigma-Aldrich, catalog number: R2625)
    38. SAG (Sigma-Aldrich, catalog number: 566660) 
    39. Sterile 1x PBS (see Recipes)
    40. Sterile ultrapure water (see Recipes)
    41. Human iNPC Proliferation Media (see Recipes)
    42. Human iAstrocyte Differentiation Media (see Recipes)
    43. Mouse Embryonic Stem Cell (mESC) Proliferation Media (see Recipes)
    44. Mouse Embryonic Bodies (mEB) Differentiation Media (see Recipes)
    45. Mouse Motor Neuron (mMN) Media (see Recipes)
    46. EB Dissociation Buffer (see Recipes)

Equipment

  1. 4 °C fridge
  2. -20 °C freezer
  3. -80 °C freezer
  4. Autoclave
  5. Class II biosafety cabinet (NuAire, catalog number: NU-437-400E)
  6. CO2 Incubator (Sanyo, catalog number: MCO-19AIC)
  7. Echo 550 liquid handler (Labcyte, catalog number: Echo 550)
  8. Haemocytometer
  9. Harrier 15/80 benchtop centrifuge (MSE, catalog number: MSB.080.CX1.5) 
  10. In Cell Analyzer 2000 (GE Healthcare, catalog number: 52-851714-001)
  11. Liquid nitrogen dewar
  12. Mechanical pipettes (P10, P50, P200, P 1000)
  13. Multichannel mechanical pipettes (P10, P50)
  14. MultiPod Controller (Roylan Developments, catalog number: SPOD0012)
  15. Mechanical pipette gun
  16. PK120 centrifuge with T336 rota (ALC, catalog number: 11200030), and buckets for microplates (ALC, catalog number: 11210267)
  17. Polystyrene float for water bath
  18. StoragePod Enclosure (Roylan Developments, catalog number: SPOD0010)
  19. Water bath
  20. Sterile culture hood

Software

  1. Echo Liquid Handler Software (Labcyte)
  2. Echo Plate Reformat (Labcyte)
  3. In Cell Analyzer 2000 (GE Healthcare)
  4. Columbus Image Data Storage and Analysis System (PerkinElmer)
  5. Excel 2016 (Microsoft)
  6. GraphPad Prism 7.0 (GraphPad)

Procedure

Part I: Hb9-GFP Mouse Embryonic Stem Cell (mESC) Maintenance Protocol

  1. Prepare in advance of mESC maintenance protocol
    Prepare Human iAstrocyte Differentiation Media, Mouse Embryonic Stem Cell (mESC) Proliferation Media, and sterile 1x PBS according to the recipes below in a sterile culture hood.

  2. Defrost Primary Mouse Embryonic Fibroblasts (MEFs) the day before splitting or defrosting Hb9-GFP mESC in a sterile culture hood (unless stated otherwise)
    1. Place Human iAstrocyte Differentiation Media in a 37 °C water bath.
    2. Prepare fibronectin-coated tissue culture plates.
      1. Prepare 18 ml of 2.5 µg/ml Fibronectin Coating Solution per 10 cm plate by adding the 1 mg/ml Human Fibronectin to room temperature PBS in a 1:400 ratio.
      2. Mix the 2.5 µg/ml Fibronectin Coating Solution and transfer 6 ml to each of 3 x 10 cm tissue culture plates using a 10 ml pipet.
      3. Incubate the plates with the 2.5 µg/ml Fibronectin Coating Solution at room temperature for a minimum of 5 min.
    3. Defrost and seed MEFs into Human iAstrocyte Differentiation Media.
      1. Take one vial of MEFs from the liquid nitrogen dewar, and defrost the vial in a 37 °C water bath using a float.
      2. Remove Fibronectin Coating Solution from 10 cm tissue culture plates using a 10 ml pipet, and add 10 ml warm Human iAstrocyte Differentiation Media to each 10 cm tissue culture plate.
      3. Transfer 1 ml of the defrosted MEFs from the vial to 2 ml warm Human iAstrocyte Differentiation Media in a 15 ml Falcon using a P1000 (1 ml) mechanical pipette, mix and then transfer 1 ml of this MEF mixture to each 10 cm tissue culture plate. 
      4. Rock the plates containing the MEFs back and forward, then side to side to mix.
      5. Incubate the plates in a 37 °C/5% CO2 incubator overnight, ready to add defrosted mESC or split mESC the following day.

  3. Defrost or split Hb9-GFP mESC in a sterile culture hood (unless stated otherwise)
    1. Place Mouse Embryonic Stem Cell (mESC) Proliferation Media and 1x Trypsin solution in a 37 °C water bath.
    2. Change the media on the 10 cm plates containing MEFs.
      1. Prepare 45 ml of mESC Proliferation Media + LIF by adding 6 µl LIF to 45 ml warm mESC Proliferation Media in a 50 ml Falcon tube using a P10 (10 µl) mechanical pipette.
      2. Remove Human iAstrocyte Differentiation Media from MEFs using a 10 ml pipet.
      3. Add 15 ml warm mESC Media + LIF to each 10 cm plate containing MEFs using a 25 ml pipet.
      4. Incubate the plates of MEFs in a 37 °C/5% CO2 for a minimum of 30 min, or until mESC are ready to plate.
    3. Defrost and seed mESC onto MEF feeder plates.
      1. Take one vial of mESC from the liquid nitrogen dewar, and defrost the vial in a 37 °C water bath using a float.
      2. Transfer 1 ml of the defrosted MEFs from the vial to 2 ml warm mESC Proliferation Media in a 15 ml Falcon using a P1000 (1 ml) mechanical pipette, mix and then transfer 1 ml of this mESC mixture to each 10 cm plate of MEFs. 
      3. Rock the plates containing the mESC back and forward, then side to side to mix.
      4. Incubate the mESC plates in a 37 °C/5% CO2 incubator.
    4. Alternatively to Step C3, the mESC may already be in culture, and can be split (instead of being defrosted) onto the MEFs (according to the ‘split the mESC’ section below).

  4. Change the media on mESC every day in a sterile culture hood (unless stated otherwise)
    1. Place mESC Proliferation Media in a 37 °C water bath.
    2. Change the media on the 10 cm plates containing mESC.
      1. Prepare 45 ml of mESC Media + LIF by adding 6 µl LIF to 45 ml warm mESC Proliferation Media in a 50 ml Falcon tube using a P10 (10 µl) mechanical pipette.
      2. Remove most of the media from mESCs plates using a 25 ml pipet, leaving a few ml to cover the mESC.
      3. Add 15 ml warm mESC Media + LIF to each mESC plate using a 25 ml pipet.
      4. Incubate the mESC plates in a 37 °C/5% CO2 for 24 h.
      5. Repeat steps 1 and 2a-2d every day.

  5. Split the mESC every 3-4 days in a sterile culture hood
    1. Place mESC Proliferation Media and 1x Trypsin solution in a 37 °C water bath.
    2. Split Hb9-GFP mESC into mEB Differentiation Media in a sterile culture hood (unless stated otherwise).
      1. Remove all mESC Proliferation Medium from mESC plates using a 25 ml pipet, and wash the 10 cm plate of cells in 8 ml room temperature PBS using a 10 ml pipet.
      2. Remove all the PBS wash using a 10 ml pipet, and add 1 ml 1x trypsin to each plate of mESC using a P1000 (1 ml) mechanical pipette.
      3. Incubate the mESC with trypsin in a 37 °C incubator for 5 min.
      4. Incubate longer if necessary-ideally mESC come off in suspension with some MEFs as well.
      5. Add 10 ml mESC Proliferation Media to each plate using a 10 ml pipet.
      6. For each plate of mESC, inspire the mESC and media using a 10 ml pipet, and release the suspension back into the 10 cm tissue culture plate whilst pressing the pipet nozzle to the bottom of the plate and applying pressure. Repeat this step 10 times. This process helps to break up the mESC colonies into single cells.
      7. Transfer the contents of each 10 cm plate into separate, clean 10 cm tissue culture plates using a 10 ml pipet, and incubate in a 37 °C/5% CO2 incubator for 30 min.
      8. For each plate, inspire the mESC suspension and wash the plate a few times with the mESC suspension using a 10 ml pipet, and then transfer all mESC suspension to a 50 ml Falcon tube. Combine up to three 10 cm plates of mESC suspension in one 50 ml Falcon tube.
      9. On the bench, centrifuge the mESC suspension at 200 x g for 4 min at room temperature.
      10. Remove the supernatant using a 25 ml pipet, and then add 4 ml mESC Proliferation Media for each 10 cm plate of mESC that was transferred to the Falcon tube.
      11. Re-suspend the mESC pellet using a 10 ml pipet.
      12. Place a sterile 70 µm cell strainer in a 50 ml Falcon tube, and then transfer the mESC suspension through the strainer using a 10 ml pipet.
      13. Rinse the strainer with 1 ml mESC Proliferation Media.
      14. Add 150-200 µl of the mESC suspension to each 10 cm plate of MEF in mESC Media + LIF using a P200 (200 µl) mechanical pipette.
      15. Rock the plates containing the mESC back and forward, then side to side to mix.
      16. Incubate the mESC plates in a 37 °C/5% CO2 incubator.

Part II: Human iNPC Maintenance Protocol

  1. Prepare in advance of Human iNPC maintenance protocol
    Prepare Human iNPC Proliferation Media and sterile 1x PBS according to the recipes below in a sterile culture hood.

  2. Split iNPC
    1. Place Human iNPC Proliferation Media and 1x Accutase solution in a 37 °C water bath.
    2. Prepare fibronectin-coated tissue culture plates for iNPC maintenance.
      1. Prepare 6 ml of 5 µg/ml Fibronectin Coating Solution per 10 cm tissue culture plate by adding the 1 mg/ml Human Fibronectin to room temperature PBS in a 1:200 ratio.\
      2. Mix the 5 µg/ml Fibronectin Coating Solution and transfer 6 ml to each 10 cm tissue culture plate using a 10 ml pipette.
      3. Incubate the plates with the 5 µg/ml Fibronectin Coating Solution at room temperature for a minimum of 5 min.
    3. Split iNPC into Human iNPC Proliferation Media in a sterile culture hood (unless stated otherwise).
      1. Remove all Human iNPC Proliferation Media from iNPC using a 10 ml pipet, and wash the cells in 5 ml room temperature PBS using a 10 ml pipet.
      2. Remove all the PBS wash using a 10 ml pipet, and add 1 ml Accutase to the iNPC using a P1000 (1 ml) mechanical pipette.
      3. Incubate the iNPC with Accutase in a 37 °C incubator for 4 min.
      4. Gently tap the iNPC plate to dislodge the cells from the plate completely.
      5. Add 5 ml room temperature PBS to the lifted iNPC, mix and transfer to a 15 ml Falcon tube using a 10 ml pipet.
      6. On the bench, centrifuge the iNPCs at 200 x g for 4 min at room temperature.
      7. Pour off the supernatant, and re-suspend the iNPC pellet in an appropriate amount of Human iNPC Proliferation Media.
      8. Remove the Fibronectin Coating Solution from the 10 cm tissue culture plates using a 10 ml pipet.
      9. Add 12 ml warm Human iNPC Proliferation Media to the 10 cm tissue culture plates coated with 5 µg/ml Fibronectin Coating Solution using a 10 ml pipet.
      10. Add an appropriate volume of the re-suspended iNPC solution to the Human iNPC Proliferation Media in the 10 cm tissue culture plates.
      11. Incubate the iNPC proliferation plates in a 37 °C/5% CO2 incubator for 2-4 days, or until the iNPCs reach 80-90% confluency, and then split the iNPCs again as described above.

Part III: Human iAstrocyte-mouse Hb9-GFP+ Motor Neuron Co-culture Protocol

  1. Prepare in advance of co-culture protocol
    1. Prepare Human iNPC Proliferation Media, Human iAstrocyte Differentiation Media, Mouse Embryonic Stem Cell (mESC) Proliferation Media, Mouse Embryonic Bodies (mEB) Differentiation Media, sterile 1x PBS, and sterile ultrapure water according to the recipes below in a sterile culture hood.
    2. Prepare compound library in 100% anhydrous DMSO on 384-well PP source plates, with DMSO resistant foil lid. Store in the StoragePod Enclosure and maintain dry nitrogen atmosphere using the MultiPod Controller.
    3. Write compound transfer protocol using the Echo liquid handler software. Briefly, set the source plate to ‘384PP_DMSO’, set the destination plate to ‘Griener_384PS_781096’, and use a custom mapping mode. Set the protocol to transfer 40 nl of compound in DMSO from the source plate to each well on the destination plate. Given there will be 40 µl media in each well on the destination plate, the final DMSO concentration will be 0.1% (v/v). Set the protocol to deliver 6 technical replicate wells for each test compound, and 12-16 technical replicate wells for the positive control and 12-16 technical replicate wells for the negative control (DMSO only). We discovered a compound in house that increased Hb9-GFP+ mMN survival in the co-culture assay, which we will refer to as compound A, and used that as our positive control. For a comprehensive dose response of select test compounds, we recommend testing a 7-point dose response at every half-log, i.e., 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM.
    4. Write an ‘Hb9-GFP MN’ protocol for imaging the Hb9-GFP+ motor neurons on the In Cell Analyzer 2000 software. Briefly, set the following criteria: objective = Nikon 10x; number of fields = 4 per well; wavelength = FITC (Ex490 nm/Em525 nm); exposure = 0.5 s; focus = laser autofocus at 10% power; deconvolution = enhanced ratio method, 5 cycles; plate temperature = 37 °C. Note that other high-throughput fluorescence microscopes and software could be used instead of the In Cell Analyzer 2000 and In Cell Analyzer 2000 software respectively, however, the imaging protocols would need adapting accordingly.

  2. Differentiate Human iNPC into Human iAstrocytes (Day 0)
    1. Place Human iNPC Proliferation Media, Human iAstrocyte Differentiation Media, Mouse Embryonic Bodies (mEB) Differentiation Media, Mouse Embryonic Stem Cell (mESC) Proliferation Media, 1x Trypsin solution and 1x Accutase solution in a 37 °C water bath.
    2. Prepare fibronectin-coated tissue culture plates for iAstrocyte differentiation.
      1. Prepare 6 ml of 2.5 µg/ml Fibronectin Coating Solution per 10 cm plate by adding the 1 mg/ml Human Fibronectin to room temperature PBS in a 1:400 ratio.
      2. Mix the 2.5 µg/ml Fibronectin Coating Solution and transfer 6 ml to each 10 cm tissue culture plate using a 10 ml pipet.
      3. Incubate the plates with the 2.5 µg/ml Fibronectin Coating Solution at room temperature for a minimum of 5 min.
    3. Prepare fibronectin-coated tissue culture plates for iNPC maintenance (see Human iNPC Maintenance Protocol above).
    4. Split iNPC into Human iAstrocyte Differentiation Media or Human iNPC Proliferation Media in a sterile culture hood (unless stated otherwise).
      1. Remove all Human iNPC Proliferation Media from iNPC using a 10 ml pipet, and wash the cells in 5 ml room temperature PBS using a 10 ml pipet.
      2. Remove all the PBS wash using a 10 ml pipet, and add 1 ml Accutase to the iNPC using a P1000 (1 ml) mechanical pipette.
      3. Incubate the iNPC with Accutase in a 37 °C incubator for 4 min.
      4. Gently tap the iNPC plate to dislodge the cells from the plate completely.
      5. Add 5 ml room temperature PBS to the lifted iNPC, mix and transfer to a 15 ml Falcon tube using a 10 ml pipet.
      6. On the bench, centrifuge the iNPCs at 200 x g for 4 min at room temperature.
      7. Pour off the supernatant, and re-suspend the iNPC pellet in an appropriate amount of Human iNPC Proliferation Media.
      8. Remove the Fibronectin Coating Solution from the 10 cm tissue culture plates using a 10 ml pipet.
      9. Add 12 ml warm Human iAstrocyte Differentiation Media to the 10 cm tissue culture plates coated with 2.5 µg/ml Fibronectin Coating Solution using a 10 ml pipet.
      10. Add 12 ml warm Human iNPC Proliferation Media to the 10 cm tissue culture plates coated with 5 µg/ml Fibronectin Coating Solution using a 10 ml pipet.
      11. Add an appropriate volume of the re-suspended iNPC solution to the Human iAstrocyte Differentiation Media in the 10 cm tissue culture plates.
      12. Add an appropriate volume of the re-suspended iNPC solution to the Human iNPC Proliferation Media in the 10 cm tissue culture plates.
      13. Incubate the iAstrocyte differentiation plates in a 37 °C/5% CO2 incubator for 3 days.
      14. Incubate the iNPC proliferation plates in a 37 °C/5% CO2 incubator for 2-4 days, or until the iNPCs reach 80-90% confluency, and then split the iNPCs as described above.

  3. Differentiate mESC into mEB (Day 0)
    1. Change the media on the 10 cm plates containing MEFs (as described in the Hb9-GFP mESC Maintenance Protocol above).
    2. Split the Hb9 mESC into mEB Differentiation Media in a sterile culture hood (unless stated otherwise).
      1. Split the mESC (as described in the Hb9-GFP mESC Maintenance Protocol above).
      2. Add 18 ml warm mEB Differentiation media to a 9 cm Petri dish, then add 1 ml of the mESC suspension using a P1000 (1 ml) mechanical pipette to start mESC differentiation into motor neurons via mEB. Each 10 cm plate of mEB will yield approximately 4 x 106 Hb9-GFP mouse motor neurons after 7 days of differentiation.
      3. Incubate the plates of mEB in a 37 °C/5% CO2 for 24 h.

  4. Change media on mEBs (Days 1-6)
    1. Place mEB Differentiation Media in a 37 °C water bath.
    2. Change media on EBs.
      1. Transfer mEB and media from 9 cm Petri dish to a 50 ml Falcon using a 25 ml pipet.
      2. Incubate the Falcon tubes for 10 min in a Falcon rack in the hood, allowing the mEB to sink to the bottom of the Falcon tube.
      3. Remove the media in the Falcon from the top downwards, leaving the final 5 ml at the bottom of the Falcon tube that contains the soft pellet of mEBs.
      4. Add 15 ml fresh mEB Differentiation Media to each Falcon tube.
      5. On Days 2-6 only, add 9 µl 4 mM Retinoic Acid (in ethanol) and 18 µl 1 mM SAG to each Falcon tube of mEBs using a P20 (20 µl) mechanical pipette.
      6. Mix the mEB and media using a 25 ml pipet, and then transfer to a fresh 9 cm Petri dish.
      7. Incubate the plates of mEBs in a 37 °C/5% CO2 incubator for 24 h.
      8. Repeat steps 1 and 2a-2g every day until day of EB dissociation.

  5. Change media on iAstrocytes (Day 3)
    1. Place Human iAstrocyte Differentiation Media in a 37 °C water bath.
    2. Remove all Human iAstrocyte Differentiation Media from iAstrocytes using a 10 ml pipet.
    3. Add 12 ml fresh Human iAstrocyte Differentiation Media to each 10 cm tissue culture plate of iAstrocytes.
    4. Incubate the plates in a 37 °C/5% CO2 incubator for 2 days.

  6. Seed iAstrocytes onto 384-well plates (Day 5)
    1. Place Human iAstrocyte Differentiation Media and 1x Accutase solution in a 37 °C water bath.
    2. Prepare fibronectin-coated 384-well plates.
      1. Prepare 2 ml of 2.5 µg/ml Fibronectin Coating Solution per 384-well plate by adding the 1 mg/ml Human Fibronectin to room temperature PBS in a 1:400 ratio.
      2. Mix the 2.5 µg/ml Fibronectin Coating Solution and transfer it to a 9 cm Petri dish to act as a reservoir.
      3. Transfer 5 µl of the Fibronectin Coating Solution to each well of the 384-well plate excluding the outermost wells using a P10 (10 µl) multichannel mechanical pipette.
      4. Transfer 40 µl of PBS to each of the outermost wells on the 384-well plate to act as a firewall using a P50 (50 µl) multichannel mechanical pipette.
      5. Incubate the plates with the 2.5 µg/ml Fibronectin Coating Solution at room temperature for a minimum of 5 min.
    3. Lift iAstrocytes and seed 2,000 iAstrocytes/well onto 384-well plates in a sterile culture hood (unless stated otherwise).
      1. Remove all Human iAstrocyte Differentiation Media from iAstrocytes using a 10 ml pipet, and wash the cells in 5 ml room temperature PBS using a 10 ml pipet.
      2. Remove all the PBS wash using a 10 ml pipet, and add 1 ml Accutase to the iAstrocytes using a P1000 (1 ml) mechanical pipette.
      3. Incubate the iAstrocytes with Accutase in a 37 °C incubator for 4 min.
      4. Gently tap the iAstrocyte plate to dislodge the cells from the plate completely.
      5. Add 5 ml room temperature PBS to the lifted iAstrocyte, mix and transfer to a 15 ml Falcon tube using a 10 ml pipet.
      6. On the bench, centrifuge the iAstrocytes at 200 x g for 4 min at room temperature.
      7. Pour off the supernatant, flick the bottom of the Falcon tube to gently vortex the iAstrocyte pellet, and re-suspend the iAstrocytes in an appropriate amount of Human iAstrocyte Differentiation Media.
      8. Prepare a Haemocytometer, and transfer 10 µl of the iAstrocyte suspension onto the counting grid.
      9. Count the number of iAstrocytes, and calculate the cells/mL in suspension.
      10. Dilute the iAstrocytes to 5.7 x 104 iAstrocytes/ml in Human iAstrocyte Differentiation Media, and transfer the iAstrocyte suspension to a 9 cm Petri dish to act as a reservoir.
      11. Transfer 35 µl of the 5.7 x 104 iAstrocytes/ml suspension to each well of the 384-well plate–excluding the outermost wells–using a P50 (50 µl) multichannel mechanical pipette.
      12. Centrifuge the 384-well plates at 400 x g for 60 s using a PK120 centrifuge with T336 rota and buckets for microplates to collect media and cells to base of wells. 
      13. Incubate the 384-well plates in a 37 °C/5% CO2 incubator for 24 h to allow iAstrocytes to adhere to the plates.

  7. Treat iAstrocytes with compounds (Day 6)
    1. De-pressurise the StoragePod Enclosure using the MultiPod Controller, unlock and take the 384-well PP source plate out of the StoragePod Enclosure.
    2. Calibrate and focus the Echo 550 liquid handler.
    3. Centrifuge the 384-well PP source plate at 1,200 x g for 120 s using a PK120 centrifuge with T336 rota and buckets for microplates to de-gas the compounds in DMSO. 
    4. Survey the 384-well PP source plate containing the compounds in DMSO. There should be adequate volume to deliver compounds from the source plate to the destination plate using the desired compound transfer protocol (described in the ‘prepare in advance of co-culture protocol’ section above). In addition, the compounds should have low water content-below 70% DMSO is unacceptable and drugs should be refreshed on the source plate.
    5. Transfer compounds in DMSO from 384-well PP source plate to 384-well plate’s destination plate containing iAstrocytes using the compound transfer protocol using the Echo 550 liquid handler. The final concentration of DMSO should not exceed 0.5% (v/v) in the iAstrocyte media and should be consistent in all wells in any given experiment. Include DMSO only wells as negative controls.
    6. Centrifuge the 384-well plates at 400 x g for 60 s using a PK120 centrifuge with T336 rota and buckets for microplates to collect media and cells to base of wells. 
    7. Incubate the 384-well plates in a 37 °C/5% CO2 incubator for 24 h.
    8. Put the DMSO resistant foil lid back on the 384-well PP source plate and return to the StoragePod Enclosure, lock and then re-pressurise the StoragePod Enclosure with nitrogen using the MultiPod Controller.

  8. Dissociate mEBs and seed mouse GFP+ motor neurons in co-culture with compound-treated iAstrocytes (Day 7)
    1. Prepare EB Dissociation Buffer according to the recipe below in a sterile tissue culture hood.
    2. Prepare an appropriate volume of Mouse Motor Neuron (mMN) Media according to the recipe below in a sterile tissue culture hood.
    3. Place mMN Media, EB Dissociation Buffer, and FBS in a 37 °C water bath.
    4. Dissociate mEBs
      1. Transfer the mEBs and media from one or two mEB plates into a 50 ml Falcon tube.
      2. On the bench, centrifuge the mEBs at 200 x g for 2 min at room temperature.
      3. Remove the supernatant using a 25 ml pipet, and re-suspend the mEB in 10 ml PBS.
      4. On the bench, centrifuge the mEBs at 200 x g for 2 min at room temperature.
      5. Remove the PBS wash using a 10 ml pipet.
      6. Add 4.75 ml warm EB Dissociation Buffer and 100 µl 200 U/ml Papain to each Falcon of mEBs.
      7. Gently pipette up and down ten times using a P1000 (1 ml) mechanical pipette.
      8. Incubate the Falcon tubes in a 37 °C water bath for 3 min.
      9. Remove the tube and gently shake, then return to the 37 °C water bath for 2 min.
      10. Gently pipette the mEB solution up and down ten times using a P1000 (1 ml) mechanical pipette.
      11. Repeat steps h-j up to 3 more times, or until the solution is cloudy with no large mEB clumps.
      12. Depending on the activity of the papain, more 200 U/ml Papain may need to be added to the Falcons to completely dissociate the mEBs.
      13. Equally, avoid excessive papain treatment because the cells will lyse.
      14. On the bench, centrifuge the mEBs at 300 x g for 5 min at room temperature.
      15. For each 50 ml Falcon tube, prepare 2.7 ml EB Dissociation Buffer and add 300 µl FBS and 150 µl 0.5 mg/ml DNase I using a P200 (200 µl) mechanical pipette, and mix the FBS/DNAse I solution using a 10 ml pipet.
      16. Remove supernatant from the dissociated mEBs using a 10 ml pipet, and add 3 ml of the FBS/DNAse I solution using a 10 ml pipet.
      17. Gently pipette the mEB solution up and down five times using a P1000 (1 ml) mechanical pipette.
      18. Add 5 ml FBS very slowly to the bottom of the 50 ml Falcon containing the dissociated mEBs using a 10 ml pipet. This creates a cushion, and an interface between the mEB solution (upper fraction) and the FBS (lower fraction) should be clearly visible.
      19. On the bench, centrifuge the mEBs at 100 x g for 6 min at room temperature, to pellet the dissociated cells from the mEBs whilst leaving debris in the upper supernatant fraction.
      20. Remove the supernatant using a 10 ml pipet, and then gently re-suspend the cells in an appropriate volume of mMN Media.
      21. Gently pipette the mMN solution up and down five times using a P1000 (1 ml) mechanical pipette.
      22. Place a sterile 40 µm cell strainer in a 50 ml Falcon tube, and then transfer the mMN suspension through the strainer using a 5 ml pipet.
      23. Rinse the strainer with 1 ml mMN Media using a P1000 (1 ml) mechanical pipette.
      24. Prepare a Haemocytometer, and transfer 10 µl of the mMN suspension onto the counting grid.
      25. Count the bright round cells, exclude the darker rough-edged cells and potential debris (Figure 2), and then calculate the neuronal cells/ml in suspension.
      26. Dilute the mMN to 2.5 x 106 neuronal cells/ml in mMN Media, and transfer the mMN suspension to a 9 cm Petri dish to act as a reservoir.
      27. Transfer 10 µl of the 2.5 x 106 neuronal cells/ml suspension to each well of the 384-well plate containing the compound-treated iAstrocytes-excluding the outermost wells-using a P50 (50 µl) multichannel mechanical pipette.
      28. Centrifuge the 384-well plates at 400 x g for 60 s using a PK120 centrifuge with T336 rota and buckets for microplates to collect media and cells to base of wells. 
      29. Incubate the 384-well Human iAstrocyte-Mouse Motor Neuron co-culture plates in a 37 °C/5% CO2 incubator for 24 h.


        Figure 2. Counting cells derived from the Mouse Embryonic Bodies using a haemocytometer. The bright and round cells yielded from the Mouse Embryonic Bodies (mEBs) are included in the neuronal cell count (the four small arrows indicate examples). The darker rough-edged cells and potential debris are excluded from the neuronal cell count (the two large chevrons indicate examples). Scale bar = 100 µm.

  9. Add media to co-culture plates (Day 8)
    1. Prepare an appropriate volume of Mouse Motor Neuron (mMN) Media according to the recipe below in a sterile tissue culture hood.
    2. Place mMN Media in a 37 °C water bath.
    3. Add 15 µl warm mMN Media to each well of the 384-well plate containing the iAstrocyte-MN co-culture-excluding the outermost wells using a P50 (50 µl) multichannel mechanical pipette. 

  10. Image the Hb9-GFP+ MN on a high throughput microscope (Days 8 and 10)
    1. Turn on the illuminator and pre-heat the In Cell Analyzer 2000 to 37 °C.
    2. Once the illuminator is ready, run the ‘Hb9-GFP MN’ protocol (described in the ‘prepare in advance of co-culture protocol’ section above) to image Hb9-GFP+ motor neurons in the co-culture plates using an In Cell Analyzer 2000 (Figure 3). For downstream analysis, the Hb9-GFP+ motor neurons imaged at Days 8 and 10 of the co-culture protocol are simply referred to as Day 1 and 3 respectively.
    3. Incubate the co-culture plates in a 37 °C/5% CO2 incubator.


      Figure 3. Example images of human iAstrocyte-Hb9-GFP+ mMN co-cultures. Representative images of human iAstrocytes and Hb9-GFP+ mMN in co-culture at 1 and 3 days post-mMN seeding, imaged on an In Cell Analyzer 2000 using the GFP channel. iAstrocytes were treated with either DMSO only (-ve control), or compound A (+ve control) 24 h prior to mMN seeding in co-culture. After 3 days of co-culture, there is greater mMN survival in the wells treated with compound A compared to wells treated with DMSO only. Scale bars = 200 µm.

Data analysis

  1. Motor neuron viability assessment
    1. Import the .XCDE/.tif files captured on the In Cell Analyzer 2000 to the Columbus Image Data Storage and Analysis System.
    2. Write an analysis protocol on the Columbus Image Data Storage and Analysis System that can identify and count GFP+ mMN. The analysis protocol is made up of several ‘building blocks’ or steps in the Image Analysis section on the Columbus Image Data Storage and Analysis System, which are detailed and described below: 
      1. Input Image. Here, the images are loaded into the analysis protocol (Figure 4A). Set stack processing as ‘individual planes’ and set no ‘flat field correction’ and no ‘quick tune’.
      2. Find Nuclei. Identify the GFP+ cell bodies of the motor neurons (Figure 4B). Use method ‘M’ on the GFP channel. Method ‘M’ is a robust method provided on the Columbus Image Data Storage and Analysis System used to detect round objects, termed ‘nuclei’, from a range of different types of images.
      3. Calculate Morphology Properties. This step allows the calculation of standard morphological properties such as area or roundness of the GFP+ cell bodies or ‘nuclei’ in the images. Use the ‘standard method’ in this step, which allows the images to be filtered based on cell area size.
      4. Select Population (1). Select the population of GFP+ cell bodies that are greater than 120 pixels2, and also have a width to length ratio of greater than 0.2 (Figure 4C). This filters out small GFP+ items such as debris.
      5. Calculate Intensity Properties. This step allows the calculation of standard intensity properties, such as the average pixel intensity of the GFP+ cell bodies or ‘nuclei’ in the images. Use the ‘standard method’ option in the Columbus Image Data Storage and Analysis System in this step.
      6. Select Population (2). Select the population of GFP+ cell bodies that have an average pixel intensity greater than 2,000 (arbitrary units) (Figure 4D). This filters out dim GFP+ items such as debris.
      7. Find Neurites. This step identifies GFP+ neurites that are growing from the GFP+ cell bodies (defined by steps b-f) (Figure 4E). Use the ‘CSIRO Neurite Analysis 2’ analysis method, which is done by an algorithm developed by the Australian CSIRO research institute. The exact parameters used in this step may require some slight modification for different experimental repeats.
      8. Select Population (3). Select the population of GFP+ cell bodies (defined by steps b-f) that have at least one neurite (defined in step g) connected (Figure 4F). This population is the number of viable GFP+ mMN. GFP+ cell bodies that do not have an axon attached are dead, and therefore filtered out of the viable GFP+ mMN count.
      9. Define Results. This step performs statistical analysis on multiple parameters including the number of GFP+ cell bodies, the number of GFP+ cell bodies with at least one neurite (i.e., viable GFP+ mMN), and other morphological and intensity properties from the images.
    3. Count the number of viable GFP+ mMN in the images from Days 1 and 3 using the analysis protocol described in step 2 using the batch analysis section of the Columbus Image Data Storage and Analysis System.
    4. Export result file to Excel 2016.
    5. Plot the number of viable GFP+ mMN at Day 3 in GraphPad 7.0.


      Figure 4. Analysis protocol on the Columbus Image Data Storage and Analysis System to identify and count GFP+ mMN. A. The images from the GFP channel are loaded into the Columbus Image Analysis software. Scale bar = 200 µm. B. Round GFP+ items are identified and then highlighted in multiple different colors to distinguish the separate items. These Round GFP+ items include the mMN cell bodies. C. GFP+ items that are greater than 120 pixels2 in size are selected (green), whilst GFP+ items that are smaller than 120 pixels2 are excluded (red). D. GFP+ items that have an average intensity greater than 2,000 are selected (green), whilst GFP+ items that have an average pixel intensity of less than 2,000 are excluded (red). E. GFP+ neurites that are growing from the GFP+ cell bodies are identified and then highlighted in multiple different colors to distinguish the separate neurites. F. The number of GFP+ cell bodies with at least one neurite attached (green) are counted, whilst GFP+ cell bodies with no neurites are excluded (red). The area highlighted with the dashed white box within each image is magnified 2x in the box inset at the top right of each image.

  2. Statistical analysis
    The exact statistical analysis required depends on the experimental set-up. Typically, One-way ANOVA with Dunnett’s post-hoc test (comparing each condition to the DMSO control) is performed when screening multiple compounds at a single dose, or multiple doses of a single compound in one iAstrocyte cell line. Statistical analysis is performed in Graphpad 7.0 (Figure 5).


    Figure 5. Example Hb9-GFP+ mMN counts at day 3 from one 384-well plate of the iAstrocyte-Hb9-GFP+ mMN co-culture assay. –ve control = DMSO only, +ve control = compound A.

Notes

  1. Note the lot number of serum used because this will affect the co-culture results. We recommend serum with the same lot number be used for any given batch of work, for example, screening a library of compounds.
  2. The mESC should appear as large bright colonies with clearly defined edges when viewed on a light microscope. If the colonies become darker grey, or have rough or poorly defined edges, the mESC have differentiated and will produce a lower yield of Hb9-GFP+ MNs after differentiation.
  3. The mESC are grown in culture until passage 18, and then replaced with younger passage stocks. This is because when the mESCs reach a higher passage, the yield of Hb9-GFP+ MN they can produce becomes lower.
  4. On Day 7 of the co-culture protocol, observe the mEBs using a green fluorescence microscope. If the mEB have differentiated well, there should be a high percentage of GFP+ cells with a high level of GFP expression. If the mEB are dim, or there is a low level of GFP expression, the MN yield will be low, so it is advisable not to use the mEB in co-culture.
  5. When performing high-throughput drug screening experiments using the iAstrocyte-Hb9-GFP+ MN co-culture assay, we recommend using a minimum of 12 technical replicates (separate wells on the 384-well plate) for both negative controls (DMSO only) and positive controls (compound A identified in house). For experimental conditions, 1 technical replicate is sufficient, but we recommend using 6 technical replicates. Additionally, in secondary validation experiments, we strongly recommend using 6 technical replicates for each experimental condition (and a minimum of 12 technical replicates for the positive and negative controls). A minimum of 3 experimental repeats is also recommended for both high-throughput screens and secondary validation experiments.
  6. When coating the 10 cm tissue culture plates with either the 2.5 or 5.0 µg/ml Fibronectin Coating Solution, there is no optimal length of time for the incubation. However, we recommend a minimum of 5 min at room temperature, or alternatively, overnight at 4 °C.


Recipes

  1. Sterile1x PBS
    Add 5 PBS tablets to 500 ml Milli-Q ultrapure water and autoclave at 121 °C for 15 min
    Allow the PBS to cool to room temperature before using
  2. Sterile ultrapure water
    Autoclave 500 ml Milli-Q ultrapure water at 121 °C for 15 min
    Allow the water to cool to room temperature before using
  3. Human iNPC Proliferation Media
    DMEM/F-12, GlutaMAX
    500 ml
    N-2
    5 ml
    B-27
    5 ml
    4 mg/ml FGF
    5 µl
  4. Human iAstrocyte Differentiation Media
    DMEM
    500 ml
    N-2
    1 ml
    FBS
    50 ml
    Pen-Strep
    5 ml
  5. Mouse Embryonic Stem Cell (mESC) Proliferation Media
    KnockOut DMEM
    400 ml
    ESC FBS
    75 ml
    L-glutamine
    5 ml
    Non-Essential Amino Acids
    5 ml
    2-Mercaptoethanol
    3.6 µl
  6. Mouse Embryonic Bodies (mEB) Differentiation Media
    KnockOut DMEM
    222.5 ml
    Ham’s F-12 Nutrient Mix
    222.5 ml
    KnockOut Serum Replacement
    50 ml
    L-glutamine
    2.5 ml
    30% (w/v) filtered glucose
    2.5 ml
    N-2
    5 ml
    2-Mercaptoethanol
    4 µl
    Filter using a steritop filter
  7. Mouse Motor Neuron (mMN) Media
    mEB Differentiation Media
    40 ml
    200 µg/ml BDNF
    4 µl
    200 µg/ml CNTF
    4 µl
    200 µg/ml GDNF
    4 µl
  8. EB Dissociation Buffer
    Sterile Milli-Q ultrapure water
    31.472 ml
    1 M NaCl
    4.64 ml
    1 M KCl
    0.216 ml
    1 M NaHCO3
    1.04 ml
    0.1 M NaH2PO4
    0.4 ml
    0.1 M CaCl2
    0.6 ml
    0.1 M MgSO4
    0.4 ml
    30% (w/v) filtered glucose
    1 ml
    0.5 M EDTA
    0.04 ml
    25 mg/ml L-Cysteine
    0.192 ml
    Filter using a 0.2 µm Syringe Filter

Acknowledgments

We would like to thank all the ALS patients who have kindly donated samples for this study. Hb9-GFP mESC were a kind gift from Prof. Thomas Jessell. The iNPC reprogramming and iAstrocyte differentiation protocols were developed by Dr Kathrin Meyer and Dr Laura Ferraiuolo in Prof. Brian Kaspar’s laboratory. This work was funded by a Motor Neuron Disease Association Senior Fellowship award to S.P.A. (grant number 956-799, MNDA-registered charity number 294354). L.F. and M.J.S. are funded by BenevolentAI. L.F. is funded by the Academy of Medical Sciences (SBF002\1142).

Competing interests

M.J.S. and L.F. are funded by the biotech company BenevolentAI.

Ethics

Informed consent was obtained from all human subjects before skin sample collection (Study number STH16573, Research Ethics Committee reference 12/YH/0330). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

References

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简介

肌萎缩侧索硬化症(als)是一种以运动神经元丧失为特征的成人神经系统疾病,可导致进行性肌肉萎缩并最终死亡。星形胶质细胞在疾病发病中起着关键作用。然而,由于缺乏可持续的高通量人类细胞模型,研究als中星形胶质细胞对运动神经元的支持能力受到限制。此外,由于缺乏强有力的方法学,评估药物治疗或营养补充如何影响运动神经元的星形胶质细胞支持的能力受到了阻碍。我们开发了一种高通量星形胶质细胞运动神经元共培养实验,通过使用hb9 gfp+运动神经元,研究人员可以评估als如何影响384孔板中星形胶质细胞支持运动神经元的能力。此外,星形胶质细胞功能可以通过营养补充或药物治疗来控制,以确定可能的治疗靶点。
【背景】肌萎缩侧索硬化症(als)是一种导致上下运动神经元变性,导致神经肌肉系统进行性衰竭的神经系统疾病。由于缺乏有效的治疗,死亡通常发生在症状出现后2-3年。虽然运动神经元的死亡是als的一个关键特征,但该疾病是非细胞自主性的,邻近细胞如星形胶质细胞、小胶质细胞和少突胶质细胞起着关键作用(ferraiuolo等,2011b;ferraiuolo等,2016;frakes等,2017;vandoorne等人,2018年)。星形胶质细胞在中枢神经系统中起着至关重要的代谢作用,因为它们是脑糖原的主要来源,而星形胶质细胞乳酸可以被运动神经元吸收并用作能量来源(Pellerin和Magisterti,1994)。星形胶质细胞介导的运动神经元死亡涉及多种机制,包括一氧化氮和前列腺素e2的释放、谷氨酸转运体表达的改变、乳酸释放的减少和mirna胞外小泡介导的释放的减少(lin等,1998;ferraiu等)。Olo等人,2011a;Ferraioulo等人,2011b;Allen等人,2019年;Varcianna等人,2019年)。
直到最近,研究患者来源的星形胶质细胞如何影响运动神经元功能的能力一直受到技术限制的阻碍。初步的开创性研究(Haidet Phillips等人,2011年,Re等人,2014年)表明,人死后散发和家族性患者来源的星形胶质细胞可在体外诱导运动神经元死亡。尽管这种方法很优雅,但它在有限的可用性、可伸缩性方面存在局限性,并且代表了疾病的结束阶段。Yamanaka及其同事(Takahashi和Yamanaka,2006年)开发了将成纤维细胞重新编程为多能干细胞(IPSC)的技术,从根本上提高了我们在体外研究人类中枢神经系统疾病的能力(Myszczynska和Ferriuolo,2016年)。随着这一技术的进步,直接将小鼠成纤维细胞重编程为诱导神经前体细胞(inpcs)的方法学得到了发展,同时Meyer和Ferriuolo开发了一种新的方法,将成年人成纤维细胞从对照组和als组重编程为inpcs。项目(Kim等,2011年;Meyer等,2014年)。直接重编程的这一进展,克服了IPSC克隆变异引起的表型不一致的挑战,显著缩短了重编程时间,降低了成本,增加了吞吐量(Myszczynska和Ferraioulo,2016)。此外,直接重编程方法的另一个有益特点是,细胞的表观遗传状态没有重置,因此供体成纤维细胞固有的任何老化表型都应该保留(Mertens等,2015年)。这一随后的发展使als研究人员能够在体外评估疾病对患者源性细胞的影响,减少对死后组织和可能缺乏转化效率的疾病动物模型的依赖。
在本文所述的共培养方法中,inpcs来源于先前所述的als患者成纤维细胞和健康供体对照成纤维细胞(meyeret al,2014)。inpcs通过在iastrocyte培养基中培养至少5天而分化为iastrocytes(图1)。如前所述,在HB9运动神经元特异性启动子(从现在起称为HB9 gfp+mn)下表达绿色荧光蛋白(gfp)的小鼠运动神经元通过胚胎体(mebs)与小鼠胚胎干细胞(mescs)分化(witterle等,2002;haidet)。-Phillips等人,2011年)。与使用死后材料或ipscs的现有共培养方法相比,这种方法的优势在于,在包括药物治疗或营养补充在内的多种条件下,可以以高通量、成本效益高的方式测量运动神经元的iastrocyte支持。此外,ALS领域中使用的基于细胞的高通量筛选方法侧重于单一培养中疾病的动物或人类细胞模型,最近的综述见(McGown和Stopford,2018)。在这里采用的方法学方法中,iastrocytes可以在一周内与inpcs重复分化,并且它们对运动神经元存活的影响可以在共培养中测量。存活率可以通过监测运动神经元随时间变化的荧光和计算存活运动神经元的数量来评估(Meyeret al,2014;Hautbergueet al,2017;Allenet al,2019)。此外,也可以评估经处理或未经处理的iastrocyte条件培养基对运动神经元存活的影响(varcianna等,2019年)。因此,与传统的高通量单一培养方法相比,该方法具有更具体内状态生理学代表性的优点,因此具有更大的翻译潜力。

关键字:ALS, 星形胶质细胞, 运动神经元, 药物开发, 高通量药物筛选

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图1。高通量人iastrocyte-hb9-gfp+小鼠运动神经元共培养实验的时间轴

材料和试剂

  1. 材料
    1. 10cm组织培养板(Thermo Scientific,目录号:150350)
    2. 9cm培养皿(科学实验室用品,目录号:PET2000)
    3. 384个井板(Greiner Bio,目录号:781091)
    4. 384井聚丙烯源板(Labcyte,目录号:PP-0200)
    5. 5毫升一次性吸管(Fisher Scientific,目录号:13-676-10H)
    6. 10毫升一次性吸管(Fisher Scientific,目录号:13-676-10J)
    7. 25毫升一次性吸管(Fisher Scientific,目录号:13-678-11)
    8. 10微升吸管头(Fisher Scientific,目录号:02-707-441)
    9. 200微升吸管头(Fisher Scientific,目录号:02-707-422)
    10. 1000微升吸管头(Fisher Scientific,目录号:02-707-402)
    11. 15毫升Falcon试管(Greiner Bio,目录号:188271)
    12. 50毫升Falcon试管(Greiner Bio,目录号:227261)
    13. 0.2微米注射器过滤器(Sartorius,目录号:16534K)
    14. 耐二甲基亚砜箔盖(布鲁克斯生命科学,目录号:4TI-0512)
    15. 硬顶过滤器(微孔,目录号:S2GPt05re)
    16. 无菌细胞过滤器,40μm筛孔(Fisher Scientific,目录号:11587522)
    17. 无菌细胞过滤器,70μm网(康宁,目录号:431751)
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  2. 试剂
    1. 原代小鼠胚胎成纤维细胞(Sigma-Aldrich,目录号:PMEF-CF)
    2. 笔杆(Lonza,目录号:DE17-603E)
    3. 10x胰蛋白酶(lonza,目录号:BE02-007E)
    4. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
    5. Accutase(Gibco,目录号:11599686)
    6. B-27(Gibco,目录号:11530536)
    7. bdnf(peprotec,目录号:450-02)
    8. CACL(Sigma-Aldrich,目录号:C1016)
    9. CNTF(PEPROTEC,目录号:450-13)
    10. D-(+)-葡萄糖(Sigma-Aldrich,目录号:G7021)
    11. DMEM(Lonza,目录号:12-741F)
    12. DMEM/F-12,谷氨酰胺(Gibco,目录号:11524436)
    13. 无水二甲基亚砜(Sigma-Aldrich,目录号:276855)
    14. DNA酶I(苹果生命科学,目录号:A3778)
    15. EDTA(热科学,目录号:17892)
    16. 电子稳定控制系统FBS(Gibco,目录号:11500526)
    17. FBS(Biosra,目录号:FB-1090)
    18. FGF(PEPROTEC,目录号:100-18B)
    19. GDNF(PEPROTEC,目录号:450-10)
    20. 火腿F-12营养素混合物(Gibco,目录号:15172529)
    21. 人纤维粘连蛋白(sigma-aldrich,目录号:fc010-10mg)
    22. KCL(Sigma-Aldrich,目录号:P5405)
    23. 淘汰DMEM(Gibco,目录号:10389172)
    24. 敲除血清置换(GIGBO,目录号:11520366)
    25. L-半胱氨酸(Sigma-Aldrich,目录号:C7352)
    26. L-谷氨酰胺(Lonza,目录号:BE-17-605E)
    27. MGSO4(Sigma-Aldrich,目录号:M2643)
    28. 鼠标LIF(Sigma-Aldrich,目录号:ESG1106)
    29. N-2(Gibco,目录号:11520536)
    30. NaCl(Fisher Scientific,目录号:11904061)
    31. NaH2Po4(Sigma-Aldrich,目录号:S3139)
    32. NAHCO3(Sigma-Aldrich,目录号:S5761)
    33. 氮(无氧)(BOC,目录号:44-Y)
    34. 非必需氨基酸(Gibco,目录号:12084947)
    35. 木瓜蛋白酶(Sigma-Aldrich,目录号:P4762)
    36. PBS片剂(Thermo Scientific,目录号:BR0014G)
    37. 维甲酸(Sigma-Aldrich,目录号:R2625)
    38. SAG(Sigma-Aldrich,目录号:566660)
    39. 无菌1X PBS(见配方)
    40. 无菌超纯水(见配方)
    41. 人类inpc增殖培养基(见配方)
    42. 人iastrocyte分化培养基(见配方)
    43. 小鼠胚胎干细胞(mesc)增殖培养基(见配方)
    44. 小鼠胚胎体(MEB)分化培养基(见配方)
    45. 小鼠运动神经元(mmn)培养基(见配方)
    46. EB解离缓冲液(见配方)

设备

  1. 4°C冰箱
  2. -20°C冷冻柜
  3. -80°C冷冻柜
  4. 高压灭菌器
  5. 二级生物安全柜(Nuaire,目录号:NU-437-400E)
  6. CO2孵化器(三洋,目录号:MCO-19AIC)
  7. Echo 550液体处理器(Labcyte,目录号:Echo 550)
  8. 血细胞仪
  9. Harrier 15/80台式离心机(MSE,目录号:msb.080.cx1.5)
  10. 2000细胞内分析仪(GE Healthcare,目录号:52-851714-001)
  11. 液氮杜瓦
  12. 机械移液管(p10,p50,p200,p 1000)
  13. 多通道机械移液管(p10,p50)
  14. 多功能控制器(Roylan开发,目录号:SPOD0012)
  15. 机械移液枪
  16. PK120离心机,配有T336旋转式离心机(ALC,目录号:11200030)和微孔板吊桶(ALC,目录号:11210267)
  17. 水浴用聚苯乙烯浮子
  18. 存储舱外壳(Roylan开发,目录号:SPOD0010)
  19. 水浴
  20. 无菌培养罩

软件

  1. 回声液体处理软件(Labcyte)
  2. 回声板重整术(Labcyte)
  3. 2000细胞内分析仪(GE Healthcare)
  4. 哥伦布图像数据存储与分析系统(Perkinelmer)
  5. Excel 2016(微软)
  6. graphpad棱镜7.0(graphpad)

程序

第一部分:hb9-gfp小鼠胚胎干细胞(mesc)维持方案
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  1. 提前准备mesc维护协议
    根据以下配方,在无菌培养箱中制备人iastrocyte分化培养基、小鼠胚胎干细胞(mesc)增殖培养基和无菌1x pbs。
  2. 在无菌培养箱中分离或解冻hb9-gfp-mesc前一天解冻原代小鼠胚胎成纤维细胞(mefs)(除非另有说明)
    1. 将人iastrocyte分化培养基置于37℃水浴中。
    2. 制备纤维连接蛋白涂层组织培养板。
      1. 以1:400的比例向室温PBS中添加1 mg/ml人纤维粘连蛋白,制备每10 cm板18 ml 2.5μg/ml纤维粘连蛋白涂层溶液。
      2. 混合2.5微克/毫升纤维粘连蛋白涂层溶液,并使用10毫升移液管将6毫升移到3 x 10厘米的组织培养板上。
      3. 在室温下,用2.5μg/ml纤维连接蛋白涂层溶液培养平板至少5分钟。
    3. 将mefs解冻并接种到人iastrocyte分化培养基中。
      1. 从液氮杜瓦中取出一瓶mefs,用浮子在37℃水浴中解冻。
      2. 用10毫升移液管从10厘米的组织培养板上移去纤维粘连蛋白涂层溶液,并在每个10厘米的组织培养板上加入10毫升温热的人iastrocyte分化培养基。
      3. 使用p1000(1 ml)机械移液管将1 ml解冻的mef从小瓶移到15 ml Falcon中的2 ml温热的人造血干细胞分化培养基中,混合,然后将1 ml该mef混合物移到每个10 cm组织培养板上。
      4. 前后摇动装有MEF的盘子,然后左右混合。
      5. 在37℃/5%co2培养箱中培养过夜,准备在第二天添加解冻的mesc或分离的mesc。
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  3. 在无菌培养箱中解冻或分离HB9 gfp-mesc(除非另有说明)
    1. 将小鼠胚胎干细胞(mesc)增殖培养基和1x胰蛋白酶溶液置于37℃水浴中。
    2. 更换含有MEF的10cm板上的介质。
      1. 用p10(10微升)机械吸管将6微升lif添加到50毫升Falcon试管中的45毫升热mesc增殖培养基中,制备45毫升mesc增殖培养基+lif。
      2. 用10毫升移液管从mefs中取出人iastrocyte分化培养基。
      3. 用25毫升移液管将15毫升热mesc培养基+lif加入含有mefs的10厘米板中。
      4. 在37℃/5%co2中培养mefs板至少30分钟,或直到mesc准备好进行培养。
    3. 在MEF进料板上解冻和播种MESC。
      1. 从液氮杜瓦中取出一瓶mesc,用浮子在37℃水浴中解冻。
      2. 使用p1000(1 ml)机械移液管将1 ml解冻的mef从小瓶移到15 ml Falcon中的2 ml温热的人造血干细胞分化培养基中,混合,然后将1 ml该mef混合物移到每个10 cm组织培养板上。
      3. 前后摇动装有MEF的盘子,然后左右混合。
      4. 在37℃/5%co2培养箱中培养过夜,准备在第二天添加解冻的mesc或分离的mesc。
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  4. 在无菌培养箱中解冻或分离HB9 gfp-mesc(除非另有说明)
    1. 将小鼠胚胎干细胞(mesc)增殖培养基和1x胰蛋白酶溶液置于37℃水浴中。
    2. 更换含有MEF的10cm板上的介质。
      1. 用p10(10微升)机械吸管将6微升lif添加到50毫升Falcon试管中的45毫升热mesc增殖培养基中,制备45毫升mesc增殖培养基+lif。
      2. 用10毫升移液管从mefs中取出人iastrocyte分化培养基。
      3. 用25毫升移液管将15毫升热mesc培养基+lif加入含有mefs的10厘米板中。
      4. 在37℃/5%co2中培养mefs板至少30分钟,或直到mesc准备好进行培养。
    3. 在MEF进料板上解冻和播种MESC。
      1. 从液氮杜瓦中取出一瓶mesc,用浮子在37℃水浴中解冻。
      2. 使用p1000(1 ml)机械移液管将1 ml解冻的mefs从小瓶转移到15 ml Falcon中的2 ml热mesc增殖培养基中,混合,然后将1 ml该mesc混合物转移到每个10 cm的mefs板上。
      3. 前后摇动含有mesc的盘子,然后左右混合。
      4. 在37℃/5%co2培养箱中培养mesc板。
    4. 或者,到步骤c3,mesc可能已经在培养物中,并且可以被分离(而不是解冻)到mefs上(根据下面的“分离mesc”部分)。
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  5. 每天在无菌培养箱中更换mesc上的培养基(除非另有说明)
    1. 将mesc增殖培养基置于37℃水浴中。
    2. 更换含有mesc的10cm板上的介质。
      1. 用p10(10微升)机械吸管将6微升lif添加到50毫升Falcon试管中的45毫升热mesc增殖培养基中,制备45毫升mesc培养基+lif。
      2. 用25毫升移液管从mesc板上移走大部分培养基,留下几毫升盖住mesc。
      3. 用25毫升移液管向每个mesc板中加入15毫升热mesc培养基+lif。
      4. 在37℃/5%co2中培养mesc板24小时。
      5. 每天重复步骤1和2a-2d。
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  6. 每隔3-4天在无菌培养箱中分离mesc
    1. 将mesc增殖培养基和1x胰蛋白酶溶液置于37°C水浴中。
    2. 在无菌培养箱中将hb9-gfp-mesc分为meb分化培养基(除非另有说明)。
      1. 用25ml移液管从mesc培养皿中取出所有mesc增殖培养基,用10ml移液管在8ml室温pbs中清洗10cm培养皿。
      2. 用10毫升移液管移走所有PBS清洗液,并用p1000(1毫升)机械移液管将1毫升1x胰蛋白酶添加到每个mesc板上。
      3. 在37°C培养箱中用胰蛋白酶培养mesc 5分钟。
      4. 如有必要,培养更长时间,理想情况下,mesc与一些mef混悬。
      5. 用10毫升移液管向每个培养皿中加入10毫升的mesc增殖培养基。
      6. 对于每个mesc板,使用10毫升吸管激发mesc和培养基,并将悬浮液释放回10厘米组织培养板中,同时将吸管喷嘴压到板的底部并施加压力。重复此步骤10次。这个过程有助于将mesc菌落分裂成单个细胞。
      7. 将每个10cm培养板的内容物转移到单独的、干净的10cm组织培养板中,使用10ml移液管,并在37℃/5%co2培养箱中培养30分钟。
      8. 对于每个板,激发mesc悬浮液,并使用10ml移液管用mesc悬浮液清洗板几次,然后将所有mesc悬浮液转移到50ml falcon管中。在一个50毫升的Falcon试管中混合三个10厘米厚的mesc悬浮液。
      9. 在试验台上,在室温下以200x g离心mesc悬浮液4分钟。
      10. 用25毫升移液管移走上清液,然后为转移到Falcon试管中的每10厘米的mesc板添加4毫升mesc增殖培养基。
      11. 用10毫升移液管重新悬浮mesc颗粒。
      12. 将无菌70μm细胞过滤器放入50 ml Falcon管中,然后使用10 ml吸管通过过滤器转移mesc悬浮液。
      13. 用1毫升mesc增殖培养基冲洗过滤器。
      14. 使用p200(200微升)机械移液管将150-200微升的mesc悬浮液添加到mesc media+lif中的每10 cm mef板中。
      15. 前后摇动含有mesc的盘子,然后左右混合。
      16. 在37℃/5%co2培养箱中培养mesc板。
      < BR>

第二部分:人类inpc维护协议
< BR>

  1. 预先准备人类inpc维护协议
    根据以下配方在无菌培养箱中制备人inpc增殖培养基和无菌1x pbs。
  2. 分裂inpc
    1. 将人inpc增殖培养基和1x Accutase溶液置于37°C水浴中。
    2. 制备纤维粘连蛋白涂层组织培养板,用于inpc的维护。
      1. 以1:200的比例向室温PBS中加入1 mg/ml人纤维粘连蛋白,制备每10 cm组织培养板6 ml的5μg/ml纤维粘连蛋白涂层溶液。\
      2. 混合5微克/毫升纤维粘连蛋白涂层溶液,并使用10毫升移液管将6毫升移到每个10厘米的组织培养板上。
      3. 在室温下,用5μg/ml纤维连接蛋白涂层溶液培养平板至少5分钟。
    3. 在无菌培养箱中将inpc分成人类inpc增殖培养基(除非另有说明)。
      1. 用10毫升移液管从inpc中取出所有人inpc增殖培养基,用10毫升移液管在5毫升室温pbs中清洗细胞。
      2. 用10毫升移液管去除所有PBS清洗液,用p1000(1毫升)机械移液管向INPC添加1毫升Accutase。
      3. 用Accutase在37°C培养箱中培养INPC 4分钟。
      4. 轻轻敲击INPC板,将电池从板上完全取出。
      5. 向提升的inpc中加入5ml室温pbs,混合并使用10ml移液管转移到15ml falcon管中。
      6. 在试验台上,在200x g下,在室温下离心inpcs 4分钟。
      7. 倒出上清液,将inpc颗粒重新悬浮在适量的人inpc增殖培养基中。
      8. 用10毫升移液管从10厘米的组织培养板中取出纤维粘连蛋白涂层溶液。
      9. 用10毫升移液管将12毫升温热的人inpc增殖培养基加入10厘米的组织培养板中,该培养板涂有5微克/毫升纤维连接蛋白涂层溶液。
      10. 将再悬浮的inpc溶液适量加入10cm组织培养板中的人inpc增殖培养基中。
      11. 将inpc增殖板在37℃/5%co2培养箱中培养2-4天,或直到inpc达到80-90%的融合率,然后按上述方法再次分裂inpc。
        < BR>

第三部分:人iastrocyte小鼠hb9 gfp+运动神经元共培养方案 < BR>

  1. 预先准备共培养方案
    1. 按照以下配方在无菌培养箱中制备人inpc增殖培养基、人iastrocyte分化培养基、小鼠胚胎干细胞(mesc)增殖培养基、小鼠胚胎体(meb)分化培养基、无菌1x pbs和无菌超纯水。
    2. 用100%无水二甲基亚砜在384孔聚丙烯源板上制备化合物库,并盖上抗二甲基亚砜箔盖。储存在储存箱外壳中,并使用多功能控制器保持干燥的氮气环境。
    3. 使用回声液体处理器软件编写复合传输协议。简单地说,将源板设置为“384pp_dmso”,将目标板设置为“griener_384ps_781096”,并使用自定义映射模式。设置协议,将40 NL的DMSO化合物从源板转移到目的板上的每个井。考虑到目的板上的每口井将有40微升的介质,最终DMSO浓度将为0.1%(v/v)。设定方案为每个试验化合物提供6个技术复制井,阳性对照组提供12-16个技术复制井,阴性对照组提供12-16个技术复制井(仅DMSO)。我们在室内发现了一种化合物,在共培养试验中增加了hb9 gfp+mmn的存活率,我们称之为化合物a,并将其作为阳性对照。对于所选测试化合物的综合剂量响应,我们建议在每半对数,即、0.01、0.03、0.1、0.3、1、3和10μm处测试7点剂量响应。
    4. 在细胞内分析仪2000软件上编写一个“hb9-gfp-mn”协议来成像hb9-gfp+运动神经元。简单地说,设置以下标准:目标=尼康10x;场数=每孔4个;波长=FITC(EX490 nm/EM525 nm);曝光=0.5 s;聚焦=10%功率下的激光自动聚焦;反褶积=增强比率法,5个周期;板温=37°C。注意,其他高通量fluoRescence显微镜和软件可以分别代替细胞内分析仪2000和细胞内分析仪2000软件,但是成像协议需要相应的调整。
    < BR>
  2. 将人inpc分化为人iastrocytes(第0天)
    1. 将人inpc增殖培养基、人iastrocyte分化培养基、小鼠胚胎体(meb)分化培养基、小鼠胚胎干细胞(mesc)增殖培养基、1份胰蛋白酶溶液和1份淀粉酶溶液置于37℃水浴中。
    2. 制备纤维连接蛋白涂层的组织培养板,用于细胞分化。
      1. 以1:400的比例向室温PBS中添加1 mg/ml人纤维粘连蛋白,制备每10 cm板6 ml 2.5μg/ml纤维粘连蛋白涂层溶液。
      2. 将2.5微克/毫升纤维粘连蛋白涂层溶液混合,用10毫升移液管将6毫升移到每个10厘米的组织培养板上。
      3. 在室温下,用2.5μg/ml纤维连接蛋白涂层溶液培养平板至少5分钟。
    3. 准备纤维粘连蛋白涂层的组织培养板用于inpc的维护(见上面的人类inpc维护协议)。
    4. 在无菌培养箱中将inpc分为人iastrocyte分化培养基或人inpc增殖培养基(除非另有说明)。
      1. 用10毫升移液管从inpc中取出所有人inpc增殖培养基,用10毫升移液管在5毫升室温pbs中清洗细胞。
      2. 用10毫升移液管去除所有PBS清洗液,用p1000(1毫升)机械移液管向INPC添加1毫升Accutase。
      3. 用Accutase在37°C培养箱中培养INPC 4分钟。
      4. 轻轻敲击INPC板,将电池从板上完全取出。
      5. 向提升的inpc中加入5ml室温pbs,混合并使用10ml移液管转移到15ml falcon管中。
      6. 在试验台上,在200x g下,在室温下离心inpcs 4分钟。
      7. 倒出上清液,将inpc颗粒重新悬浮在适量的人inpc增殖培养基中。
      8. 用10毫升移液管从10厘米的组织培养板中取出纤维粘连蛋白涂层溶液。
      9. 用10毫升移液管将12毫升温热的人iastrocyte分化培养基加入涂有2.5微克/毫升纤维连接蛋白涂层溶液的10厘米组织培养板中。
      10. 用10毫升移液管将12毫升温热的人inpc增殖培养基加入10厘米的组织培养板中,该培养板涂有5微克/毫升纤维连接蛋白涂层溶液。
      11. 在10cm的组织培养板中加入适量的再悬浮inpc溶液到人iastrocyte分化培养基中。
      12. 将再悬浮的inpc溶液适量加入10cm组织培养板中的人inpc增殖培养基中。
      13. 将iastrocyte分化板在37℃/5%co2培养箱中培养3天。
      14. 将inpc增殖板在37℃/5%co2培养箱中培养2-4天,或直到inpc达到80-90%的融合率,然后如上所述分离inpc。
    < BR>
  3. 将mesc区分为meb(第0天)
    1. 更换含有mefs的10cm板上的介质(如上述hb9 gfp mesc维护协议所述)。
    2. 在无菌培养箱中将hb9-mesc分成meb分化培养基(除非另有说明)。
      1. 拆分mesc(如上hb9 gfp mesc维护协议所述)。
      2. 在9cm培养皿中加入18ml温性meb分化培养基,然后用p1000(1ml)机械吸管加入1ml mesc悬液,通过meb开始mesc向运动神经元分化。分化7天后,每10cm meb板将产生约4x 106hb9 gfp小鼠运动神经元。
      3. 将meb培养板在37℃/5%co2中培养24小时。
      < BR>
  4. 在MEB上更换介质(第1-6天)
    1. 将meb分化培养基置于37℃水浴中。
    2. 在EBS上更换媒体。
      1. 用25毫升移液管将meb和培养基从9厘米培养皿转移到50毫升falcon。
      2. 将猎鹰管在罩内的猎鹰架中孵育10分钟,使MEB沉入猎鹰管底部。
      3. 从顶部向下取出Falcon中的培养基,将最后的5毫升培养基留在Falcon试管底部,该试管含有MEB软颗粒。
      4. 每根falcon管加入15ml新鲜meb分化培养基。
      5. 仅在第2-6天,使用p20(20微升)机械移液管将9微升4毫米维甲酸(在乙醇中)和18微升1毫米凹形液添加到每个Falcon试管中。
      6. 用25毫升移液管将meb和培养基混合,然后转移到新鲜的9厘米培养皿中。
      7. 将mebs培养皿在37℃/5%co2培养箱中培养24小时。
      8. 每天重复步骤1和2a-2g,直到eb分离。
    < BR>
  5. 更换iastrocytes培养基(第3天)
    1. 将人iastrocyte分化培养基置于37℃水浴中。
    2. 用10毫升移液管将所有人iastrocyte分化培养基从iastrocytes中移走。
    3. 将12毫升新鲜人iastrocyte分化培养基加入每10厘米的iastrocyte组织培养板中。
    4. 将培养板在37°C/5%Co2培养箱中培养2天。
    < BR>
  6. 在384孔板上接种iastrocytes(第5天)
    1. 将人iastrocyte分化培养基和1x Accutase溶液置于37°C水浴中。
    2. 制备纤维连接蛋白涂层384孔板。
      1. 以1:400的比例向室温PBS中加入1 mg/ml人纤连蛋白,制备每384孔板2 ml的2.5μg/ml纤连蛋白涂层溶液。
      2. 混合2.5微克/毫升纤维粘连蛋白涂层溶液,并将其转移到9厘米的培养皿中作为贮存器。
      3. 使用p10(10微升)多通道机械吸管将5微升纤维连接蛋白涂层溶液转移到384孔板的每个孔中,最外层的孔除外。
      4. 使用P50(50微升)多通道机械吸管,将40微升的PBS转移到384井板上的每一个最外面的井,用作防火墙。
      5. 在室温下,用2.5μg/ml纤维连接蛋白涂层溶液培养平板至少5分钟。
    3. 将iastrocytes和种子2000 iastrocytes/孔提升到无菌培养罩中的384孔板上(除非另有说明)。
      1. 用10毫升移液管将所有人iastrocyte分化培养基从iastrocytes中移走,用10毫升移液管在5毫升室温pbs中清洗细胞。
      2. 用10毫升移液管移走所有PBS冲洗液,用p1000(1毫升)机械移液管向iAstrocels中加入1毫升Accutase。
      3. 用Accutase在37°C培养箱中培养iAstrocels 4分钟。
      4. 轻轻敲击iAstrocyte板,将细胞从板上完全取出。
      5. 将5毫升室温PBS添加到提升的iAstrocyte中,混合并使用10毫升移液管转移到15毫升Falcon试管中。
      6. 在试验台上,室温下,以200x g离心4分钟。
      7. 倒出上清液,轻拍Falcon试管底部,轻轻搅动iastrocyte小球,在适量的人iastrocyte分化培养基中重新悬浮iastrocytes。
      8. 制备血细胞仪,并将10微升的iAstrocyte悬浮液转移到计数网格上。
      9. 计算细胞数,计算悬浮液中细胞数/ml。
      10. 在人iastrocyte分化培养基中将iastrocytes稀释至5.7x 104iastrocytes/ml,并将iastrocyte悬液移入9cm培养皿中作为贮存器。
      11. 使用p50(50微升)多通道机械移液管将35微升5.7 x 104iAstrocels/ml悬浮液转移至384孔板的每个孔(不包括最外面的孔)。
      12. 在400x g下使用带有T336旋转装置的PK120离心机和微型板桶将384孔板离心60秒,以将介质和细胞收集到井底。
      13. 将384孔板在37℃/5%co2的培养箱中培养24小时,使iAstrocels粘附在板上。
        < BR>
  7. 用化合物治疗iastrocytes(第6天)
    1. 使用multipod控制器对storagepod机柜减压,解锁并将384井pp源板从storagepod机柜中取出。
    2. 校准并聚焦回声550液体处理器。
    3. 在1200x g下,使用带有T336旋转装置的PK120离心机和用于微孔板的桶将384井PP源板离心120 s,以在DMSO中去除化合物中的气体。
    4. 对384井含dmso化合物的pp源板进行了调查。应具有足够的体积,以使用所需的化合物转移协议(如上文“在共培养协议之前制备”一节所述)将化合物从源板传送到目的板。此外,该化合物的水分含量应低于70%DMSO是不可接受的,药物应在源板上更新。
    5. 使用Echo 550液体处理器,使用复合转移协议,将DMSO中的化合物从384井PP源板转移到384井板的含有iAstrocels的目的板。dmso在iastrocyte培养基中的最终浓度不应超过0.5%(v/v),并且在任何给定的实验中,所有井中的dmso浓度应一致。仅包括DMSO井作为阴性对照。
    6. 在400x g下使用带有T336旋转装置的PK120离心机和微型板桶将384孔板离心60秒,以将介质和细胞收集到井底。
    7. 将384孔板在37℃/5%co2培养箱中培养24小时。
    8. 将防DMSO箔盖放回384井PP源板上,然后返回存储箱外壳,锁定,然后使用多功能控制器用氮气重新加压存储箱外壳。
    < BR>
  8. 用复合处理的iastrocytes分离mebs和小鼠gfp+运动神经元(第7天)
    1. 根据以下配方在无菌组织培养罩中制备EB分离缓冲液。
    2. 根据以下配方,在无菌组织培养罩中制备适当体积的小鼠运动神经元(mmn)培养基。
    3. 将mmn介质、eb解离缓冲液和fbs置于37°c水浴中。
    4. 分离mebs
      1. 将MEB和介质从一个或两个MEB板转移到一个50毫升的Falcon管中。
      2. 在试验台上,在200x g下,在室温下离心mebs 2分钟。
      3. 用25毫升移液管移走上清液,并在10毫升PBS中重新悬浮MEB。
      4. 在试验台上,在200x g下,在室温下离心mebs 2分钟。
      5. 用10毫升移液管移走PBS清洗液。
      6. 将4.75 ml热eb解离缓冲液和100微升200 u/ml木瓜蛋白酶添加到每个mebs的falcon中。
      7. 用p1000(1毫升)机械吸管轻轻上下吸管十次。
      8. 将Falcon试管在37°C水浴中培养3分钟。
      9. 取下管子并轻轻摇动,然后返回37°C水浴2分钟。
      10. 用p1000(1毫升)机械吸管轻轻地上下吸管MEB溶液十次。
      11. 重复步骤h-j至多3次,或直到溶液浑浊且没有大的meb团。
      12. 根据木瓜蛋白酶的活性,可能需要向猎鹰中添加200 u/ml的木瓜蛋白酶,以完全分离mebs。
      13. 同样,避免过度的木瓜蛋白酶处理,因为细胞会溶解。
      14. 在试验台上,在300x g下,在室温下离心5分钟。
      15. 对于每50ml Falcon试管,制备2.7 ml EB解离缓冲液,并使用P200(200微升)机械移液管添加300微升FBS和150微升0.5 mg/ml DNA酶I,并使用10 ml移液管混合FBS/DNA酶I溶液。
      16. 用10毫升移液管从分离的mebs中除去上清液,并用10毫升移液管添加3毫升fbs/dnase i溶液。
      17. 用p1000(1毫升)机械吸管轻轻上下吸管五次。
      18. 用10毫升移液管将5毫升FBS非常缓慢地添加到含有分离的MEB的50毫升Falcon的底部。这就产生了缓冲,meb溶液(上部)和fbs(下部)之间的界面应该清晰可见。
      19. 在试验台上,在室温下将mebs在100x g下离心6min,使分离的细胞颗粒化,同时在上清液部分留下碎片。
      20. 用10毫升移液管移走上清液,然后在适当体积的mmn培养基中轻轻地重新悬浮细胞。
      21. 用p1000(1毫升)机械吸管轻轻地上下吸管mmn溶液五次。
      22. 将无菌的40μm细胞过滤器放入50 ml Falcon管中,然后使用5 ml吸管通过过滤器转移mmn悬浮液。
      23. 使用p1000(1 ml)机械吸管用1 ml mmn介质冲洗过滤器。
      24. 制备血细胞仪,并将10微升mmn悬浮液转移到计数网格上。
      25. 计数明亮的圆形细胞,排除较暗的粗糙边缘细胞和潜在碎片(图2),然后计算悬浮的神经元细胞/ml。
      26. 在mmn培养基中将mmn稀释至2.5x 106neuronal cells/ml,并将mmn悬液移入9cm培养皿中作为贮存器。
      27. 使用p50(50微升)多通道机械吸管将10微升2.5 x 106神经元细胞/ml悬浮液转移到384孔板的每个孔中,该孔板含有经化合物处理的iastrocytes,不包括最外层的孔。
      28. 在400x g下使用带有T336旋转装置的PK120离心机和微型板桶将384孔板离心60秒,以将介质和细胞收集到井底。
      29. 将384孔人iastrocyte小鼠运动神经元共培养板在37℃/5%co2培养箱中培养24小时。 < BR>
        图2。用血细胞仪计数来自小鼠胚胎体的细胞。来自小鼠胚胎体(mebs)的明亮圆形细胞包括在神经元细胞计数中(四个小箭头表示示例)。较暗的粗糙边缘细胞和潜在的碎片被排除在神经元细胞计数之外(两个大的V形表示例子)。比例尺=100微米。
        < BR>
  9. 将培养基添加到共培养板(第8天)
    1. 根据以下配方,在无菌组织培养罩中制备适当体积的小鼠运动神经元(mmn)培养基。
    2. 将mmn介质置于37°C水浴中。
    3. 使用p50(50微升)多通道机械移液管,向含有iAstrocyte-mn共培养物的384孔板(不包括最外面的孔)的每个孔中添加15微升暖mmn培养基。
    < BR>
  10. 在高通量显微镜上成像HB9 gfp+mn(第8天和第10天)
    1. 打开照明器并预热试管内分析仪2000至37°C。
    2. 一旦照明器准备好,使用细胞内分析仪2000(图3)运行“HB9-gfp-mn”方案(如上文“在共培养方案之前准备”一节所述)来对共培养板中的HB9-gfp+运动神经元进行成像。为了进行下游分析,在共培养方案的第8天和第10天成像的hb9 gfp+运动神经元分别称为第1天和第3天。
    3. 将共培养板培养在37℃/5%co2培养箱中。
      < BR>
      图3。人类iastrocyte-hb9-gfp+mmn共培养的示例图像。人类iastrocyte和hb9-gfp+mmn在mmn接种后1天和3天共培养的代表图像,使用gfp通道在细胞内分析仪2000上成像。在mmn共培养前24小时,用dmso处理iastrocytes,或用复合物a处理iastrocytes。在共培养3天后,与仅用dmso处理的井相比,用化合物a处理的井有更大的mmn存活率。比例尺=200微米。
      < BR>

数据分析

  1. 运动神经元活性评估
    1. 将细胞内分析器2000上捕获的.xcde/.tif文件导入Columbus图像数据存储和分析系统。
    2. 在哥伦布图像数据存储和分析系统上编写一个分析协议,可以识别和计算gfp+mmn。分析协议由Columbus图像数据存储和分析系统的图像分析部分中的几个“构建块”或步骤组成,详细描述如下:
      1. 输入图像。这里,图像被加载到分析协议中(图4a)。将堆栈处理设置为“单个平面”,不设置“平场校正”和“快速调整”。
      2. 找到细胞核。识别运动神经元的gfp+细胞体(图4b)。在gfp频道上使用方法“m”。方法“m”是哥伦布图像数据存储和分析系统上提供的一种稳健方法,用于从一系列不同类型的图像中检测称为“核”的圆形物体。
      3. 计算形态特性。这一步骤允许计算标准的形态特征,如图像中gfp+细胞体或“细胞核”的面积或圆度。在此步骤中使用“标准方法”,它允许根据单元格区域大小过滤图像。
      4. 选择人口(1)。选择大于120像素2且宽长比大于0.2的gfp+细胞体的总体(图4c)。这样可以过滤掉小的gfp+物品,如碎片。
      5. 计算强度特性。此步骤允许计算标准强度属性,例如图像中gfp+细胞体或“细胞核”的平均像素强度。在此步骤中,请使用Columbus图像数据存储和分析系统中的“标准方法”选项。
      6. 选择人口(2)。选择平均像素强度大于2000(任意单位)的gfp+细胞体的总体(图4d)。这将过滤掉暗色gfp+物品,如碎片。
      7. 找到神经突。这一步骤识别从gfp+细胞体生长的gfp+神经突(由步骤b-f定义)(图4e)。使用“CSIRO神经轴突分析2”分析方法,该方法由澳大利亚CSIRO研究所开发的算法完成。这个步骤中使用的精确参数可能需要对不同的实验重复进行一些细微的修改。
      8. 选择人口(3)。选择至少有一个突起(在步骤g中定义)相连的gfp+细胞体(由步骤b-f定义)的总体(图4f)。这个种群是存活的gfp+mmn的数量。没有轴突附着的gfp+细胞体死亡,因此从活的gfp+mmn计数中滤过。
      9. 定义结果。此步骤对图像中的多个参数进行统计分析,包括gfp+细胞体的数量、至少有一个轴突的gfp+细胞体的数量(即,活gfp+mmn)以及其他形态和强度特性。
        < BR>
        图4。哥伦布图像数据存储与分析系统的分析协议,用于识别和计数gfp+mmn。a.将gfp通道中的图像加载到哥伦布图像分析软件中。比例尺=200μm。b.圆形gfp+项目被识别,然后用多种不同颜色突出显示,以区分不同的项目。这些圆形gfp+项目包括mmn细胞体。c.选择大小大于120像素2的gfp+项目(绿色),而小于120像素2的gfp+项目被排除(红色)。d.选择平均强度大于2000的gfp+项目(绿色),而排除平均像素强度小于2000的gfp+项目(红色)。e.从gfp+细胞体生长的gfp+轴突被识别,然后用多种不同颜色突出显示,以区分不同的轴突。f.计算至少有一个轴突附着(绿色)的gfp+细胞体的数量,排除没有轴突的gfp+细胞体(红色)。在每张图像右上角插入的框中,用白色虚线框突出显示的区域放大2倍。
        < BR>
    3. 使用步骤2中描述的分析协议,使用Columbus图像数据存储和分析系统的批处理分析部分,计算第1天和第3天的图像中的可行gfp+mmn的数量。
    4. 将结果文件导出到Excel 2016。
    5. 在图7.0中绘制第3天的存活gfp+mmn数量。
    < BR>
  2. 统计分析
    所需的精确统计分析取决于实验装置。通常,当在一个iastrocyte细胞系中以单剂量或多剂量筛选多个化合物时,使用dunnet事后检验(将每个条件与dmso对照进行比较)进行单向方差分析。在图7.0中进行统计分析(图5)。
    < BR>
    图5。例HB9-gfp+mmn在第3天从iAstrocyte-HB9-gfp+mmn共培养分析的384孔板中计数。-ve control=dmso only,+ve control=compound a.
    < BR>

笔记

  1. 注意使用的血清批次,因为这会影响共培养结果。我们推荐相同批次的血清用于任何给定批次的工作,例如,筛选化合物库。
  2. 在光学显微镜下观察时,mesc应显示为大而明亮的菌落,边缘清晰。如果菌落变成深灰色,或边缘粗糙或不清晰,mesc已经分化,分化后将产生较低的hb9 gfp+mns产量。
  3. mesc在培养过程中一直生长到第18代,然后被较年轻的后代所取代。这是因为当mesc达到较高的传代率时,它们所能产生的hb9 gfp+mn的产量就会降低。
  4. 在共培养方案的第7天,使用绿色荧光显微镜观察mebs。如果meb分化良好,那么gfp+细胞的比例应该很高,并且gfp的表达水平也很高。如果meb较暗,或gfp表达水平较低,则mn产量较低,不宜在共培养中使用。
  5. 当使用iastrocyte-hb9-gfp+mn共培养法进行高通量药物筛选试验时,我们建议阴性对照组(仅dmso)和阳性对照组(内部鉴定的化合物a)至少使用12个技术复制品(384孔板上的单独孔)。对于实验条件,1个技术复制就足够了,但我们建议使用6个技术复制。此外,在二次验证实验中,我们强烈建议每个实验条件使用6个技术复制品(阳性和阴性对照至少使用12个技术复制品)。对于高通量屏幕和二次验证实验,也建议至少重复3次实验。
  6. 当用2.5或5.0微克/毫升纤维连接蛋白涂层溶液覆盖10cm组织培养板时,没有最佳的培养时间。但是,我们建议在室温下至少5分钟,或者在4°C下过夜。

食谱

  1. Sterile1x PBS
    将5片PBS片加入500毫升Milli-Q超纯水中,并在121℃高压灭菌15分钟
    使用前让PBS冷却至室温
  2. 无菌超纯水
    在121°C下高压灭菌500毫升Milli-Q超纯水15分钟
    使用前让水冷却至室温
  3. 人inpc增殖培养基 class=“ke zeroborder”bordercolor=“000000”style=“width:350px;”border=“0”cellspacing=“0”cellpadding=“2”>dmem/f-12,谷氨酰胺
    500毫升
    n-2
    5毫升
    B-27
    5毫升
    4毫克/毫升FGF
    5微升
  4. 人iastrocyte分化培养基 class=“ke zeroborder”bordercolor=“000000”style=“width:350px;”border=“0”cellspacing=“0”cellpadding=“2”>dmem
    500毫升
    n-2
    1毫升
    < >fbs
    < >50毫升
    笔迹
    < >5毫升
  5. 小鼠胚胎干细胞(mesc)增殖培养基 class=“ke zeroborder”bordercolor=“000000”style=“width:350px;”border=“0”cellspacing=“0”cellpadding=“2”>击倒dmem
    < > < > 400毫升
    esc fbs
    < >75毫升
    谷氨酰胺
    5毫升
    非必需氨基酸
    < >5毫升
    < >2-巯基乙醇
    < >3.6微升
  6. 小鼠胚胎体(meb)分化培养基
    class=“ke zeroborder”bordercolor=“000000”style=“width:350px;”border=“0”cellspacing=“0”cellpadding=“2”>击倒dmem
    222.5毫升
    火腿的F-12营养素混合物
    222.5毫升
    敲除血清置换< BR>50毫升
    谷氨酰胺
    2.5毫升
    30%(w/v)过滤葡萄糖
    2.5毫升
    n-2
    5毫升
    2-巯基乙醇
    4微升
    使用Steritop过滤器过滤
  7. 小鼠运动神经元培养基 class=“ke zeroborder”bordercolor=“000000”style=“width:350px;”border=“0”cellspacing=“0”cellpadding=“2”>meb分化培养基
    40毫升
    200微克/毫升bdnf
    4微升
    200微克/毫升cntf
    4微升
    200微克/毫升GDNF
    4微升
  8. eb解离缓冲液
    class=“ke zeroborder”bordercolor=“000000”style=“width:400px;”border=“0”cellspacing=“0”cellpadding=“2”>无菌Milli-Q超纯水
    31.472毫升
    1 m氯化钠
    4.64毫升
    1米KCl
    0.216毫升
    1米NaHCO3
    1.04毫升
    0.1 m NaH2Po4
    0.4毫升
    0.1米cacl2
    0.6毫升
    0.1米mgso4
    0.4毫升
    30%(w/v)过滤葡萄糖
    1毫升
    0.5米EDTA
    0.04毫升
    25 mg/ml L-半胱氨酸
    0.192毫升
    使用0.2μm注射器过滤器过滤

致谢

我们要感谢所有为这项研究捐赠样本的als患者。HB9gfp-mesc是托马斯·杰塞尔教授的一份厚礼。inpc重编程和iastrocyte分化方案是由kathrin meyer博士和laura ferraiuolo博士在brian kaspar教授的实验室中开发的。这项工作由S.P.A.的运动神经元疾病协会高级研究员奖(赠款编号956-799,MNDA注册慈善机构编号294354)资助。L.F.和M.J.S.由Benevolentai资助。L.F.由医学科学院(SBF002\1142)资助。

相互竞争的利益

M.J.S.和L.F.由生物技术公司Benevolentai提供资金。

伦理学

在采集皮肤样本之前,所有受试者均获得知情同意(研究编号STH16573,研究伦理委员会参考12/YH/0330)。遵循所有适用的国际、国家和/或机构动物护理和使用指南。

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引用:Stopford, M. J., Allen, S. P. and Ferraiuolo, L. (2019). A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery. Bio-protocol 9(17): e3353. DOI: 10.21769/BioProtoc.3353.
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ethan tara
dr
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2020/7/14 2:31:49 回复