Co-culture of Human Stem Cell Derived Neurons and Oligodendrocyte Progenitor Cells
人干细胞源性神经元与少突胶质祖细胞的共培养   

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Abstract

Crosstalk between neurons and oligodendrocytes is important for proper brain functioning. Multiple co-culture methods have been developed to study oligodendrocyte maturation, myelination or the effect of oligodendrocytes on neurons. However, most of these methods contain cells derived from animal models. In the current protocol, we co-culture human neurons with human oligodendrocytes. Neurons and oligodendrocyte precursor cells (OPCs) were differentiated separately from pluripotent stem cells according to previously published protocols. To study neuron-glia cross-talk, neurons and OPCs were plated in co-culture mode in optimized conditions for additional 28 days, and prepared for OPC maturation and neuronal morphology analysis. To our knowledge, this is one of the first neuron-OPC protocols containing all human cells. Specific neuronal abnormalities not observed in mono-cultures of Tuberous Sclerosis Complex (TSC) neurons, became apparent when TSC neurons were co-cultured with TSC OPCs. These results show that this co-culture system can be used to study human neuron-OPC interactive mechanisms involved in health and disease.

Keywords: Human iPSC (人类诱导性多能干细胞), Neuron (神经), Oligodendrocyte (少突胶质细胞), Co-culture (共培养), Myelin (髓磷脂), Neuron-glia interaction (神经胶质相互作用)

Background

The human brain consists of an immense complex organization of cells that we are only recently starting to identify, and that cannot be studied in animal models. The human brain also contains a high white matter content, which is suggested to account for higher brain functions, such as social and cognitive learning (Maldonado and Angulo, 2015; Almeida and Lyons, 2016; Kougioumtzidou et al., 2017). Single cell expression studies in animals indicate that oligodendrocytes form heterogeneous populations of cells (Marques et al., 2016). This supports the notion that oligodendrocytes fulfill more complex functions than solely isolating axons. As the human comprises of higher diversity of neurons compared to rodent brain, and considering the role of white matter in complex functions in learning and cognition, we could expect an even more complex diversity of oligodendrocyte lineage cells in the human brain. Therefore, we need to identify oligodendrocyte-neuron crosstalk in the human brain. Next to the classic white matter disorders, such as multiple sclerosis and the leukodystrophies (van der Knaap and Bugiani, 2017), white matter abnormalities are consistently been found in psychiatric disorders (Haroutunian et al., 2014). Increasing evidence shows that crosstalk between neurons and oligodendrocytes is important for proper neural network functioning (Bergles et al., 2000; Velez-Fort et al., 2010; Maldonado and Angulo, 2015) and myelin formation (Almeida and Lyons, 2016; Kougioumtzidou et al., 2017). Therefore, to study the involvement of neuron-oligodendrocyte interactions in the normal and diseased brains, we are in need of human-based model systems. As current assays mostly involve non-human cells (Cui et al., 2010; Hill et al., 2014; Clark et al., 2017; Pang et al., 2018; Treichel and Hines, 2018), we developed human induced pluripotent stem cell (iPSC)-based co-culture models to study crosstalk between human neurons and human oligodendrocyte progenitor cells (OPCs). The presented co-culture method was used to study neuron-OPC interactions in Tuberous Sclerosis Complex (TSC) (Nadadhur et al., 2019), a genetic multisystem disorder that shows both grey and white matter abnormalities in the brain. Although some neuronal abnormalities were present in mono-cultures of TSC neurons, in the presence of OPCs increased axonal density and hypertrophy became apparent (Nadadhur et al., 2019). This suggests that specific neuronal phenotypes can only be studied when oligodendrocytes are present. Vice versa oligodendrocyte maturation is highly dependent on neuronal signaling. Therefore these culture systems can be applied to study multiple processes in health and disease in which complex neuron-oligodendrocyte interactions are involved, and provide prospects for the development of drug screening platforms for all-human cells, e.g., patient iPSCs. To conclude, this novel human neuron-OPC co-culture model can be used to study neuron-OPC crosstalk in health and disease.

Materials and Reagents

  1. 12-well plate (VWR, catalog number: 665180)
  2. 10 μl filter tips (Thermo Fisher, catalog number: 11977714)
  3. 100 μl filter tips (Thermo Fisher, catalog number: 11953466)
  4. 1000 μl filter tips (Thermo Fisher, catalog number: 11973466)
  5. 6-well plate (VWR, catalog number: 734-2323)
  6. 18 mm coverslips (VWR, catalog number: 631-0153)
  7. 5 ml pipette (VWR, catalog number: 606180)
  8. 10 ml pipette (VWR, catalog number: 607180)
  9. 15 ml tube (VWR, catalog number: 525-0400)
  10. Microscope slide (VWR, catalog number: 631-0108)
  11. Syringe needle (BD Biosciences, catalog number: 300400)
  12. 4’,6-Diamidino-2-Phenylindole (DAPI) (Sigma-Aldrich, catalog number: D9542-5MG)
  13. Accutase (Merck-Millipore, catalog number: sf006)
  14. Anti-MAP2 antibody (Abcam, catalog number: AB5392)
  15. Anti-MBP antibody (Covance, catalog number: SMI-99P)
  16. Anti-Olig2 antibody (Merck-Millipore, catalog number: AB9610)
  17. Anti-SMI312 antibody (Eurogentec, catalog number: SMI-312P-050)
  18. Arabinosylcytosine (AraC) (Merck-Millipore, catalog number: 251010)
  19. β-mercaptoethanol (Thermo Fisher Scientific, catalog number: 21985023)
  20. B27 with vitamin A (Thermo Fisher Scientific, catalog number: 17504-044)
  21. B27 without vitamin A (Thermo Fisher Scientific, catalog number: 12587-010)
  22. Basic fibroblast growth factor (bFGF) (Peprotech, catalog number: 100-18B-50ug)
  23. Brain-derived neurotrophic factor (BDNF) (Peprotech, catalog number: 450-02)
  24. Biotin (Sigma-Aldrich, catalog number: B4501-100MG)
  25. Bovine Serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418)
  26. Cyclic adenosine monophosphate (cAMP) (Sigma, catalog number: D0260-5MG)
  27. Defined Trypsin Inhibitor (DTI) (Thermo Fisher Scientific, catalog number: R007100)
  28. DMEM/F12 with Glutamax (Life Technologies, catalog number: 21331-020)
  29. DMEM/F12 without L-glutamine (Life Technologies, catalog number: 21331-046)
  30. Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650)
  31. Dorsomorphin (Tocris Bioscience, catalog number: 3093/10)
  32. Epidermal growth factor (EGF) (Peprotech, catalog number: AF-100-15-500ug)
  33. Ethylenediaminetetraacetic acid (EDTA) (Invitrogen, catalog number: 15575-038)
  34. Fetal Bovine Serum (FBS) (ThermoFisher Scientific, catalog number: 16140063)
  35. Fluoromount G (Southern Biotech, catalog number: 0100-01)
  36. Glial-cell derived neurotrophic factor (GDNF) (Peprotech, catalog number: 450-10)
  37. Geltrex (Life Technologies, catalog number: A1413302)
  38. Glutamax (Thermo Fisher Scientific, catalog number: 35050-038)
  39. Goat anti-rat/mouse/rabbit/chicken/guinea pig Alexa Fluor antibodies (Life Technologies)
  40. Heparin (Sigma-Aldrich, catalog number: H3393-50KU)
  41. HEPES (Thermo Fisher Scientific, catalog number: 15630-056)
  42. Human Sonic Hedgehog (hSHH) (Peprotech, catalog number: 100-45-500ughSHH)
  43. Insulin-like growth factor 1 (IGF1) (Peprotech, catalog number: 100-11-100ug)
  44. Insulin (Sigma-Aldrich, catalog number: I9278)
  45. KCl (Sigma-Aldrich, catalog number: P5405-250gr)
  46. KH2PO4 (Sigma-Aldrich, catalog number: P5379)
  47. L-glutamine (Thermo Fisher Scientific, catalog number: 25030-024)
  48. Mouse laminin (mLaminin) (Sigma-Aldrich, catalog number: L2020-1mg)
  49. N2 supplement (Thermo Fisher Scientific, catalog number: 17502-048)
  50. Na2HPO4 (Sigma-Aldrich, catalog number: S7907-500gr)
  51. NaCl (VWR, catalog number: S9888-1Kg)
  52. Neurobasal medium (Thermo Fisher Scientific, catalog number: 21103-049)
  53. Neurotrophin 3 (NT3) (Peprotech, catalog number: 450-03-100ug)
  54. Noggin (Peprotech, catalog number: 120-10C)
  55. Non-essential amino acids (NEAA) (Thermo Fisher Scientific, catalog number: 11140-035)
  56. Normal goat serum (NGS) (Life Technologies, catalog number: 16210-064)
  57. Penicillin/streptomycin (Pen/Strep) (Sigma-Aldrich, catalog number: P0781)
  58. Paraformaldehyde (PFA) 16% (Electron Microscopy Sciences, catalog number: 15710-S)
  59. Poly-L-ornithine (PLO) (Sigma-Aldrich, catalog number: P3655-100mg)
  60. Rock Inhibitor (RI) (Y27632; Selleckchem, catalog number: S1049)
  61. Retinoic Acid (RA) (Sigma-Aldrich, catalog number: R2625-100MG)
  62. SB431542 (Selleckchem, catalog number: S1067)
  63. Triiodothyronine (T3) (Sigma-Aldrich, catalog number: T6397-100MG)
  64. TeSRE8 (Stem Cell Technologies, catalog number: 5940)
  65. Triton X-100 (Sigma-Aldrich, catalog number: T8787-100ml)
  66. TryplE (Life Technologies, catalog number: 12563-029)
  67. Valproic acid (VPA) (Sigma-Aldrich, catalog number: P4543-10G)
  68. Vitamin C/Ascorbic Acid (Sigma-Aldrich, catalog number: A4544-25G)
  69. N1 supplement (Sigma-Aldrich, catalog number: N6530-5ML)
  70. Neuroglia co-culture medium (see Recipes)
  71. Neural Maintenance Medium (NMM) with Vitamin A (see Recipes)
  72. Neural Maintenance Medium (NMM) without Vitamin A (see Recipes)
  73. N2 medium (see Recipes)
  74. NB medium (see Recipes)
  75. Blocking Buffer (see Recipes)
  76. PBS (see Recipes)
  77. PLO/mLaminin coating (see Recipes)
  78. Geltrex coating (see Recipes)

Equipment

  1. Pipette controller (BD Biosciences, model number: Falcon Express)
  2. Incubator (Binder, model number: 9140-0044; 5% CO2, 20% O2)
  3. Tabletop Centrifuge (Eppendorf, model number: centrifuge 5810)
  4. -80 °C freezer (Thermo Fisher Scientific; model number ULT1786-6-V49)
  5. Bright field microscope (Zeiss, model number: Axiovert 40C.)
  6. Fluorescent microscope (Leica Microsystems, model number: Leica DM6000B)

Software

  1. Columbus 2.5 online software (Perkin Elmer)
  2. Leica Application Suite Advanced Fluorescence (Leica)

Procedure

  1. Neuronal differentiation
    Neurons are differentiated according to previously published protocols (Shi et al., 2012; Nadadhur et al., 2017), shortly:
    1. hiPSCs are passaged onto Geltrex-coated 12-well plates in 1 ml TesRE8 medium with 10 μM RI per well.
    2. Refresh all medium every day.
    3. After 2 days (or when cells are 100% confluent), add 1 ml neural maintenance medium (NMM) with vitamin A + 1 μM Dorsomorphin + 10 μM SB431542.
    4. Refresh all medium every day until Day 12.
      Note: Between Days 8 and 12 a uniform neuroepithelial sheet should appear (see Figure 1).
    5. Prepare a PLO/mLaminin coated 6-well plate as described in the Recipes.
    6. Collect neuroepithelial rosette cells (NES cells) by manually cutting them (see description in note) and plate them in the 6-well plate.
      Note: Prepare fresh pre-warmed NMM with vitamin A + 1 μM Dorsomorphin + 10 μM SB431542 + 10 μM RI. Remove the medium from the 12-well plate, wash the wells with PBS once and add 1 ml of the freshly prepared medium. Sterilize the microscope area with 70% Ethanol and cut the rosettes using a 10 μl tip to mark their borders and to lift them up. Collect the floating rosettes with a 5 ml pipette and move them to the PLO/mLaminin coated 6-well plate with NMM with vitamin A + 1 μM Dorsomorphin + 10 μM SB431542 + 10 μM RI. All rosettes from 1 well of a 12-well plate are plated in 1 well of a 6-well plate.


      Figure 1. Brightfield picture showing neural rosette structures that form between Days 8 and 12. Scale bar = 200 μm.

    7. On Day 13, change the medium to 2 ml pre-warmed NMM with vitamin A + 20 ng/ml bFGF + 20 ng/ml EGF.
    8. Every day remove 1 ml of medium and add 1 ml pre-warmed NMM with vitamin A + 40 ng/ml bFGF + 40 ng/ml EGF. When confluent, passage the cells with TrypLE in a 1:2 or 1:3 ratio as described in the note below.
      Notes:
      1. Cells can be kept in NES cell stage for 1-4 passages. 
      2. Growth factor concentration is double when only half the medium is changed: together with the medium left in the well the concentration will be similar to the concentration in Step A7.
      3. TrypLE passage: Remove all medium from the well and add 300 μl pre-warmed TrypLE. Swirl it around the well and incubate for 2 min at room temperature (RT). Add 600 μl of pre-warmed DTI or medium to stop the reaction and collect the cells into a tube containing 5 ml NMM. Spin down at 300 x g for 5 min at RT and re-suspend the pellet in desired medium.
    9. To start the neuronal induction, plate the NES cells after a TrypLE passage in a PLO/mLaminin-coated 12-well plate in pre-warmed 1 ml NMM + 20 ng/ml bFGF + 20 ng/ml EGF per well.
    10. When cells reach 80%-90% confluence, switch half of the medium (500 μl/well) to pre-warmed N2 medium + 800 ng/ml hSHH.
      Note: This is considered Day 1 of the differentiation protocol.
    11. Until Day 4, refresh half of the medium (500 μl/well) daily with pre-warmed N2 medium + 800 ng/ml hSHH.
    12. On Day 5, switch half of the medium (500 μl/well) to pre-warmed NB medium + 40 μM VPA.
    13. Until Day 7, refresh half of the medium (500 μl/well) daily with pre-warmed NB medium + 40 μM VPA.
    14. Prepare PLO/mLaminin-coated 12-well plates before Day 8 as described in the Recipes.
    15. On Day 8, passage the neural progenitors with accutase 1:2 or 1:3 to a new well (see note for explanation). Plate cells in pre-warmed 1.5 ml NB medium + 20 ng/ml BDNF + 10 ng/ml GDNF + 10 ng/ml IGF1 + 1 μM cAMP.
      Accutase treatment: Remove all medium, add 300 μl of accutase per well of a 12-well plate and return the plate to the incubator for 5-7 min. By gently tapping the plate, the cells will visibly come off. At this point, collect the cells in a tube, add 5 ml fresh pre-warmed medium, spin at 300 x g for 5 min at RT and re-suspend the pellet in the desired medium. Resuspend carefully; do not break clumps into single cells.
    16. Until Day 18 refresh medium three times a week by removing 1 ml of medium and adding 1 ml of pre-warmed NB medium + 30 ng/ml BDNF + 15 ng/ml GDNF + 15 ng/ml IGF1 + 1.5 μM cAMP. 
    17. On Day 12, prepare a 12-well sandwich plate as described in the note, Figure 2 and Video 1. Coat the plate with Geltrex as described in the Recipes. Plate 25,000 primary rat astrocytes in each well containing pre-warmed DMEM/F12 medium with Glutamax + 10% FBS + 1x NEAA + 1x Pen/Strep.
      Note: Sandwich plate preparation: Take a 12-well plate and heat up a sterile syringe needle in flame. Punch in 4 corners of each well to make uniform small bumps in the plastic bottom of the well. These bumps will help suspend a coverslip on them, separated by a small distance from the cells in the bottom of the well. See Figure 2 and Video 1.

      Video 1. Sandwich plate

    18. Before Day 18, prepare the desired amount of 12-well plates containing 18 mm diameter coverslips and coat with PLO/laminin as described in the Recipes.
      Note: Coverslips can be stored by sealing the plate tightly with parafilm for maximum 1 week at 4 °C.
    19. On Day 18, make a single-cell suspension of the neurons using accutase as described in the note. Plate 1.5 million cells per 12-well plate on the pre-coated PLO/mLaminin coverslips in pre-warmed 1 ml NB medium + 20 ng/ml BDNF + 10 ng/ml GDNF + 10 ng/ml IGF1 + 1 μM cAMP.
      Note: Remove all medium, add 300 μl of accutase per well of a 12-well plate and return the plate to the incubator for 10-15 min. By gently tapping the plate, the cells will visibly come off. Dissociate the cells in the accutase using a 1000 μl pipette very slowly and pipet into a 15 ml tube with 5 ml of neurobasal medium. Spin at 300 x g for 5 min at RT. Remove supernatant and very gently resuspend the pellet in 1 ml of NB medium and try breaking the clumps of cells very gently using a 1000 μl pipette (pipet up and down maximum 3-4 times). Then leave the tube for 2-5 min to let the big clumps settle down at the bottom. Take the supernatant into another tube; thrash the pellet (the big clumps will not have mature cells but mainly proliferating precursor cells).


      Figure 2. Sandwich plate preparation. A. Take a 12-well plate and a sterile syringe needle. Flame the needle until it is hot. With the hot needle, make a bump on 4 sides of the well (B-D). The astrocytes will be plated on the bottom of the plate, and a coverslip with neurons can be inverted on top. The coverslip will lean on the bumps. See also Video 1.

    20. On Day 19, turn the coverslips with adhered neurons upside down over the bumps on the astrocyte plates.
      Note: By now, the astrocytes should have reached 80% confluence in order to support the neurons. 
    21. Change half of the medium (500 μl/well) twice a week with pre-warmed NB medium + 40 ng/ml BDNF + 20 ng/ml GDNF + 20 ng/ml IGF1 + 2 μM cAMP.
    22. On the day before the second refresh (Day 24/25), add 1 μM AraC to cultures to stop proliferation.
    23. Next day (Day 25/26) refresh half of the medium (500 μl/well) with pre-warmed NB medium + 40 ng/ml BDNF + 20 ng/ml GDNF + 20 ng/ml IGF1 + 2 μM cAMP.
    24. Keep cultures until Day 37 (see Figure 3), while refreshing medium twice a week as described in Step 21.


      Figure 3. Brightfield picture of Day-37 neurons prior to co-culture showing neuronal network formation in sandwich culture. Scale bar = 100 μm.

  2. OPC differentiation
    OPCs are generated according to previously published protocol (Izrael et al., 2007), shortly:
    1. hiPSCs are passaged with EDTA and plated on an anti-adherent 6-well plate in 3 ml pre-warmed NMM with Vit A + 20 ng/ml EGF + 4 ng/ml bFGF + 10 μM RI + 20 ng/ml T3 per well for embryoid body (EB) formation (see Figure 3).
      Note: Use 2 wells of iPSCs for 1 well of EBs.
    2. The next day (Day 1), refresh ⅔ of the medium: swirl cells to the middle of the wells and carefully aspirate 2 ml of medium. Add 2 ml of pre-warmed NMM with Vit A + 20 ng/ml EGF + 4 ng/ml bFGF + 10 μM RI + 20 ng/ml T3.
    3. On Day 2, refresh ⅔ of the medium as described in Step B2 with pre-warmed NMM with Vit A + 20 ng/ml EGF + 4 ng/ml bFGF + 10 μM RA.
    4. Repeat every other day.
    5. On Day 10, plate EBs on Geltrex-coated plate as described in the note. If EBs become very dark, or decrease in density, plate EBs sooner than on Day 10, but not before Day 4. In our experience, Day 8 is mostly ideal (see Figure 4).
      Note: Plate EBs on geltrex coated plate by collecting the medium with EBs in a 15 or 50 ml tube. Leave the tube for 2-3 min to let the EBs settle. Carefully aspirate most of the medium, and resuspend EBs in the appropriate amount of desired medium (1 ml for a 12-well plate; 2 ml for a 6-well plate). On Days 4-9, EBs are plated in pre-warmed NMM with Vit A + 20 ng/ml EGF + 4 ng/ml bFGF + 10 μM RA + 20 ng/ml T3. If EBs are plated on Day 10, NMM with Vit A + 20 ng/ml EGF + 20 ng/ml T3 should be used instead.


      Figure 4. Brightfield pictures showing embryoid body formation (A) and outgrowth of cells from embryoid bodies after plating on a geltrex-coated plate (B). Scale bars = 500 μm.

    6. On Day 10, switch all medium to pre-warmed NMM with Vit A + 20 ng/ml EGF + 20 ng/ml T3.
    7. From now on: refresh ⅔ of the medium every other day and split cells when they reach 90% density to a new Geltrex-coated plate using accutase.
      Note: Add RI to medium after passaging to help the cells recover.
    8. On Day 18, change medium to NMM without Vit A + 20 ng/ml EGF; keep refreshing and splitting cells as described in Step B7.
    9. On Day 37, change medium to NMM without Vit A + 5 ng/ml bFGF + 5 ng/ml EGF + 1 μg/ml mLaminin + 50 μg/ml Vit C; keep refreshing and splitting cells as described in Step B7.
    10. On Day 39, change medium to NMM without Vit A + 5 ng/ml bFGF + 5 ng/ml EGF + 1 μg/ml mLaminin + 50 μg/ml Vit C + 50 ng/ml noggin; keep refreshing and splitting cells as described in Step B7.
    11. On Day 42, change medium to NMM without Vit A + 1 μg/ml mouse laminin + 50 μg/ml Vit C + 50 ng/ml noggin; keep refreshing and splitting cells as described in Step B7.
    12. Keep OPC cultures in the medium of Step B11 until Day 65 (see Figure 5).


      Figure 5. OPC differentiation. At the end of the OPC differentiation protocol, cells show a glial morphology (A, Day 45) and express OPC markers like OLIG2 (B, Day 65). Scale bars = 100 μm.

  3. Neuron-OPC co-culture
    1. At day 1 of co-culture, move the Day 37 neurons and plate Day 65 OPCs on top:
      1. Take a new 12-well plate. 
      2. Pick the Day 37 neuronal coverslips up with a forceps from the rat astrocyte plate and plate in a new 12-well plate (with neurons on top, so flip coverslip compared to how it was on astrocyte plate).
        Note: No coating is necessary for the new plate as the neuronal coverslips already contain the coating. 
      3. Add 1 ml of pre-warmed neuroglia co-culture medium. 
      4. Get a plate with Day 65 OPCs.
      5. Remove medium and wash plate once with PBS.
      6. Remove PBS and add 750 μl accutase to every well (6-well plate).
      7. Incubate the plate for 5-10 min at 37 °C.
      8. Check if cells are detaching from the plate by gentle tapping; if not, incubate longer.
      9. When cells are detaching, collect cell suspension in a 15 ml tube with 9 ml pre-warmed DMEM/F12.
      10. Spin down 5 min at 300 x g at RT.
      11. Resuspend cells in 1 ml of pre-warmed neuroglia co-culture medium.
      12. Remove 10 μl of cell suspension for cell count.
      13. Plate 4 x 105 OPCs per coverslip on a 12-well plate. 
    2. Refresh ½ of the medium twice a week: remove 500 μl of medium and add 500 μl of new pre-warmed neuroglia co-culture medium.
    3. At Day 28 of co-culture, fix cells with 4% PFA for 15-20 min and use coverslips for immunostaining immediately (see Figure 6).


      Figure 6. Immunostaining of co-cultures. (A) shows many OLIG2-positive OPCs and MBP-positive mature oligodendrocytes with MAP2-positive dendrites. (B) shows oligodendrocytes (MBP-positive) closely interacting with dendrites (MAP2-positive) Scale bars = 25 μm.

  4. Immunostaining
    1. Wash plate 6 x 5 min with PBS.
    2. Remove PBS.
    3. Add 300 μl blocking buffer per well.
    4. Incubate for 1 h at RT.
    5. Remove blocking buffer.
    6. Add primary antibodies diluted in 300 μl blocking buffer. 
    7. Incubate for 1 h at RT, then overnight at 4 °C.
    8. Wash 6 x 5 min with PBS.
    9. Add secondary antibodies diluted 1:1,000 in 300 μl blocking buffer. 
    10. Incubate for 2 h at RT.
    11. Wash 6 x 5 min with PBS.
    12. Remove PBS.
    13. Add DAPI 1:1000 diluted in PBS.
    14. Incubate for 2 min at RT.
    15. Wash 1 x with PBS.
    16. Mount coverslips with Fluoromount G upside down on a microscopical slide.

Data analysis

  1. Coverslips can be stained with markers for OPC maturation (for example OLIG2, MBP) and for markers of neuronal morphology (for example MAP2, SMI312). In our experience, for a control line there will be about 10% OLIG2+ cells, 1-2% MBP+ cells and about 40% of the cells in the culture are MAP2+ neurons.
    Note: OLIG2 and MBP do not label all human oligodendrocyte lineage cells.
  2. Cell properties can be analyzed by automated software packages, for example Columbus 2.5 online software (see https://www.perkinelmer.com/nl/product/image-data-storage-and-analysis- system-columbus for more information about the Columbus software and Figure 7 for example pictures of analysis) To analyze neuronal morphology, algorithms for morphology, soma recognition and co-localization can be used. More precisely, based on MAP2 and nuclear staining the dendritic density and average dendritic density per neuron can be determined. Similar analysis based on SMI312 staining can be used to analyze axonal density. 
  3. OPC maturation can be assessed using immunostaining for mature oligodendrocyte markers like MBP. Because different cell lines might grow at different speeds, and have different efficiencies in oligodendrocyte generation, it is best to correlate the number of MBP-positive to the number of OLIG2- positive OPCs.
  4. The correct statistical analysis depends on the research question of interest. For example, this also depends on the culture set-up, i.e., when culturing 1 control oligodendrocyte line with neurons from different lines of patients and controls, vs. all combinations of control/patient oligodendrocytes with control/patient neurons. See Nadadhur et al. (2019) for how we performed statistical analysis.


    Figure 7. Example of cell analysis in Columbus. Fluorescent images can be imported into Columbus (A) which has preprogrammed settings to find nuclei (B) and trace neurites (C). The output includes parameters as the maximum neurite length, total neurite length and number of extremities per cell.

Notes

As some variation is common in iPSC differentiation protocols, we recommend some quality checks before the start of a neuron-OPC co-culture.

  1. Proper OPC differentiation can be verified by checking for OLIG2 expression around Days 57-60 of the differentiation protocol.
  2. Proper neuronal differentiation can be verified by staining for MAP2/beta-III tubulin/NeuN in the cultures on Day 37. If GFAP expression is observed, it is advised to discard cultures, as glia contamination could interfere with mixed co-cultures from patient and control iPSCs. See Nadadhur et al. (2017) for more glial free neuronal culture images.

Recipes

  1. Neuroglia co-culture medium
    480 ml DMEM/F12
    480 ml Neurobasal
    10 ml N1 supplement
    20 ml B27 supplement
    10 ml NEAA
    60 ng/ml T3
    100 ng/ml Biotin
    10 ng/ml NT3
    1 μg/ml mLaminin
    20 ng/ml BDNF
    1 μM cAMP
  2. Neural Maintenance Medium (NMM) with Vitamin A
    482 ml DMEM/F12 + glutamax
    482 ml Neurobasal
    10 ml B27 supplement with vitamin A
    5 μg/ml Insulin
    5 ml Glutamax
    5 ml N2 supplement
    5 ml NEAA
    10 ml Pen/Strep
    10 μM β-mercaptoethanol
  3. Neural Maintenance Medium (NMM) without Vitamin A
    482 ml DMEM/F12 + glutamax
    482 ml Neurobasal
    10 ml B27 supplement without vitamin A
    5 μg/ml Insulin
    5 ml Glutamax
    5 ml N2 supplement
    5 ml NEAA
    10 ml Pen/Strep
    10 μM β-mercaptoethanol
  4. N2 medium
    48 ml DMEM/F12 without L-glutamine
    500 μl 100x N2
    500 μl Non-essential amino acids (10 mM stock)
    500 μl L-glutamine (200 mM stock)
    2 μg/ml Heparin
    500 μl Pen/Strep
  5. NB medium
    475 ml Neurobasal
    10 ml B27
    18 mM HEPES
    1.25 ml Glutamax
    5 ml Pen/strep
  6. Blocking Buffer
    95 ml PBS
    5 ml NGS
    300 μl Triton X-100
    0.1 g BSA
  7. PBS
    8 g NaCl
    0.1 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    800 ml H2O
  8. PLO/mLaminin coating
    1. Add to plate 20 μg/ml PLO in PBS (0.5 ml for 12-well plate; 1 ml for 6-well plate)
    2. Incubate at 37 °C for 4 h or O/N
    3. Remove PLO
    4. Wash 3 times with PBS
    5. Add to plate 20 μg/ml mLaminin (same amounts as for PLO)
    6. Incubate at 37 °C for at least 2 h or O/N
    7. Remove mLaminin
    8. Add medium and cells
  9. Geltrex coating
    1. Dilute Geltrex 1:1 with cold DMEM/F12
    2. Store aliquots at -80 °C
    3. Dilute 1:50 with cold DMEM/F12
    4. Add to plate appropriate amount (0.5 ml for 12-well plate; 1 ml for 6-well plate)
    5. Incubate at 37 °C for at least 1 h
    6. After remove Geltrex, immediately add medium with cells

Acknowledgments

This study was financially supported by Amsterdam Neuroscience and EU MSCA-ITN CognitionNet (FP7-PEOPLE-2013-ITN 607508). V.M.H. is supported by ZonMw VIDI Research grant (91712343), E-Rare Joint Call project (9003037601) and a European Leukodystrophy Association (ELA) Research Grant (2014-012L1). Neuronal differentiation protocol is based on previous studies by Nadadhur et al. (2017). OPC differentiation protocol is based on the previous study of Izrael et al. (2007).

Competing interests

The authors declare no conflict of interest.

Ethics

All experiments were exempt from approval of Medical Ethical Toetsingscommissie (METC), Institutional Review Board of the VU medical center.

References

  1. Almeida, R. and Lyons, D. (2016). Oligodendrocyte development in the absence of their target axons in vivo. PLoS One 11(10): e0164432.
  2. Bergles, D. E., Roberts, J. D., Somogyi, P. and Jahr, C. E. (2000). Glutamatergic synapses on oligodendrocyte precursor cells in the hippocampus. Nature 405(6783): 187-191.
  3. Clark, A. J., Kaller, M. S., Galino, J., Willison, H. J., Rinaldi, S. and Bennett, D. L. H. (2017). Co-cultures with stem cell-derived human sensory neurons reveal regulators of peripheral myelination. Brain 140(4): 898-913.
  4. Cui, Q. L., Fragoso, G., Miron, V. E., Darlington, P. J., Mushynski, W. E., Antel, J. and Almazan, G. (2010). Response of human oligodendrocyte progenitors to growth factors and axon signals. J Neuropathol Exp Neurol 69(9): 930-944.
  5. Haroutunian, V., Katsel, P., Roussos, P., Davis, K. L., Altshuler, L. L. and Bartzokis, G. (2014). Myelination, oligodendrocytes, and serious mental illness. Glia 62(11): 1856-1877.
  6. Hill, R. A., Medved, J., Patel, K. D. and Nishiyama, A. (2014). Organotypic slice cultures to study oligodendrocyte dynamics and myelination. J Vis Exp(90): e51835.
  7. Izrael, M., Zhang, P., Kaufman, R., Shinder, V., Ella, R., Amit, M., Itskovitz-Eldor, J., Chebath, J. and Revel, M. (2007). Human oligodendrocytes derived from embryonic stem cells: Effect of noggin on phenotypic differentiation in vitro and on myelination in vivo. Mol Cell Neurosci 34(3): 310-323.
  8. Kougioumtzidou, E., Shimizu, T., Hamilton, N. B., Tohyama, K., Sprengel, R., Monyer, H., Attwell, D. and Richardson, W. D. (2017). Signalling through AMPA receptors on oligodendrocyte precursors promotes myelination by enhancing oligodendrocyte survival. Elife 6: e28080.
  9. Maldonado, P. P. and Angulo, M. C. (2015). Multiple modes of communication between neurons and oligodendrocyte precursor cells. Neuroscientist 21(3): 266-276. 
  10. Marques, S., Zeisel, A., Codeluppi, S., van Bruggen, D., Mendanha Falcao, A., Xiao, L., Li, H., Häring, M., Hochgerner, H., Romanov, R.A., Gyllborg, D., Munoz Manchado, A., La Manno, G., Lönnerberg, P., Floriddia, E.M., Rezayee, F., Ernfors, P., Arenas, E., Hjerling-Leffier, J., Harkany, T., Richardson, W.D., Linnarsson, S., Castelo-Branco, G. (2016). Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science 352 (6291): 1326-1329.
  11. Nadadhur, A. G., Alsaqati, M., Gasparotto, L., Cornelissen-Steijger, P., van Hugte, E., Dooves, S., Harwood, A. J. and Heine, V. M. (2019). Neuron-glia interactions increase neuronal phenotypes in tuberous sclerosis complex patient iPSC-derived models. Stem Cell Reports 12(1): 42-56.
  12. Nadadhur, A. G., Emperador Melero, J., Meijer, M., Schut, D., Jacobs, G., Li, K. W., Hjorth, J. J. J., Meredith, R. M., Toonen, R. F., Van Kesteren, R. E., Smit, A. B., Verhage, M. and Heine, V. M. (2017). Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells. PLoS One 12(6): e0178533.
  13. Pang, Y., Simpson, K., Miguel-Hidalgo, J. J. and Savich, R. (2018). Neuron/Oligodendrocyte myelination coculture. Methods Mol Biol 1791: 131-144.
  14. Shi, Y., Kirwan, P., Smith, J., Robinson, H. P. and Livesey, F. J. (2012). Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat Neurosci 15(3): 477-486, S471.
  15. Treichel, A. J. and Hines, J. H. (2018). Development of an embryonic zebrafish oligodendrocyte-neuron mixed coculture system. Zebrafish 15(6): 586-596.
  16. van der Knaap, M. S. and Bugiani, M. (2017). Leukodystrophies: a proposed classification system based on pathological changes and pathogenetic mechanisms. Acta Neuropathol 134(3): 351-382.
  17. Velez-Fort, M., Maldonado, P. P., Butt, A. M., Audinat, E. and Angulo, M. C. (2010). Postnatal switch from synaptic to extrasynaptic transmission between interneurons and NG2 cells. J Neurosci 30(20): 6921-6929.

简介

神经元和少突胶质细胞之间的串扰对于正常的大脑功能非常重要。已经开发了多种共培养方法来研究少突胶质细胞成熟,髓鞘形成或少突胶质细胞对神经元的作用。然而,这些方法中的大多数含有源自动物模型的细胞。在目前的方案中,我们将人类神经元与人类少突胶质细胞共培养。根据先前公开的方案,神经元和少突胶质细胞前体细胞(OPCs)与多能干细胞分开分化。为了研究神经元 - 神经胶质细胞串扰,将神经元和OPC以共培养模式在优化条件下铺板另外28天,并准备用于OPC成熟和神经元形态学分析。据我们所知,这是第一个包含所有人类细胞的神经元-OPC协议之一。当TSC神经元与TSC OPCs共培养时,在Tuberous Sclerosis Complex(TSC)神经元的单培养物中未观察到特异性神经元异常变得明显。这些结果表明,该共培养系统可用于研究涉及健康和疾病的人类神经元-OPP交互机制。
【背景】人类的大脑由一个巨大的复杂细胞组织组成,我们最近才开始识别这些细胞,并且无法在动物模型中进行研究。人脑也含有高白质含量,建议用于解释更高的脑功能,如社交和认知学习(Maldonado和Angulo,2015; Almeida和Lyons,2016; Kougioumtzidou 等。,2017)。动物中的单细胞表达研究表明,少突胶质细胞形成异质细胞群(Marques et al。,2016)。这支持了少突胶质细胞实现比单独分离轴突更复杂的功能的观点。由于人类与啮齿动物大脑相比具有更高的神经元多样性,并且考虑到白质在学习和认知中的复杂功能中的作用,我们可以预期人类大脑中少突胶质细胞谱系细胞的更复杂多样性。因此,我们需要识别人脑中的少突胶质细胞 - 神经元串扰。除了典型的白质紊乱,如多发性硬化症和脑白质营养不良症(van der Knaap和Bugiani,2017),在精神疾病中始终发现白质异常(Haroutunian et al。,2014) 。越来越多的证据表明,神经元和少突胶质细胞之间的串扰对于正常的神经网络功能非常重要(Bergles et al。,2000; Velez-Fort et al。,2010; Maldonado and Angulo ,2015)和髓鞘形成(Almeida和Lyons,2016; Kougioumtzidou et al。,2017)。因此,为了研究神经元 - 少突胶质细胞相互作用在正常和患病大脑中的参与,我们需要基于人类的模型系统。由于目前的检测主要涉及非人类细胞(Cui et al。,2010; Hill et al。,2014; Clark et al。, 2017; Pang et al。,2018; Treichel和Hines,2018),我们开发了基于人诱导多能干细胞(iPSC)的共培养模型,以研究人神经元和人少突胶质细胞祖细胞之间的串扰(的OPC)。所提出的共培养方法用于研究结节性硬化症复合体(TSC)中的神经元-OPC相互作用(Nadadhur et al。,2019),这是一种遗传多系统疾病,显示灰质和白质异常。大脑。尽管在TSC神经元的单一培养物中存在一些神经元异常,但在存在OPC的情况下,轴突密度和肥大增加变得明显(Nadadhur et al。,2019)。这表明只有少突胶质细胞存在时才能研究特定的神经元表型。相反,少突胶质细胞成熟高度依赖于神经元信号传导。因此,这些培养系统可用于研究涉及复杂神经元 - 少突胶质细胞相互作用的健康和疾病的多个过程,并为全人类细胞的药物筛选平台的开发提供前景,例如,,患者iPSCs。总之,这种新型人类神经元-OPC共培养模型可用于研究健康和疾病中的神经元-OPC串扰。

关键字:人类诱导性多能干细胞, 神经, 少突胶质细胞, 共培养, 髓磷脂, 神经胶质相互作用

材料和试剂

  1. 12孔板(VWR,目录号:665180)
  2. 10μl过滤嘴(Thermo Fisher,目录号:11977714)
  3. 100μl过滤嘴(Thermo Fisher,目录号:11953466)
  4. 1000μl过滤嘴(Thermo Fisher,目录号:11973466)
  5. 6孔板(VWR,目录号:734-2323)
  6. 18毫米盖玻片(VWR,目录号:631-0153)
  7. 5毫升移液器(VWR,目录号:606180)
  8. 10毫升移液器(VWR,目录号:607180)
  9. 15毫升管(VWR,目录号:525-0400)
  10. 显微镜载玻片(VWR,目录号:631-0108)
  11. 注射器针头(BD Biosciences,目录号:300400)
  12. 4',6-二脒基-2-苯基吲哚(DAPI)(Sigma-Aldrich,目录号:D9542-5MG)
  13. Accutase(Merck-Millipore,目录号:sf006)
  14. 抗MAP2抗体(Abcam,目录编号:AB5392)
  15. 抗MBP抗体(Covance,目录号:SMI-99P)
  16. Anti-Olig2抗体(Merck-Millipore,目录编号:AB9610)
  17. 抗SMI312抗体(Eurogentec,目录编号:SMI-312P-050)
  18. 阿拉伯糖基胞嘧啶(AraC)(Merck-Millipore,目录号:251010)
  19. β-巯基乙醇(赛默飞世尔科技,目录号:21985023)
  20. B27含维生素A(赛默飞世尔科技,目录号:17504-044)
  21. 不含维生素A的B27(赛默飞世尔科技,目录号:12587-010)
  22. 碱性成纤维细胞生长因子(bFGF)(Peprotech,目录号:100-18B-50ug)
  23. 脑源性神经营养因子(BDNF)(Peprotech,目录号:450-02)
  24. 生物素(Sigma-Aldrich,目录号:B4501-100MG)
  25. 牛血清白蛋白(BSA)(西格玛奥德里奇,目录号:A9418)
  26. 环磷酸腺苷(cAMP)(西格玛,目录号:D0260-5MG)
  27. 定义胰蛋白酶抑制剂(DTI)(Thermo Fisher Scientific,目录号:R007100)
  28. 含有Glutamax的DMEM / F12(Life Technologies,目录号:21331-020)
  29. 不含L-谷氨酰胺的DMEM / F12(Life Technologies,目录号:21331-046)
  30. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  31. Dorsomorphin(Tocris Bioscience,目录号:3093/10)
  32. 表皮生长因子(EGF)(Peprotech,目录号:AF-100-15-500ug)
  33. 乙二胺四乙酸(EDTA)(Invitrogen,目录号:15575-038)
  34. 胎牛血清(FBS)(ThermoFisher Scientific,目录号:16140063)
  35. Fluoromount G(Southern Biotech,目录号:0100-01)
  36. 胶质细胞源性神经营养因子(GDNF)(Peprotech,目录号:450-10)
  37. Geltrex(Life Technologies,目录号:A1413302)
  38. Glutamax(赛默飞世尔科技,目录号:35050-038)
  39. 山羊抗大鼠/小鼠/兔/鸡/豚鼠Alexa Fluor抗体(Life Technologies)
  40. 肝素(Sigma-Aldrich,目录号:H3393-50KU)
  41. HEPES(赛默飞世尔科技,目录号:15630-056)
  42. Human Sonic Hedgehog(hSHH)(Peprotech,目录号:100-45-500ughSHH)
  43. 胰岛素样生长因子1(IGF1)(Peprotech,目录号:100-11-100ug)
  44. 胰岛素(Sigma-Aldrich,目录号:I9278)
  45. KCl(Sigma-Aldrich,目录号:P5405-250gr)
  46. KH 2 PO 4 (Sigma-Aldrich,目录号:P5379)
  47. L-谷氨酰胺(Thermo Fisher Scientific,目录号:25030-024)
  48. 小鼠层粘连蛋白(mLaminin)(Sigma-Aldrich,目录号:L2020-1mg)
  49. N2补充剂(Thermo Fisher Scientific,目录号:17502-048)
  50. Na 2 HPO 4 (Sigma-Aldrich,目录号:S7907-500gr)
  51. NaCl(VWR,目录号:S9888-1Kg)
  52. Neurobasal培养基(赛默飞世尔科技,目录号:21103-049)
  53. Neurotrophin 3(NT3)(Peprotech,目录号:450-03-100ug)
  54. Noggin(Peprotech,目录号:120-10C)
  55. 非必需氨基酸(NEAA)(赛默飞世尔科技,目录号:11140-035)
  56. 正常山羊血清(NGS)(Life Technologies,目录号:16210-064)
  57. 青霉素/链霉素(Pen / Strep)(西格玛奥德里奇,目录号:P0781)
  58. 多聚甲醛(PFA)16%(电子显微镜科学,目录号:15710-S)
  59. Poly-L-ornithine(PLO)(Sigma-Aldrich,目录号:P3655-100mg)
  60. Rock Inhibitor(RI)(Y27632; Selleckchem,目录号:S1049)
  61. 维甲酸(RA)(西格玛奥德里奇,目录号:R2625-100MG)
  62. SB431542(Selleckchem,目录号:S1067)
  63. 三碘甲状腺原氨酸(T3)(西格玛奥德里奇,目录号:T6397-100MG)
  64. TeSRE8(干细胞技术,目录号:5940)
  65. Triton X-100(Sigma-Aldrich,目录号:T8787-100ml)
  66. TryplE(Life Technologies,目录号:12563-029)
  67. 丙戊酸(VPA)(Sigma-Aldrich,目录号:P4543-10G)
  68. 维生素C /抗坏血酸(Sigma-Aldrich,目录号:A4544-25G)
  69. N1补充剂(Sigma-Aldrich,目录号:N6530-5ML)
  70. Neuroglia共培养基(参见食谱)
  71. 含有维生素A的神经维持培养基(NMM)(见食谱)
  72. 不含维生素A的神经维持培养基(NMM)(见食谱)
  73. N2培养基(见食谱)
  74. NB介质(见食谱)
  75. 阻塞缓冲液(见食谱)
  76. PBS(见食谱)
  77. PLO / mLaminin涂层(见食谱)
  78. Geltrex涂层(见食谱)

设备

  1. 移液器控制器(BD Biosciences,型号:Falcon Express)
  2. 培养箱(粘合剂,型号:9140-0044; 5%CO 2 ,20%O 2 )
  3. 培养箱(粘合剂,型号:9140-0044;5%CO2,20%O2)
  4. 片剂顶部离心机(Eppendorf,型号:离心机5810)
  5. -80-C冷冻柜(Thermo Fisher Scientific;型号ULT1786-6-V49)
  6. 亮场显微镜(蔡司,型号:Axiovert 40C.)
  7. 荧光显微镜(徕卡微系统,型号:徕卡DM6000B)

软件

  1. 哥伦布2.5在线软件(Perkin Elmer)
  2. 徕卡高级荧光应用套件(徕卡)

程序

  1. 神经分化
    神经元根据先前发表的协议进行分化(Shi等人,2012年;Nadadhur等人,2017年),很快:
    1. HIPSC在1毫升的Tesre8培养基中通过盖尔特涂层的12孔板,每孔10毫米ri。
    2. 每天刷新全部中等。
    3. 两天后(或当细胞100%汇合时),加入1毫升含有维生素A+1毫米背吗啡+10毫米SB431542的神经维持培养基(NMM)。
    4. 每天刷新所有媒体,直到第12天。
      注意:在第8天到第12天之间,应出现均匀的神经上皮层(见图1)。
    5. 按照配方中的说明,准备PLO/MLaminin涂层6孔板。
    6. 通过手工切割收集神经上皮玫瑰花结细胞(NES细胞)(见注释中的描述)并将其置于6孔板中。< BR> 注:用维生素A+1毫米多索芬+10毫米SB431542+10毫米碘化锂制备新鲜的预热NMM。从12孔板上取出培养基,用PBS清洗一次,然后加入1毫升新制备的培养基。用70%乙醇消毒显微镜区域,并用10毫米的尖端切割玫瑰花结,以标记其边缘并将其提起。用5毫升移液管收集漂浮的玫瑰花结,并将其移到pLO/mLaminin涂层的6孔板上,用维生素A+1毫米多索芬+10毫米SB431542+10毫米ri的NMM。12孔板的1孔的所有玫瑰花结都镀在6孔板的1孔中。
      < BR>
      图1。Brightfield图片显示了在第8天到第12天之间形成的神经丛结构。比例尺=200米。
      < BR>
    7. 第13天,将培养基改为2毫升预热的NMM,加入维生素A+20 ng/ml bFGF+20 ng/ml EGF。
    8. 每天取出1毫升培养基,加入1毫升预先加热的NMM和维生素A+40 ng/ml bFGF+40 ng/ml EGF。当细胞汇合时,用锥虫以1:2或1:3的比例传代细胞,如下所述。
      注:
      1. 细胞可在NES细胞期保存1-4代。
      2. 当只改变一半培养基时,生长因子浓度加倍:与留在井中的培养基一起,浓度将与步骤a7中的浓度相似。
      3. 锥虫通道:从井中取出所有培养基,加入300毫升预热锥虫。在井周围旋转,在室温(RT)下培养2分钟。加入600毫升预热DTI或培养基以停止反应,并将细胞收集到含有5毫升NMM的试管中。在室温下以300 x g的速度旋转5分钟,然后将颗粒重新悬浮在所需的介质中。
    9. 为了开始神经诱导,将锥状通道后的NES细胞置于PLO/mLaminin包被的12孔板中,预先加热,每孔1毫升NMM+20 ng/ml bFGF+20 ng/ml EGF。
    10. 当细胞达到80%-90%汇合时,将一半培养基(500 m/井)切换到预热的氮气培养基+800ng/ml HSHH。
      注:这被视为差异化协议的第一天。
    11. 在第4天之前,每天用预热的N2培养基+800 ng/ml HSHH刷新一半培养基(500 m/井)。
    12. 在第5天,将一半的培养基(500米/井)切换到预热NB培养基+40毫米VPA。
    13. 在第7天之前,每天用预热的NB培养基+40毫米VPA刷新一半培养基(500米/井)。
    14. 如食谱所述,在第8天之前准备好涂有plo/mlaminin的12孔盘子。
    15. 在第8天,用Accutase1:2或1:3将神经祖细胞传至一个新井(解释见注释)。在预热的1.5ml NB培养基+20ng/ml BDNF+10ng/ml GDNF+10ng/ml IGF1+1μm cAMP中培养板细胞。
      Accutase处理:取下所有培养基,每孔12孔培养皿中加入300μl Accutase,将培养皿放回培养箱5-7分钟。轻轻敲击培养皿,细胞会明显脱落。此时,将细胞收集在试管中,加入5毫升新鲜预热培养基,在室温下以300×g旋转5分钟,并将颗粒重新悬浮在所需培养基中。小心地重新使用;不要将块分成单个单元格。
    16. 直到第18天,每周三次刷新培养基,去除1毫升培养基,加入1毫升预热NB培养基+30 ng/ml BDNF+15 ng/ml GDNF+15 ng/ml IGF1+1.5μm cAMP。
    17. 在第12天,准备一个12孔夹心板,如注释、图2和视频1所述。如食谱所述,在盘子上涂上盖尔特。在每个含有预热DMEM/F12培养基(谷氨酰胺+10%FBS+1x NeAA+1x Pen/Strep)的孔中放置25000个原代大鼠星形胶质细胞。
      注:夹心板准备:取12孔板,用火焰加热无菌注射器针头。在每口井的4个角上打孔,使井的塑料底部均匀地出现小凸起。这些凸起将有助于在其上悬挂一个盖玻片,与井底的细胞相隔一小段距离。参见图2和视频1。
      < BR> 视频1.夹层板
      < BR>
    18. 在第18天之前,准备所需数量的12孔板,其中包含直径为18 mm的盖玻片,并按照配方中的说明涂上plo/层粘连蛋白。
      注意:盖片可在4°C下用副膜密封板,最多保存1周。
    19. 在第18天,如注释所述,用Accutase使神经元单细胞悬浮。在预先加热的1毫升NB培养基+20纳克/毫升BDNF+10纳克/毫升GDNF+10纳克/毫升IGF1+1μm cAMP中,在预先涂层的PLO/mLaminin盖玻片上每12孔板上放置150万个细胞。
      注:取下所有培养基,每孔12孔板中加入300μl的Accutase,并将培养板放回培养箱10-15分钟。轻轻敲击培养板,细胞会明显脱落。用1000μl移液管缓慢分离Accutase中的细胞,并用5 ml神经基础培养基移液管移到15 ml管中。在室温下,以300 x g旋转5分钟。取出上清液,非常轻地将颗粒重新放入1 ml NB培养基中,并尝试使用1000μl移液管非常轻地打破细胞团(上下移液管最多3-4次)。然后离开管子2-5分钟,让大团块在底部沉淀下来。将上清液放入另一个试管中;搅拌颗粒(大团不会有成熟细胞,但主要是增殖前体细胞)。
      < BR>
      图2。三明治板准备。a.取12孔板和无菌注射器针头。把针点燃,直到它变热。用滚烫的针头在井(B-D)的四个侧面做一个凸起。星形胶质细胞将被镀在板的底部,一个有神经元的盖玻片可以被倒置在板的顶部。床单会靠在颠簸处。另请参见视频1。
      < BR>
    20. 第19天,翻转盖玻片,将粘附的神经元倒置在星形胶质细胞板上的突起上。< BR> 注意:到目前为止,星形胶质细胞应该已经达到80%的汇合,以便支持神经元。
    21. 用预先加热的nb培养基+40ng/m l bdnf+20ng/ml gdnf+20ng/ml igf1+2μm camp,每周更换一半培养基(500μl/孔)。
    22. 在第二次刷新前一天(24/25天),向培养物中添加1μm arac以阻止增殖。
    23. 第二天(第25/26天)用预热的NB培养基+40 ng/m l BDNF+20 ng/ml GDNF+20 ng/ml IGF1+2μm cAMP刷新一半培养基(500μl/孔)。
    24. 将培养基保存到第37天(见图3),同时按步骤21所述每周刷新培养基两次。
      < BR>
      图3。共培养前37天神经元的Brightfield图像,显示三明治培养中神经元网络的形成。比例尺=100μm。
      < BR>
  2. opc差异
    OPC是根据先前发布的协议(Izrael等人,2007)生成的,很快:
    1. 用EDTA穿过HIPSC,并在3毫升预热NMM中的抗粘附6孔板上镀上Vit A+20 ng/ml EGF+4 ng/ml bFGF+10μm Ri+20 ng/ml T3/孔,以形成胚状体(EB)(见图3)。
      注:对于1口EBS井,使用2口IPSC井。
    2. 第二天(第1天),刷新培养基:将细胞旋转到孔的中间,小心地吸出2毫升培养基。加入2毫升预热的NMM,Vit A+20 ng/ml EGF+4 ng/ml bFGF+10μm Ri+20 ng/ml T3。
    3. 在第2天,用预先加热的NMM(含维生素A+20 ng/ml EGF+4 ng/ml bFGF+10μm RA)刷新步骤B2中所述的培养基。
    4. 每隔一天重复一次。
    5. 在第10天,如注释所述,将EBS板放在盖尔特涂层板上。如果EBS变得非常暗,或者密度降低,则在第10天之前,但不是在第4天之前,放置EBS。根据我们的经验,第8天最理想(见图4)。
      注:通过在15或50毫升的试管中收集带有EBS的介质,在盖尔特涂层板上放置EBS。离开管子2-3分钟,让电子束稳定下来。小心地吸出大部分培养基,并在适当数量的所需培养基中重新使用EBS(12孔板为1毫升,6孔板为2毫升)。在第4-9天,在预热的NMM中用维生素A+20 ng/ml EGF+4 ng/ml bFGF+10μm RA+20 ng/ml T3镀EBS。如果在第10天电镀EBS,则应使用含有维生素A+20 ng/ml EGF+20 ng/ml T3的NMM。
      < BR>
      图4。Brightfield图片显示胚胎体形成(A)和胚胎体细胞在盖尔特涂层板(B)上电镀后的生长。比例尺=500μm。
      < BR>
    6. 第10天,将所有培养基切换至预热NMM,维生素A+20 ng/ml EGF+20 ng/ml T3。
    7. 从现在起:每隔一天刷新培养基,当细胞达到90%的密度时,用Accutase将其分裂到一个新的盖尔特涂层板上。< BR> 注:传代后将ri加入培养基中,帮助细胞恢复。
    8. 在第18天,将培养基改为不含vit a+20ng/ml egf的nmm;如步骤b7所述,保持细胞的新鲜和分裂。
    9. 第37天,将培养基改为不含维生素A+5 ng/ml bfgf+5 ng/ml egf+1μg/ml mlaminin+50μg/ml vit c的nmm;如步骤B7所述,保持细胞的新鲜和分裂。
    10. 第39天,将培养基改为不含维生素A+5 ng/ml bfgf+5 ng/ml egf+1μg/ml mlaminin+50μg/ml vit c+50 ng/ml noggin的nmm;如步骤B7所述保持细胞的新鲜和分裂。
    11. 在第42天,将培养基改为不含维生素A+1μg/ml小鼠层粘连蛋白+50μg/ml维生素C+50 ng/ml诺金的NMM;如步骤B7所述保持细胞的新鲜和分裂。
    12. 将opc培养物保存在步骤b11的培养基中直到第65天(见图5)。
      < BR>
      图5。opc分化。在opc分化方案结束时,细胞呈现胶质形态(a,45天),并表达opc标记物,如olig2(b,65天)。比例尺=100μm。
      < BR>
  3. 神经元opc共培养
    1. 在共培养的第1天,移动第37天的神经元并将第65天的opcs固定在顶部:
      1. 取一个新的12孔板。
      2. 用钳子从大鼠星形胶质细胞板上取下第37天的神经元覆盖物,并将其放入一个新的12孔板中(顶部有神经元,因此将覆盖物翻转至星形胶质细胞板上的情况进行比较)。
        注意:由于神经元盖玻片中已经含有涂层,因此无需对新板进行涂层。
      3. 加入1毫升预热的神经胶质细胞共培养基。
      4. 用第65天的OPC做一个盘子。
      5. 取出培养基,用PBS清洗板一次。
      6. 去除PBS,向每个孔(6孔板)添加750μL Accutase。
      7. 将培养板在37°C下培养5-10分钟。
      8. 轻轻敲击,检查细胞是否从培养板上脱落;如果没有,则继续培养。
      9. 当细胞分离时,用预先加热的9毫升DMEM/F12在15毫升试管中收集细胞悬浮液。
      10. 在300x g下旋转5分钟。
      11. 在1毫升预热神经胶质细胞共培养基中再培养细胞。
      12. 取10μl细胞悬液进行细胞计数。
      13. 板4 x 10512孔板上的每个盖片的OPC。
    2. 每周更新培养基的一半两次:取出500μl培养基,加入500μl新的预热神经胶质细胞共培养基。
    3. 在共培养的第28天,用4%pfa固定细胞15-20分钟,并立即用盖玻片进行免疫染色(见图6)。
      < BR>
      图6。共培养物的免疫染色。(a)显示许多olig2阳性的opcs和mbp阳性的成熟少突胶质细胞有map2阳性的树突。(b)显示少突胶质细胞(MBP阳性)与树突(MAP2阳性)紧密相互作用,比例尺=25μm。
      < BR>
  4. 免疫染色
    1. 用PBS清洗板6 x 5分钟。
    2. 拆下PBS。
    3. 每口井加入300μl封闭缓冲液。
    4. 室温下孵育1h。
    5. 拆下阻塞缓冲器。
    6. 加入稀释在300μl封闭缓冲液中的一级抗体。
    7. 在室温下孵育1h,然后在4℃下过夜。
    8. 用PBS清洗6 x 5分钟。
    9. 在300μl封闭缓冲液中加入稀释1:1000的二级抗体。
    10. 室温下孵育2小时。
    11. 用PBS清洗6 x 5分钟。
    12. 拆下PBS。
    13. 加入1:1000稀释在PBS中的DAPI。
    14. 室温下孵育2分钟。
    15. 用PBS清洗1个。
    16. 将盖玻片和荧光支架G倒置安装在显微镜玻片上。

数据分析

  1. 盖玻片可以用OPC成熟标记(例如OLIG2、MBP)和神经元形态学标记(例如MAP2、SMI312)染色。根据我们的经验,对于一个对照系,大约有10%的olig2+细胞,1-2%的mbp+细胞和大约40%的细胞是map2+神经元。 注:OLIG2和MBP并不标记所有人类少突胶质细胞系细胞。
  2. 单元属性可以通过自动化软件包进行分析,例如Columbus 2.5在线软件(请参见https://www.perkinelmer.com/nl/product/image-data-storage-and-analysis-system columbus有关columbus软件的更多信息,以及图7(例如分析图片)用于分析神经元形态学的算法,可以使用形态学、soma识别和共同定位。更准确地说,基于map2和核染色可以确定每个神经元的树突密度和平均树突密度。基于SMI312染色的类似分析可用于分析轴突密度。
  3. opc成熟度可以通过免疫染色来评估成熟的少突胶质细胞标志物,如mbp。因为不同的细胞系可能以不同的速度生长,并且在少突胶质细胞的产生中有不同的效率,所以最好将MBP阳性的数量与OLIG2阳性的OPC的数量联系起来。
  4. 正确的统计分析取决于感兴趣的研究问题。例如,这也取决于培养设置,即,当用来自不同患者和对照系的神经元培养1个对照少突胶质细胞系时,与所有对照/患者少突胶质细胞与对照/患者神经元的组合相比。关于我们如何进行统计分析,请参见Nadadhur等人(2019年)。
    < BR>
    图7。哥伦布细胞分析的例子。荧光图像可以导入到哥伦布(a)中,哥伦布(a)具有预先编程的设置,以查找细胞核(b)和跟踪轴突(c)。输出包括最大轴突长度、总轴突长度和每个细胞的末端数等参数。

笔记

由于IPSC分化协议中存在一些常见的变异,我们建议在神经元OPC共培养开始之前进行一些质量检查。

  1. 通过在分化方案的57-60天左右检测olig2的表达,可以验证opc的正确分化。
  2. 在培养的第37天,map2/beta-iii微管蛋白/neun染色可以证实神经元的分化是正确的。如果观察到gfap的表达,建议丢弃培养物,因为胶质细胞污染可能干扰患者和对照ipscs的混合共培养物。更多无神经胶质细胞的神经元培养图像,请参见Nadadhur等人(2017年)。

食谱

  1. 神经胶质细胞共培养基
    480ml dmem/f12
    480ml神经基底膜
    10毫升n1补充剂
    20毫升B27补充剂
    10毫升NEAA
    60 ng/ml t3
    100 ng/ml生物素
    10 ng/ml NT3
    1μg/ml盐酸米林
    20 ng/ml bdnf
    1μm营地
  2. 含维生素a的神经维持培养基(nmm)
    482毫升DMEM/F12+谷氨酰胺
    482毫升神经基底膜
    10毫升含维生素A的B27补充剂
    5μg/ml胰岛素
    5毫升谷氨酰胺
    5毫升氮气补充
    5毫升NEAA
    10毫升钢笔/链球菌
    10μmβ-巯基乙醇
  3. 不含维生素a的神经维持培养基(nmm)
    482毫升DMEM/F12+谷氨酰胺
    482毫升神经基底膜
    10毫升不含维生素A的B27补充剂
    5μg/ml胰岛素
    5毫升谷氨酰胺
    5毫升氮气补充
    5毫升NEAA
    10毫升钢笔/链球菌
    10μmβ-巯基乙醇
  4. 氮气介质
    48毫升DMEM/F12,不含L-谷氨酰胺
    500μl 100x氮气
    500μl非必需氨基酸(10 mM储备)
    500μl谷氨酰胺(200 mm库存)
    2μg/ml肝素
    500μl钢笔/链球菌
  5. nb中等
    475毫升神经基底膜
    10毫升B27
    18毫米HEPES
    1.25毫升谷氨酰胺
    5毫升钢笔/链球菌
  6. 阻塞缓冲区
    95毫升PBS
    5毫升天然气
    300毫米Triton X-100
    0.1克牛血清白蛋白
  7. pbs
    8g氯化钠
    0.1克kcl
    1.44克钠2hpo4
    0.24克kh2po4
    800毫升H2O
  8. plo/mlaminin涂层
    1. 加入20 mg/ml PLO和PBS(12孔板0.5 ml;6孔板1 ml)
    2. 37℃孵育4小时或O/N
    3. 删除plo
    4. 用PBS清洗3次
    5. 加入20 mg/ml盐酸米拉明(与PLO的用量相同)
    6. 在37℃下培养至少2小时或O/N
    7. 去氨胺
    8. 添加培养基和单元格
  9. 盖尔特涂层
    1. 用冷DMEM/F12稀释Geltrex 1:1
    2. 大等份-80'C
    3. 用冷DMEM/F12稀释1:50
    4. 加入适量(12孔板0.5ml,6孔板1ml)
    5. 在37℃下孵育至至少1小时
    6. 去除凝胶剂后,立即加入含有细胞的培养基

致谢

这项研究得到了阿姆斯特丹神经科学和欧盟msca-itn认知网(fp7-people-2013-itn 607508)的资助。V.M.H.由Zonmw Vidi研究基金(91712343)、E-Rare联合呼叫项目(9003037601)和欧洲白质营养协会(ELA)研究基金(2014-012L1)资助。神经元分化方案基于Nadadhur等(2017)的先前研究。opc鉴别协议是基于以色列等(2007)的先前研究。

相互竞争的利益

作者声明没有利益冲突。

伦理学

所有的实验都免于医学伦理审查委员会(METC)的批准,该委员会是VU医学中心的机构审查委员会。

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引用:Dooves, S., Nadadhur, A. G., Gasparotto, L. and Heine, . M. (2019). Co-culture of Human Stem Cell Derived Neurons and Oligodendrocyte Progenitor Cells. Bio-protocol 9(17): e3350. DOI: 10.21769/BioProtoc.3350.
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