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Feb 2018
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Isolation of Neural Stem Cells from the Embryonic Mouse Hippocampus for in vitro Growth or Engraftment into a Host Tissue
从胚胎小鼠海马分离神经干细胞用于体外生长或植入宿主组织   

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Abstract

For both stem cell research and treatment of the central nervous system disorders, neural stem/progenitor cells (NSPCs) represent an important breakthrough tool. In the expanded stem cell-based therapy use, NSPCs not only provide a powerful cell source for neural cell replacement but a useful model for developmental biology research. Despite numerous approaches were described for isolation of NSPCs from either fetal or adult brain, the main issue remains in extending cell survival following isolation. Here we provide a simple and affordable protocol for making viable NSPCs from the fetal mouse hippocampi, which are capable of maintaining the high viability in a 2D monolayer cell culture or generating 3D neuro-spheroids of cell aggregates. Further, we describe the detailed method for engraftment of embryonic NSPCs onto a host hippocampal tissue for promoting multilinear cell differentiation and maturation within endogenous environment. Our experimental data demonstrate that embryonic NSPCs isolated using this approach show the high viability (above 88%). Within a host tissue, these cells were capable of differentiating to the main neural subpopulations (principal neurons, oligodendrocytes, astroglia). Finally, NSPC-derived neurons demonstrated matured functional properties (electrophysiological activity), becoming functionally integrated into the host hippocampal circuits within a couple of weeks after engraftment.

Keywords: Embryonic neural stem/progenitor cells (NSPCs) (胚胎神经干/祖细胞), Mouse hippocampus (小鼠海马), Cell viability (细胞活力), Adherent 2D monolayer cell culture (贴壁二维单层细胞培养), 3D neuro-spheroids (三维神经球体), Organotypic hippocampal tissue (器官海马组织)

Background

Multipotent stem cells of neural origin, neural stem/progenitor cells (NSPCs), represent a versatile tool for enabling nerve cell replacement in a number of neurological disorders characterized by the loss of specialized neural subpopulations. The unlimited lifespan and directed NSPC differentiation into the key neural subtypes (neurons, astrocytes, and oligodendrocytes) make these cells the most attractive candidate for emerging stem cell-based therapies for presently incurable brain disorders (Kopach and Pivneva, 2018). Stem cells exist in the developing and mature nervous system and can be isolated from either fetal brain (embryonic NSPCs) or, as established more recently, from mature brain (in adults, both subventricular and subgranular zones in dentate gyrus of the hippocampus, forebrain, cerebellum and olfactory bulb) (Pagano et al., 2000; Roy et al., 2000; Klein et al., 2005; Behnan et al., 2017; Kempermann et al., 2018). Despite that adult stem cells bear expectations for an advanced translational approach (Kempermann et al., 2018), a limited number of rigorous evidence supports this research avenue at the moment.

Accumulating evidence for the properties of fetal NSPCs and phenotype specifications, confirmed by dedicated clinical trials, retains a focus on this stem cell type. Furthermore, embryonic NSPCs represent an essential tool for developmental biology research. Various methodological approaches were probed to isolate NSPCs from the fetal brains and to maintain cells in conditions determining their high viability, hence, progeny. Such conditions largely dictate phenotyping of differentiating progenitors to acquire the competitive NSPC-derived cells. The two main strategies for preserving the high viability of isolated NSPCs exist in i) cell culture in vitro and ii) grafting into a host tissue. A classical 2D culture, an adherently expanded monolayer of cells, has been routinely used for stem cell growth in vitro and/or for directed phenotypic differentiation (Conti et al., 2005; Pollard et al., 2006). In parallel, newer developed approach of generating 3D cultures, neuro-spheroids of an assembly of NSPCs at various phenotypic and developmental stages, has been emerged to represent more physiological conditions pertinent to neuronal differentiation rather than glial (Ignatova et al., 2002; Suslov et al., 2002; Makri et al., 2010). This approach can potentially enhance the therapeutic potential of NSPCs in vitro. Another alternative–grafting of viable NSPCs into brain tissue–fulfill a strategy for promoting the multi-lineage differentiation of progenitors within a host tissue, regulated by endogenous environment. Such an approach has recently been implemented in the treatment of the ischemia-damaged hippocampal tissue (Tsupykov et al., 2014; Kopach et al., 2018).

Here we provide the detailed protocol that we routinely use for isolation of viable NSPCs from the fetal mouse hippocampus. This protocol was also utilized for engraftment of undifferentiated cells to the hippocampal tissue for monitoring maturation of NSPC-derived cells within a host environment in our previous studies (Kopach et al., 2018). We describe all the details on how to test the viability of the obtained embryonic NSPCs and maintain the cells in multiple approaches for further proposed use.

Materials and Reagents

  1. Materials
    1. 15 ml and 50 ml centrifuge tubes (Corning, catalog numbers: 430790, 430828)
    2. 1.5 ml Eppendorf tubes (Sigma-Aldrich, catalog number: T9661)
    3. Glass Pasteur pipette (1 mm diameter) (Fisher Scientific, catalog number: 13-678-6A)
    4. 35 mm, 60 mm, and 100 mm tissue culture dishes (CELLSTAR, catalog numbers: 627160, 628160, 664160)
    5. Falcon® 40-µm cell strainer (Corning, Falcon®, catalog number: 352340)
    6. 0.45-µm sterile filter (Fisher Scientific, MerckTM, catalog number: 12279299) 
    7. 6-well and 24-well tissue culture plates (Greiner Bio-One, catalog numbers: 657160, 662160)
    8. 13 mm glass coverslips (Henz Herenz, catalog number: 1051204)
    9. SuperFrost® glass slides (VWR, Thermo Fisher Scientific, catalog number: 631-0706)
    10. Plastic serological pipettes (5 ml, 10 ml, and 25 ml) (Corning® Costar® Stripette®, catalog numbers: 4487, 4488, 4489)
    11. Pipette tips (TipOne, STARLAB, catalog numbers: S1111-3700, S1111-1706, S1111-6701)
    12. Millipore® Millicell® cell culture plate inserts (Sigma-Aldrich, catalog number: Z353086)
    13. Whatman® qualitative filter paper (Sigma-Aldrich, catalog number: WHA1001110)
    14. Scalpel blades (Fisher Scientific, Swann-MortonTM, catalog number: 11772724)
    15. Scalpel with retractable blade (Eickemeyer®, catalog number: 100504)
    16. Parafilm® M (Sigma-Aldrich, catalog number: P7793)

  2. Animals
    1. Pregnant FVB-Cg-Tg(GFPU) 5Nagy/J GFP mice (obtained from the animal facilities of State Institute of Genetic and Regenerative Medicine)
    2. Embryos of FVB-Cg-Tg(GFPU) 5Nagy/J GFP mice at the developmental stage of E16-E17
    3. Pups of FVB mice (8 to 9-days old)

  3. Reagents
    1. HBBS without calcium and magnesium (Sigma-Aldrich, catalog number: 55021C)
    2. Paraformaldehyde (PFA, Sigma-Aldrich, catalog number: P6148)
    3. 0.25% trypsin-EDTA (Sigma-Aldrich, catalog number: T4049)
    4. 22% Percoll (Sigma-Aldrich, catalog number: 17-0891-01)
    5. Neurobasal® medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049)
    6. B-27® supplement (50x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044)
    7. GlutaMAXTM supplement (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061)
    8. N-acetyl-L-cysteine (NAC) (Sigma-Aldrich, catalog number: A7250)
    9. Penicillin-streptomycin (P/S) solution (100,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122)
    10. FGF-2 (Sigma-Aldrich, catalog number: SRP4037)
    11. Matrigel (BD Biosciences, catalog number: 354234)
    12. Triton® X-100 (Sigma-Aldrich, catalog number: T8787)
    13. Accutase® solution (Sigma-Aldrich, catalog number: A6964)
    14. 0.4% Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061)
    15. Antibodies
      1. Primary
        1. Mouse anti-Nestin antibody (1:100) (Santa Cruz Biotechnology, catalog number: sc-23927)
        2. Rabbit anti-β-tubulin III (1:500) (Sigma-Aldrich, catalog number: T2200)
        3. Mouse anti-GFAP (1:200) (Sigma-Aldrich, catalog number: AMAB91033)
        4. Rabbit anti-olig-2 (1:200) (Abcam, catalog number: ab136253)
        5. Mouse anti-NeuN (1:100) (Millipore, catalog number: MAB377)
      2. Secondary
        1. Donkey anti-mouse Alexa Fluor 555 (1:1,000) (Thermo Fisher Scientific, catalog number: A-31570)
        2. Donkey anti-rabbit Alexa Fluor 647 (1:1,000) (Thermo Fisher Scientific, catalog number: A-31573) 
        3. Nuclei tracer Hoechst 33342 (1:5,000) (Sigma-Aldrich, catalog number: 14533)
    16. ImmunoHistoMountTM (Sigma-Aldrich, catalog number: I1161)
    17. 70% Ethanol (Sigma-Aldrich, catalog number: 459836)
    18. Sodium dihydrogen phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S0751)
    19. Disodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S0876)
    20. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045)
    21. Minimum Essential Medium (MEM) (Sigma-Aldrich, catalog number: M2249)
    22. Tris (Sigma-Aldrich, catalog number: 252859)
    23. Sodium bicarbonate (Sigma-Aldrich, catalog number: 792519)
    24. HEPES (Thermo Fisher Scientific, catalog number: 15630080)
    25. D(+)-glucose (Sigma-Aldrich, catalog number: G8270)
    26. Horse serum (Sigma-Aldrich, catalog number: H1138)
    27. Phosphate buffered saline (PBS), pH 7.4 (Sigma-Aldrich, catalog number: P3813)
    28. BD Cell Wash buffer (BD Biosciences, catalog number: 349524)
    29. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 05470)
    30. 7-AAD (BD PharmingenTM, catalog number: 559925)
    31. 0.2 M Phosphate buffer (pH 7.4) (see Recipes)
    32. 4% Paraformaldehyde (PFA) in 0.1 M phosphate buffer (see Recipes)
    33. Growth medium (see Recipes)
    34. Tissue dissecting medium (pH 7.3) (see Recipes)
    35. Tissue culture medium (pH 7.2) (see Recipes)
    36. Antibody solution (see Recipes)
    37. Blocking solution (see Recipes)

Equipment

  1. Pipettes (Eppendorf, Research® Plus, catalog numbers: EP 3123000012; EP 3123000098; EP 3123000055; EP 3120000062)
  2. 250 ml Graduated cylinder (Karter Scientific, catalog number: 213I13)
  3. Dry heat sterilizing cabinet (Grande, model: GIO-072)
  4. Fine tools for dissection and manipulating embryonic brains
    1. Dissecting scissors (Dumont, catalog number: 14090-09)
    2. Small spatula (Dumont, catalog number: 10090-13)
    3. Forceps (Dumont, catalog number: 91197-00)
    4. Angled forceps (Dumont, catalog number: 00125-11)
  5. C-Chip disposable hemocytometer/Neubauer (Labtech International, Heathfield, UK, catalog number: DHC-N01)
  6. Refrigerated centrifuge (Thermo Fisher Scientific, model: IEC-CL30R)
  7. Water bath (Biosan, model: WB-4MS)
  8. Biosafety Cabinet Class II Type A2 (Labconco, model: Purifier Logic)
  9. CO2 incubator (Thermo Fisher Scientific, model: SteriCycle 371)
  10. Flow cytometer (Becton Dickinson, model: BD FACSAria сell sorter)
  11. Tissue chopper (McIlwain, model: MTC/2E)
  12. Alcohol burner (Sigma-Aldrich, catalog number: Z509604)
  13. Inverted microscope (Olympus, model: IX71, equipped with camera DP-20)
  14. Confocal microscope (Olympus, model: FV1000)
  15. Stereo microscope (Olympus, model: SZX7)
  16. 4 °C refrigerator (Haier Biomedical, model: HYC-940)

Software

  1. QuickPHOTO software (PROMICRA, s.r.o., Czech Republic)
  2. FV10-ASW software (Olympus, Japan)
  3. BD FACSDivaTM 6.1.2 software (BD Biosciences, USA)

Procedure

  1. Set-up (general preparation)
    Note: All steps, including harvesting the embryos, are performed in a flow hood.
    1. Sterilization of the working area
      Expose the biosafety cabinet hood to UV light for 1 h before starting to use it. Wipe all working surfaces and the tools with 70% ethanol.
    2. Prepare the Matrigel-coated coverslips for cell cultures
      Dissolve Matrigel in a cold Neurobasal medium (10 mg/ml) by pipetting up and down to mix the Matrigel and medium very well. Put a sterile glass coverslip at each well of a 24-well plate. Add the prepared mixture onto each coverslip in a volume of approximately 0.5 ml per coverslip, making sure the surface of each coverslip is well covered. Keep coverslips for 40 min at room temperature to let them be coated with Matrigel. Afterward, remove Matrigel and wash the coverslips with Neurobasal medium 3 times. Avoid letting the Matrigel-coated coverslips become completely dry after washing.
      Note: For monolayer cell cultures, prepare the Matrigel-coated coverslips at a 24-well plate. The number of coverslips/wells plated depends on experimental design and density of desired cultures and may need to be adjusted accordingly. Seeding cells onto glass coverslips is also preferable for the purpose of further immunostaining. Glass coverslips may be prepared in advance. In such a case, seal the 24-well plate containing the Matrigel-coated coverslips very well with parafilm. The Matrigel-coated coverslips should not be kept longer than for 24 h.
    3. Polishing and sterilizing glass Pasteur pipettes
      Prepare the glass Pasteur pipettes by polishing the pipette tips over flame of an alcohol burner. To obtain pipettes with tips of various sizes, rotate each pipette slowly over flame from few to ten seconds until the glass of pipette tip’s edge become polished that can be visually seen (Figure 1). The smallest-sized tips have an internal diameter of approximately 0.2-0.3 mm, the medium-sized–approximately 0.5-0.6 mm, and the large-sized–about 0.9 mm (Figure 1B). Before use, sterilize the flame-polished pipettes at 180 °C for 1-2 h.


      Figure 1. Snapshots of the glass Pasteur pipettes with flame-polished tips of different sizes. The pipettes are used for mechanical dissociation of the cells from the fetal mouse hippocampi, starting using the pipette with the largest-sized internal tip diameter (from left to right on A and B).

  2. Harvesting embryos and dissecting the fetal hippocampi
    1. Sacrifice a pregnant female mouse at the desired stage (we use E16-E17) according to the procedures approved by your Institute.
    2. Wipe the abdominal skin with 70% ethanol and make a longitudinal incision to open the abdominal wall. Use another pair of sterile tools (forceps and scissors) for opening.
    3. Cut the uterus and carefully take the embryos out using a stereo microscope to visually control the procedure. If needed, use PBS to wash out the tissue. Place the embryos in a sterile 100 mm Petri dish containing a cold PBS (approximately 10 ml).
    4. Dissect out the brains from all embryos and carefully transfer to a 35 mm Petri dish containing 1 ml of HBBS. The dish should be kept on ice to ensure well-chilled medium. 
    5. Very carefully dissect both hippocampi out of the brain by dissecting first the olfactory bulbs and the cerebellum and splitting then the hemispheres either. For each of the hemisphere, remove the thalamus and very gently pull the hippocampus out of the brain with the help of a spatula.
    Note: To prepare a suspension of NSPCs containing approximately 1 x 106 cells, take at least five to six embryos (ten to twelve hippocampi, at the developmental stage E16-E17).

  3. Isolating NSPCs
    1. Take 0.5 ml of 0.25% trypsin-EDTA to a 15 ml Falcon tube and warm it in a CO2 incubator (35 °C, for 20-30 min). 
    2. Enzymatic dissociation of hippocampal cells
      Transfer the isolated hippocampi into a warm 0.25% trypsin-EDTA. Incubate in a CO2 incubator for 5-7 min. After incubation, stop the enzymatic digestion by adding Neurobasal medium in a volume of 1 ml (room temperature). Give a gentle flick to the tube containing the tissue to mix it up.
      Note: Each hippocampus may be cut to several parts using a sterile scalpel in order to facilitate enzymatic digestion of the tissue. Also, we recommend shaking the tube over tissue incubation period (2-3 times each minute).
    3. Mechanical dissociation of the cells
      Pipette the hippocampal tissue up and down using the flame-polished glass Pasteur pipettes. Start to pipette the tissue using the pipette with the largest-sized internal tip diameter first, followed by the medium-sized one. Finally, pipette the dissociated cells using the pipette with the smallest-sized tip. All steps are performed at room temperature.
      Note: Use each pipette for up and down pipetting not more than 10 times in order to increase the cell viability upon mechanical dissociation of the cells. Gently apply very small pressure to minimize cell damage while breaking cell aggregates. Avoid bubbles at any step of cell isolation.
    4. Take 5 ml of Neurobasal medium and add to the obtained cell suspension, gently mixing while adding the medium.
    5. Let the mixture of isolated cells in Neurobasal medium go through a 40-µm cell strainer into a 50 ml centrifuge tube.
    6. Centrifuge the obtained suspension at 200 x g for 10 min (room temperature).
    7. Aspirate the supernatant, leaving the cell pellet intact. Add 10 ml of Neurobasal medium to the pellet and gently resuspend it.
    8. Centrifuge cells at 200 x g for 10 min, as before (Figure 2).
    9. Aspirate the supernatant, leaving the cell pellet intact. Add 1 ml of PBS to the pellet and gently re-suspend the cell pellet.
    10. Centrifugation in a density gradient
      1. Take 1 ml of the obtained cell suspension and transfer to the surface of 22% Percoll solution (total volume: 10 ml). 
      2. Centrifuge the mixture at 450 x g for 10 min (room temperature). Remove the supernatant, leaving the cell pellet intact (Figure 2). 
      3. Add 10 ml of Neurobasal medium to the pellet and gently re-suspend it. 
    11. Centrifuge the obtained NSPCs at 200 x g for 10 min (room temperature). Remove the supernatant, add 1 ml of growth medium to the pellet and gently resuspend it.
      Note: Use a 1 ml pipette for mixing and resuspending the cell suspension. This minimizes cell damage and increases the viability of isolated NSPCs.


      Figure 2. Schematic illustration of the main steps for NSPC isolation from the fetal mouse hippocampus through controlled enzymatic digestion and subsequent mechanical dissociation of the cells, followed by a number of centrifugation/resuspension steps

  4. Testing the viability of isolated stem cells
    1. Trypan blue staining
      Once the suspension of NSPCs has been obtained, perform testing the cell viability using trypan blue. For this, take 20 µl of cell suspension and add 20 µl of 0.4% trypan blue solution to a droplet. Mix the sample well. Load the mixture into a glass hemocytometer very gently. Let the cells fill the chamber of hemocytometer by capillary action. Count the viable cells versus damaged cells (considering those brightly stained with trypan blue). It is advised that the proportion of viable cells shall to get over at least 75%.
      Note: For the assessment of cell viability, another dye can be else used. We probed propidium iodide, a commonly used red-fluorescent DNA counterstain, to mark dead cells in a population. This approach requires equipment for detection of the propidium iodide-mediated fluorescence.
    2. FACS-based analysis
      Take a suspension of the obtained NSPCs (approximately 5 x 105 cells) and resuspend it in 100 µl of BD Cell Wash buffer. Transfer the mixture to a flow cytometer and add 7-AAD (5 µl). Mix carefully. Incubate the cells for 10 min at room temperature. After incubation, perform analysis (we use BD FACSDivaTM 6.1.2 software). Typically, viable cells count up to ~88% (see Figure 3). This confirms the high viability of embryonic NSPCs obtained from mouse hippocampi using the described protocol.


      Figure 3. FACS-based analysis of embryonic NSPCs, freshly isolated from the fetal mouse hippocampi. A. Dot plot with gate of 7-AAD-positive non-viable (red) cells to show the proportion of dead cells after isolation. B. Distribution of viable (green) and non-viable (red) cells by morphology on the dot plot FSC vs. SSC. At least 5 x 104 cells were analyzed for each sample using BD FACSDivaTM software.

  5. Growing embryonic NSPCs in a 2D culture
    1. Take the obtained NSPC suspension (1 x 105 cells/ml) and add 250 µl of FGF-2 (20 ng/ml) to the cells. We seed the cells on the Matrigel-coated coverslips at a 24-well plate by diluting the mixture to get the total volume of 50 ml.
      Note: The total volume depends on the desired cell density (number of coverslips) to grow.
    2. To seed NSPCs in a 2D culture, take 0.5 ml of the mixture and transfer the cells onto the Matrigel-coated coverslips at a 24-well plate.
      Note: For a 2D culture, seed NSPCs by distributing cells evenly across the coverslip surface, since cell aggregates dramatically influence growth and subsequent differentiation of NSPCs in a monolayer cell culture.
    3. Place the plate in a CO2 incubator (35 °C, 5% CO2) and incubate for the next 24 h. 
    4. Next day, check the cell survival using an inverted microscope. At this point, change the growth medium (0.5 ml/well) (see Recipes) by gently aspirating the entire medium from each well containing coverslips with the cells.
    5. Change the growth medium every two days. While performing the changes, visually assess cultures for cell growth. We present an example of embryonic stem cells shortly after plating in a 2D culture (see Figure 4A). Undifferentiated cells can be confirmed by immunostaining for nestin, a specific marker of undifferentiated neural stem cells, showing typically up to 97% nestin-positive staining in 2D cultures (see Figure 4B).


      Figure 4. Example images of embryonic NSPCs in a 2D culture. A. Transmitted light image of undifferentiated NSPCs isolated from the fetal mouse hippocampi shortly after plating in an adherent 2D culture (snapshot taken on Day 1 in vitro). B. Confocal image of immunostained NSPCs for nestin (green) and Hoechst 33342 (blue) staining. Scale bars, 30 µm.

  6. Passaging NSPCs in a 2D culture
    1. Passage the cells once NSPCs reach about 80% confluency. 
    2. Aspirate all media from each well containing the cells at a 24-well plate. Gently wash the attached cells with a warm PBS (see Recipes). 
    3. Take 200 µl Accutase (1x solution, thawed in advance at room temperature) and add it to the cells (each well of the plate). Incubate the cells with Accutase in a CO2 incubator (at 35 °C) for 5 min.
    4. Check under a microscope that cells are detached and then add 1 ml of growth medium to the cells (to dilute/deactivate Accutase).
      Note: If cells are not detached after 5 min of incubation with Accutase, return the 24-well plate into a CO2 incubator (up to 5 min), by checking every minute or two if the cells have detached.
    5. Collect the detached cells to a 1.5 ml Eppendorf tube and centrifuge the suspension at 200 x g for 10 min (room temperature). Discard the supernatant and re-suspend the pellet in 1 ml of growth medium.
      Note: Perform a check of the NSPC viability for each of the obtained passage (we use trypan blue staining, but another approach can also be used). Also, estimate the density of the obtained suspension before seeding the cells. If the density is higher than 1 x 105 cells/ml, dilute the suspension accordingly (typically, 1:2). 
    6. Take 0.5 ml of the obtained suspension and transfer the cells onto each of the Matrigel-coated coverslips in a 24-well plate for seeding the next passage of NSPCs.
      Note: We recommend seeding NSPCs in a 2D culture in the density at least 3 x 104 cells per coverslip, as we have established experimentally.

  7. Generating 3D neuro-spheroids by embryonic NSPCs
    1. Formation of the adherent neuro-spheroids (neurospheres)
      For the formation of 3D neurospheres take a suspension of NSPCs (8 x 104 cells per coverslip) and resuspend the cells in the culture medium (without supplementing FGF-2). As we have observed routinely, removing FGF-2 from the culture medium boosts spontaneous differentiation of embryonic NSPCs and facilitates the formation of the adherent neuro-spheroids. 
    2. Transfer cells onto the Matrigel-coated coverslips at a 24-well plate in a volume of 0.5 ml per coverslip. Place the plate in a CO2 incubator (35 °C, 5% CO2).
      Note: For the formation of adherent neuro-spheroids, coverslips need to be coated with Matrigel. There is no requirement to coat coverslips if experimental design includes formation of floating neuro-spheroids.
    3. Neurospheres will become forming within first 24-48 h after plating the cells. There is no need to change the culture medium during that period of time in order to not disrupt the formations. Neurospheres progressively grow over the time in a 3D culture. After 6-7 days in a 3D culture, embryonic NSPCs generate stable neuro-spheroids, which can develop up to 150-200 µm (see Figure 5).


      Figure 5. Typical neuro-spheroids generated by embryonic mouse NSPCs after 5 days in an adherent 3D culture. Scale bar: 50 µm.

    Note: We recommend not growing embryonic NSPCs in a 3D culture for over 9-10 days since 3D neurospheres facilitate spontaneous cell differentiation. At that time-point, 3D neuro-spheroids can reach above 300 µm that could promote cell death within the formation core. Therefore, it is strongly advised passaging NSPCs before 3D neuro-spheroids reach overgrowth.

  8. Immunostaining of NSPCs in 2D or 3D cultures
    1. Aspirate all the media from 2D or 3D cultures of embryonic NSPCs. Wash the cells with PBS (pre-warmed to room temperature) to remove dead cells and any debris.
    2. Add 500 µl of 4% PFA in 0.1 M phosphate buffer to each well containing the cells. Incubate for 30 min at room temperature. 
    3. Wash with PBS at least 2 times.
      Note: We recommend using ~0.5 ml of the buffer to ensure sufficient washing. At this step, cells may be stored until proceeding with immunostaining (up to a week time). In such a case, seal the plate containing coverslips with the cells hermetically with parafilm to avoid drying and keep it at 4 °C.
    4. For blocking unspecific staining incubate the cells (or neuro-spheroids) in blocking solution containing 0.3% Triton X-100 and 0.5% BSA (see Recipes) for 1.5 h at room temperature.
    5. Incubate with primary antibodies in antibody solution containing 0.3% Triton X-100 and 0.5% BSA (see Recipes) for overnight at 4 °C. We use 200 µl of antibody solution per well.
      Note: Shaking the plate in a slow rate is useful (but not required) during incubation in blocking solution or with antibodies for facilitating the treatment.
    6. Wash the cells/neurospheres 3 times in PBS, each for 15 min.
    7. Incubate with secondary antibody(ies) in antibody solution containing 0.5% BSA (without Triton X-100) for 1 h at room temperature in the dark. We use 200 µl of the solution per well. 
    8. Wash 3 times in PBS for 15 min. 
    9. Incubate with Hoechst 33342 (1:5,000 in PBS) for 3-5 min at room temperature in the dark. 
    10. Wash 3 times in PBS. Then wash in ddH2O to rinse out all salts. The cells/spheroids are ready for imaging.
    11. Imaging
      We carry out imaging of the immunostained cells on a glass slide. For this, mount the coverslips containing a 2D culture (or 3D neuro-spheroids) onto the SuperFrost® glass slides, using a mounting medium (ImmunoHistoMount). Then let the slides air-dry for overnight at room temperature. Keep the samples at 4 °C. With such an approach, the immunostained cell preparations can be stored as long as needed. We present the example images for immunostaining of i) the adherent neuro-spheroids generated by embryonic NSPCs for both nestin and β-tubulin III staining (see Figure 6) and ii) an adherent monolayer of embryonic NSPCs for both nestin and Hoechst staining in a 2D culture (see Figure 4B). Staining for other proteins can also be performed using this method.


      Figure 6. Immunofluorescent staining of neuro-spheroids generated from mouse embryonic NSPCs in a 3D culture. A and B. Confocal images of neuro-spheroids generated by embryonic NSPCs merged for nestin (green) and β-tubulin III (red) staining on Day 5 in a 3D culture. Scale bars, 50 µm (A) and 40 µm (B).

  9. Engraftment of mouse embryonic NSPCs onto organotypic hippocampal tissue
    1. Preparation of the hippocampal slices
      Sacrifice pups of FVB mice at P8-P9 according to the procedures approved by your Institute. Decapitate the animals, dissect out the brains and remove both hippocampi into a cold dissecting medium (see Recipes), as we have described above and previously (Rybachuk et al., 2017). Place each hippocampus on a tissue chopper disc, mounted on a tissue chopper (McIlwain), and initiate chopping to cut the tissue to the transverse slices of 350-μm thick. Flash hippocampal slices into a Petri dish containing a cold dissecting medium and turn slices over to make their medial side faces up. Collect only hippocampal slices those have a well-recognized hippocampal-like morphology.
      Note: For ‘good’ slicing the tissue, we recommend to put a filter paper above the chopper disc and place the hippocampus on it to avoid slipping and wobbling the tissue by the blade. Also, remove excess medium around the tissue before initiating chopping. 
    2. Organotypic hippocampal tissue cultures
      Transfer slices using a glass Pasteur pipette with the flame-polished large-sized tip (inner diameter of near 3 mm) onto a surface of 0.4-μm membrane inserts placed at a 6-well plate. We typically plate four slices on a 0.4-μm membrane insert. Remove the spare medium using a glass Pasteur pipette of a small-sized tip (inner diameter of around 0.8 mm). Add 1 ml of culture medium (see Recipes) to each well containing slices on membrane insert. Place the plate in a CO2 incubator (35°C, 5% CO2). Change the culture medium on Day 2 after plating. Afterward, change the media every two days.
      Note: A number of membrane inserts (wells at a tissue culture plate) depend on the amount of slices cut ‘good’ and may be adjusted from a 6-well plate to a 24-well plate. 
    3. Engraftment of embryonic NSPCs
      Take 50 µl of a suspension of freshly isolated NSPCs and place the cells onto the surface of organotypic hippocampal tissue using a standard pipette (Eppendorf, Research® Plus). We grafted embryonic NSPCs onto hippocampal tissue at 7 days post-plating, but any time-point can be used. Any timing needs to be adjusted accordingly to experimental design. Transfer NSPCs by very carefully distributing the cells evenly across the tissue surface, not forming cell aggregates. Also, make sure that NSPCs settle exclusively on the tissue surface. Do not move the plate sharply to prevent the freshly engrafted cells drop down on membrane inserts. At Day 2 after engraftment, change the culture medium to wash out NSPCs not attached to a host tissue.
      Note: We recommend engraftment of NSPCs at the density of 0.25 x 105 cells per slice. NSPCs from 2D or 3D cultures may also be engrafted after careful dissociation of the cells to ensure homogeneous distribution across the surface of a host tissue.
    4. Growth of embryonic stem cells within a host organotypic tissue.
      Maintain organotypic hippocampal tissue with NSPC grafts in a CO2 incubator (35 °C, 5% CO2) until use. Change the culture medium every two days.
      Note: We recommend changing the culture medium by aspirating it under membrane inserts.
    5. Check the growth of differentiating NSPCs within a host tissue. This can be performed at any time-point due to simple tracing of the GFP-labeled NSPCs within the tissue. After 3 days, the grafted NSPCs began to incorporate into a host tissue (see Figure 7). Immunostaining in organotypic hippocampal tissue is performed similarly to the procedure described for cell cultures above and in our previous works (Rybachuk et al., 2017; Kopach et al., 2018).


      Figure 7. Example of embryonic mouse NSPCs grafted onto a host hippocampal tissue. A and B. Confocal images of immunostained organotypic hippocampal tissue with GFP-labeled NSPCs (green, A) and merge image of GFP-labeled NSPCs (green) and GFAP (red) staining (B) on Day 3 after engraftment. Scale bars, 100 µm.

Data analysis

The obtained suspension of embryonic NSPCs can be used to grow and/or differentiation of the cells in vitro or within endogenous environment of a host tissue.

  1. In a 2D culture, the growth of the cells can be monitored visually and confirmed through immunostaining for nestin (red) and Hoechst 33342 (blue), as shown in Figure 4. See also Kopach et al. (2018), Figure 1B, for immunostaining images of NSPC fraction at 1 d in vitro and also Figure 6A for statistical analysis of the proportion of nestin-positive NSPCs (undifferentiated progenitors) in different experimental conditions.
  2. In 3D cultures, NSPCs can follow a robust multi-lineage differentiation. NSPCs isolated from the fetal mouse hippocampi can generate neuro-spheroids, the formation that enhances cell growth and differentiation and facilitates maintaining the high viability of differentiating progenitors. For the assessment whether neurons or glia are generated from embryonic NSPCs in a 3D culture, use of the cell type-specific markers is required. For visualization of phenotypic distribution within neuro-spheroids, we carried out immunostaining of differentiating progenitors for astrocytic marker GFAP together with neuronal marker NeuN and oligodendrocyte-specific marker olig-2. The example shown are images of phenotypic differentiation of embryonic NSPCs taken on Day 5 of 3D culture (see Figure 8).


    Figure 8. Immunofluorescent staining of 3D neuro-spheroids generated by embryonic mouse NSPCs. A. Confocal image of immunostained neuro-spheroids of embryonic hippocampal NSPCs for GFAP (cyan) and β-tubulin III (red) staining on Day 5 of 3D culture. B. Confocal image of neuro-spheroid generated by embryonic NSPCs for GFAP (red) and olig-2 (cyan) staining. C. Confocal image of the NSPC-generating neuro-spheroid for Hoechst 33342 (blue) and NeuN (red) staining. Scale bars, 50 µm (A) and 20 µm (B and C).

  3. In a host brain tissue, organotypic hippocampal slices where both morphological layer architecture and signaling pathway assemblies remained preserved for the required period of time (weeks of tissue maintenance), embryonic NSPCs displayed a prompt multi-lineage neurogenesis. Using organotypic hippocampal tissue, we enabled to trace the time-dependent maturation of NSPC-derived hippocampal neurons through monitoring neuronal excitability with electrophysiological approaches. In particular, we recorded the passive membrane properties from differentiating progenitors [see Kopach et al. (2018), Figure 1], neuronal firing [see Kopach et al. (2018), Figure 3] and synaptic events spontaneously evoked in NSPC-derived neurons [see Kopach et al. (2018), Figure 2]. Organotypic hippocampal tissue represents a useful tool to feasibly assess maturation of neurophysiological properties of differentiating NSPCs within endogenous host environment at various time-points after engraftment. Moreover, it is implementable to carry out the direct quantitative comparisons between the functional properties of maturing progenitors versus endogenous principal neurons. Glial lineage of differentiating NSPCs can be further confirmed using immunostaining for the cell type-specific markers (see the description of the protocol in Kopach et al. [2018] and Figure 6F, for images of NSPC-derived oligodendrocytes or astroglia at 2 or 3 weeks post-grafting).

Recipes

  1. 0.2 M Phosphate buffer (total volume 250 ml; pH 7.4)
    1. Take 1.56 g sodium dihydrogen phosphate (NaH2PO4) and add 50 ml ddH2
    2. Take 5.68 g disodium phosphate (Na2HPO4) and add 200 ml ddH2
    3. Mix 200 ml diluted Na2HPO4 with 50 ml diluted NaH2PO4 
    4. Store 0.2 M phosphate buffer (pH 7.4) at 4 °C
  2. 4% Paraformaldehyde (PFA) in 0.1 M phosphate buffer (total volume 100 ml)
    1. Take 4 g PFA and add it into 50 ml ddH2O while stirring at 60 °C
    2. To facilitate dilution of PFA in ddH2O, add 20 μl NaOH to the mixture while stirring 
    3. Stir the mixture for approximately 30 min
    4. Add 50 ml of 0.2 M phosphate buffer to the mixture
    5. Filter the mixture using a 0.45-μm filter 
    6. Store 4% PFA at -20 °C if not use it over the next days
  3. Growth medium (total volume 50 ml)
    1. Take 48.5 ml Neurobasal medium and add it to a 50 ml Falcon tube 
    2. Add 1 ml B-27® (50x) 
    3. Add 0.5 ml GlutaMAXTM (100x)
    4. Add 50 µl N-acetyl-L-cysteine (NAC) and 0.25 ml P/S
    5. Store the medium at 4 °C if not use immediately 
    6. Add 20 ng/ml FGF-2 to the medium just before use it
  4. Tissue dissecting medium (total volume 100 ml; pH 7.3)
    1. Take 50 ml MEM and add it to a Falcon tube 
    2. Add 25 ml HBSS
    3. Add 60 µg Tris to get its final concentration of 5 mM
    4. Add 17.5 µg NaHCO3 (final concentration 2 mM)
    5. Add 1.26 ml HEPES (final concentration 12.5 mM)
    6. Add 276.5 µg glucose (final concentration 15 mM)
    7. Add 1 ml P/S 
    8. Dilute in ddH2O to get the total volume of 100 ml
    9. Filter the prepared medium using a 0.45-µm sterile filter
    10. Store the medium at 4 °C (or freeze if not use it over the next days)
  5. Tissue culture medium (total volume 100 ml; pH 7.2)
    1. Take 50 ml MEM and add it to a Falcon tube 
    2. Add 25 ml HBSS
    3. Add 30 µg Tris (final concentration 2.5 mM)
    4. Add 17.5 µg NaHCO3 (2 mM)
    5. Add 1.26 ml HEPES (12.5 mM)
    6. Add 276.5 µg glucose (15 mM)
    7. Add 1 ml P/S 
    8. Filter the medium through a 0.45-µm sterile filter and store it at 4 °C (or freeze it if not use)
    9. Add 250 µl/ml horse serum to the medium right before using 
    10. Add 20 µl/ml B-27® supplement (50x) to the medium before using 
  6. Antibody solution
    Dissolve BSA (0.5%) in PBS just before use it
  7. Blocking solution
    Dissolve BSA (0.5%) and Triton X-100 (0.3%) in PBS before use

Acknowledgments

The work was supported by grants from the National Academy of Sciences of Ukraine.

Competing interests

Authors declare that there are no conflicts of interest or competing interests.

Ethics

All procedures were used in accordance with the protocols approved by the Animal Care and Use Committee at Bogomoletz Institute of Physiology and State Institute of Genetic and Regenerative Medicine (Kyiv, Ukraine) and were within the European Commission Directive (86/609/EEC) guidelines.

References

  1. Behnan, J., Stangeland, B., Langella, T., Finocchiaro, G., Tringali, G., Meling, T. R. and Murrell, W. (2017). Identification and characterization of a new source of adult human neural progenitors. Cell Death Dis 8(8): e2991.
  2. Conti, L., Pollard, S. M., Gorba, T., Reitano, E., Toselli, M., Biella, G., Sun, Y., Sanzone, S., Ying, Q. L., Cattaneo, E. and Smith, A. (2005). Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol 3(9): e283.
  3. Ignatova, T. N., Kukekov, V. G., Laywell, E. D., Suslov, O. N., Vrionis, F. D. and Steindler, D. A. (2002). Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro. Glia 39(3): 193-206.
  4. Kempermann, G., Gage, F. H., Aigner, L., Song, H., Curtis, M. A., Thuret, S., Kuhn, H. G., Jessberger, S., Frankland, P. W., Cameron, H. A., Gould, E., Hen, R., Abrous, D. N., Toni, N., Schinder, A. F., Zhao, X., Lucassen, P. J. and Frisen, J. (2018). Human adult neurogenesis: evidence and remaining questions. Cell Stem Cell 23(1): 25-30.
  5. Klein, C., Butt, S. J., Machold, R. P., Johnson, J. E. and Fishell, G. (2005). Cerebellum- and forebrain-derived stem cells possess intrinsic regional character. Development 132(20): 4497-4508.
  6. Kopach, O. and Pivneva, T. (2018). Cell-based therapies for neural replacement strategies in stroke-related neurodegeneration: neurophysiological insights into stem progenitor cell neurogenesis within a host environment. Neural Regen Res 13(8): 1350-1351.
  7. Kopach, O., Rybachuk, O., Krotov, V., Kyryk, V., Voitenko, N. and Pivneva, T. (2018). Maturation of neural stem cells and integration into hippocampal circuits - a functional study in an in situ model of cerebral ischemia. J Cell Sci 131(4) pii: jcs210989.
  8. Makri, G., Lavdas, A. A., Katsimpardi, L., Charneau, P., Thomaidou, D. and Matsas, R. (2010). Transplantation of embryonic neural stem/precursor cells overexpressing BM88/Cend1 enhances the generation of neuronal cells in the injured mouse cortex. Stem Cells 28(1): 127-139.
  9. Pagano, S. F., Impagnatiello, F., Girelli, M., Cova, L., Grioni, E., Onofri, M., Cavallaro, M., Etteri, S., Vitello, F., Giombini, S., Solero, C. L. and Parati, E. A. (2000). Isolation and characterization of neural stem cells from the adult human olfactory bulb. Stem Cells 18(4): 295-300.
  10. Pollard, S. M., Conti, L., Sun, Y., Goffredo, D. and Smith, A. (2006). Adherent neural stem (NS) cells from fetal and adult forebrain. Cereb Cortex 16 Suppl 1: i112-120.
  11. Roy, N. S., Wang, S., Jiang, L., Kang, J., Benraiss, A., Harrison-Restelli, C., Fraser, R. A., Couldwell, W. T., Kawaguchi, A., Okano, H., Nedergaard, M. and Goldman, S. A. (2000). In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus. Nat Med 6(3): 271-277.
  12. Rybachuk, O., Kopach, O., Krotov, V., Voitenko, N. and Pivneva, T. (2017). Optimized model of cerebral ischemia in situ for the long-lasting assessment of hippocampal cell death. Front Neurosci 11: 388.
  13. Suslov, O. N., Kukekov, V. G., Ignatova, T. N. and Steindler, D. A. (2002). Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres. Proc Natl Acad Sci U S A 99(22): 14506-14511.
  14. Tsupykov, O., Kyryk, V., Smozhanik, E., Rybachuk, O., Butenko, G., Pivneva, T. and Skibo, G. (2014). Long-term fate of grafted hippocampal neural progenitor cells following ischemic injury. J Neurosci Res 92(8): 964-974.

简介

对于干细胞研究和中枢神经系统疾病的治疗,神经干/祖细胞(NSPCs)代表了重要的突破性工具。在扩展的干细胞疗法中,NSPCs不仅为神经细胞替代提供了强大的细胞来源,而且是发育生物学研究的有用模型。尽管描述了从胎儿或成人脑中分离NSPC的许多方法,但主要问题仍然是在分离后延长细胞存活。在这里,我们提供了一种简单且经济实惠的方案,用于从胎儿小鼠海马体制备可行的NSPC,其能够维持2D单层细胞培养物中的高活力或产生细胞聚集体的3D神经球体。此外,我们描述了将胚胎NSPCs植入宿主海马组织以促进内源环境中多线性细胞分化和成熟的详细方法。我们的实验数据表明,使用这种方法分离的胚胎NSPCs显示出高活力(高于88%)。在宿主组织内,这些细胞能够分化为主要神经亚群(主要神经元,少突胶质细胞,星形胶质细胞)。最后,NSPC衍生的神经元显示出成熟的功能特性(电生理活动),在植入后的几周内功能性地整合到宿主海马回路中。
【背景】神经起源的多能干细胞,神经干/祖细胞(NSPCs),代表了一种多功能的工具,可以在许多以失去特化神经亚群为特征的神经系统疾病中实现神经细胞替代。无限的寿命和定向的NSPC分化成关键的神经亚型(神经元,星形胶质细胞和少突胶质细胞)使得这些细胞成为目前无法治愈的脑疾病的新兴干细胞疗法的最有吸引力的候选者(Kopach和Pivneva,2018)。干细胞存在于发育和成熟的神经系统中,可以从胎儿大脑(胚胎NSPCs)中分离,或者最近建立的成熟大脑(成人,海马齿状回的脑室下和颗粒下区,前脑,小脑和嗅球)(Pagano et al。,2000; Roy et al。,2000; Klein et al。,2005; Behnan et al。,2017; Kempermann et al。,2018)。尽管成体干细胞对先进的转化方法抱有期望(Kempermann et al。,2018),但目前有限数量的严谨证据支持这一研究途径。

通过专门的临床试验证实,胎儿NSPCs特性和表型规格的累积证据仍然保留了对这种干细胞类型的关注。此外,胚胎NSPCs是发育生物学研究的重要工具。探索了各种方法学方法以从胎儿脑中分离NSPC并将细胞维持在确定其高活力的条件下,从而确定后代。这些条件在很大程度上决定了分化祖细胞的表型,以获得竞争性NSPC衍生细胞。保持分离的NSPC的高活力的两种主要策略存在于i)细胞培养体外和ii)移植入宿主组织中。经典的2D培养物,一种粘附的单层细胞,已经常规用于体外的干细胞生长和/或定向表型分化(Conti et al。,2005) ; Pollard et al。,2006)。同时,新的开发方法产生3D培养物,NSPC在各种表型和发育阶段的神经球体,已经出现,以代表与神经元分化相关的更多生理条件,而不是胶质细胞(Ignatova et al。,2002; Suslov et al。,2002; Makri et al。,2010)。该方法可潜在地增强体外NSPC的治疗潜力。另一种将活的NSPC移植到脑组织中的替代方法 - 实现了促进宿主组织内祖细胞的多谱系分化的策略,其受内源环境的调节。最近已经在缺血损伤的海马组织的治疗中实施了这种方法(Tsupykov 等人,<2014; Kopach 等人,2018)。

在这里,我们提供了我们常规用于从胎儿小鼠海马中分离活NSPC的详细方案。该方案还用于将未分化细胞植入海马组织,用于在我们先前的研究中监测宿主环境中NSPC衍生细胞的成熟(Kopach 等人,2018)。我们描述了如何测试获得的胚胎NSPC的活力的所有细节,并将细胞维持在多种方法中以供进一步提出使用。

关键字:胚胎神经干/祖细胞, 小鼠海马, 细胞活力, 贴壁二维单层细胞培养, 三维神经球体, 器官海马组织

材料和试剂

  1. 材料
    1. 15毫升和50毫升离心管(康宁,目录号:430790,430828)
    2. 1.5毫升Eppendorf管(Sigma-Aldrich,目录号:T9661)
    3. 玻璃巴斯德吸管(直径1 mm)(Fisher Scientific,目录号:13-678-6A)
    4. 35毫米,60毫米和100毫米组织培养皿(CELLSTAR,目录号:627160,628160,664160)
    5. Falcon ® 40-μm细胞过滤器(Corning,Falcon ®,目录号:352340)
    6. 0.45-μm无菌过滤器(Fisher Scientific,Merck TM ,目录号:12279299)&nbsp;
    7. 6孔和24孔组织培养板(Greiner Bio-One,目录号:657160,662160)
    8. 13毫米玻璃盖玻片(Henz Herenz,目录号:1051204)
    9. SuperFrost ®载玻片(VWR,Thermo Fisher Scientific,目录号:631-0706)
    10. 塑料血清移液器(5 ml,10 ml和25 ml)(Corning ® Costar ® Stripette ®,目录号:4487,4488, 4489)
    11. 移液器吸头(TipOne,STARLAB,目录号:S1111-3700,S1111-1706,S1111-6701)
    12. Millipore ® Millicell ®细胞培养板插入物(Sigma-Aldrich,目录号:Z353086)
    13. Whatman ®定性滤纸(Sigma-Aldrich,目录号:WHA1001110)
    14. 手术刀片(Fisher Scientific,Swann-Morton TM ,目录号:11772724)
    15. 带可伸缩刀片的手术刀(Eickemeyer ®,目录号:100504)
    16. Parafilm ® M(Sigma-Aldrich,目录号:P7793)

  2. 动物
    1. 怀孕的FVB-Cg-Tg(GFPU)5Nagy / J GFP小鼠(获自国立遗传与再生医学研究所的动物设施)
    2. E16-E17发育阶段FVB-Cg-Tg(GFPU)5Nagy / J GFP小鼠的胚胎
    3. FVB小鼠(8至9日龄)的小狗

  3. 试剂
    1. 不含钙和镁的HBBS(Sigma-Aldrich,目录号:55021C)
    2. 多聚甲醛(PFA,Sigma-Aldrich,目录号:P6148)
    3. 0.25%胰蛋白酶-EDTA(Sigma-Aldrich,目录号:T4049)
    4. 22%Percoll(Sigma-Aldrich,目录号:17-0891-01)
    5. Neurobasal ®培养基(Thermo Fisher Scientific,Gibco TM ,目录号:21103049)
    6. B-27 ®补充剂(50x)(Thermo Fisher Scientific,Gibco TM ,目录号:17504044)
    7. GlutaMAX TM 补充剂(100x)(Thermo Fisher Scientific,Gibco TM ,目录号:35050061)
    8. N-乙酰基-L-半胱氨酸(NAC)(Sigma-Aldrich,目录号:A7250)
    9. 青霉素 - 链霉素(P / S)溶液(100,000 U / ml)(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
    10. FGF-2(Sigma-Aldrich,目录号:SRP4037)
    11. Matrigel(BD Biosciences,目录号:354234)
    12. Triton ® X-100(Sigma-Aldrich,目录号:T8787)
    13. Accutase ®解决方案(Sigma-Aldrich,目录号:A6964)
    14. 0.4%台盼蓝(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
    15. 抗体
      1. 小学
        1. 小鼠抗Nestin抗体(1:100)(Santa Cruz Biotechnology,目录号:sc-23927)
        2. 兔抗β-微管蛋白III(1:500)(Sigma-Aldrich,目录号:T2200)
        3. 小鼠抗GFAP(1:200)(Sigma-Aldrich,目录号:AMAB91033)
        4. 兔抗寡聚-2(1:200)(艾博抗(上海),目录号:ab136253)
        5. Mouse anti-NeuN(1:100)(Millipore,目录号:MAB377)
      2. 中学
        1. 驴抗小鼠Alexa Fluor 555(1:1,000)(Thermo Fisher Scientific,目录号:A-31570)
        2. 驴抗兔Alexa Fluor 647(1:1,000)(赛默飞世尔科技,目录号:A-31573)&nbsp;
        3. 细胞核示踪剂Hoechst 33342(1:5,000)(Sigma-Aldrich,目录号:14533)
    16. ImmunoHistoMount TM (Sigma-Aldrich,目录号:I1161)
    17. 70%乙醇(Sigma-Aldrich,目录号:459836)
    18. 磷酸二氢钠一元(NaH 2 PO 4 )(Sigma-Aldrich,目录号:S0751)
    19. 磷酸氢二钠(Na 2 HPO 4 )(Sigma-Aldrich,目录号:S0876)
    20. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
    21. 最低基本培养基(MEM)(Sigma-Aldrich,目录号:M2249)
    22. Tris(西格玛奥德里奇,目录号:252859)
    23. 碳酸氢钠(Sigma-Aldrich,目录号:792519)
    24. HEPES(赛默飞世尔科技,目录号:15630080)
    25. D(+) - 葡萄糖(Sigma-Aldrich,目录号:G8270)
    26. 马血清(Sigma-Aldrich,目录号:H1138)
    27. 磷酸盐缓冲盐水(PBS),pH 7.4(Sigma-Aldrich,目录号:P3813)
    28. BD Cell Wash缓冲液(BD Biosciences,目录号:349524)
    29. 牛血清白蛋白(BSA)(西格玛奥德里奇,目录号:05470)
    30. 7-AAD(BD Pharmingen TM ,目录号:559925)
    31. 0.2 M磷酸盐缓冲液(pH 7.4)(见食谱)
    32. 0.1%磷酸盐缓冲液中的4%多聚甲醛(PFA)(见食谱)
    33. 生长培养基(见食谱)
    34. 组织解剖培养基(pH 7.3)(见食谱)
    35. 组织培养基(pH 7.2)(见食谱)
    36. 抗体解决方案(见食谱)
    37. 阻止解决方案(见食谱)

设备

  1. 移液管(Eppendorf,Research ® Plus,目录号:EP 3123000012; EP 3123000098; EP 3123000055; EP 3120000062)
  2. 250毫升量筒(Karter Scientific,目录号:213I13)
  3. 干热消毒柜(格兰德,型号:GIO-072)
  4. 解剖和操纵胚胎大脑的精细工具
    1. 解剖剪刀(Dumont,目录号:14090-09)
    2. 小铲(Dumont,目录号:10090-13)
    3. 镊子(Dumont,目录号:91197-00)
    4. 角度钳(Dumont,目录号:00125-11)
  5. C-Chip一次性血细胞计数仪/ Neubauer(Labtech International,Heathfield,UK,目录号:DHC-N01)
  6. 冷冻离心机(Thermo Fisher Scientific,型号:IEC-CL30R)
  7. 水浴(Biosan,型号:WB-4MS)
  8. 生物安全柜II类A2(Labconco,型号:净化器逻辑)
  9. CO 2 培养箱(Thermo Fisher Scientific,型号:SteriCycle 371)
  10. 流式细胞仪(Becton Dickinson,型号:BDFACSAriaсell分选机)
  11. 组织切碎机(McIlwain,型号:MTC / 2E)
  12. 酒精燃烧器(Sigma-Aldrich,目录号:Z509604)
  13. 倒置显微镜(奥林巴斯,型号:IX71,配备摄像头DP-20)
  14. 共聚焦显微镜(奥林巴斯,型号:FV1000)
  15. 立体显微镜(奥林巴斯,型号:SZX7)
  16. 4°C冰箱(海尔生物医药,型号:HYC-940)

软件

  1. QuickPHOTO软件(PROMICRA,s.r.o。,捷克共和国)
  2. FV10-ASW软件(日本奥林巴斯)
  3. BD FACSDiva TM 6.1.2软件(BD Biosciences,USA)

程序

  1. 设置(一般准备)
    注意:所有步骤,包括收获胚胎,都是在流动罩中进行的。
    1. 工作区域灭菌
      在开始使用之前,将生物安全柜罩暴露在紫外线下1小时。用70%乙醇擦拭所有工作表面和工具。
    2. 准备用于细胞培养的Matrigel涂层盖玻片
      通过上下吸移将Matrigel溶解在冷的Neurobasal培养基(10mg / ml)中以非常好地混合Matrigel和培养基。将无菌玻璃盖玻片放在24孔板的每个孔中。将制备好的混合物以每个盖玻片约0.5ml的体积加到每个盖玻片上,确保每个盖玻片的表面被很好地覆盖。在室温下保持盖玻片40分钟,让它们涂上Matrigel。然后,去除Matrigel并用Neurobasal培养基洗涤盖玻片3次。洗完后,避免让涂有Matrigel的盖玻片完全干燥。
      注意:对于单层细胞培养,在24孔板上制备涂有Matrigel的盖玻片。镀覆的盖玻片/孔的数量取决于实验设计和所需培养物的密度,并且可能需要相应地调整。出于进一步免疫染色的目的,将细胞接种到玻璃盖玻片上也是优选的。可以预先准备玻璃盖玻片。在这种情况下,用封口膜很好地密封含有Matrigel涂层盖玻片的24孔板。涂有Matrigel涂层的盖玻片不应长于24小时。
    3. 抛光和消毒玻璃巴斯德吸管
      通过在酒精燃烧器的火焰上抛光移液器吸头来准备玻璃巴斯德吸管。为了获得具有各种尺寸尖端的移液器,将每个移液管在火焰上缓慢旋转几到十秒,直到移液管尖端的玻璃边缘变得抛光,这可以在视觉上看到(图1)。最小尺寸的尖端的内径约为0.2-0.3毫米,中等尺寸约为0.5-0.6毫米,大尺寸约为0.9毫米(图1B)。使用前,将火焰抛光的移液器在180°C下灭菌1-2小时。


      图1.玻璃巴斯德移液器的快照,带有不同大小的火焰抛光尖端。移液器用于从胎鼠海马体中机械解离细胞,开始使用最大尺寸的移液器内部尖端直径(在A和B上从左到右)。

  2. 收获胚胎和解剖胎儿海马
    1. 根据您所在研究所批准的程序,在期望的阶段(我们使用E16-E17)牺牲怀孕的雌性小鼠。
    2. 用70%乙醇擦拭腹部皮肤并做纵向切口以打开腹壁。使用另一对无菌工具(镊子和剪刀)打开。
    3. 切开子宫,用立体显微镜仔细取出胚胎,目测控制手术。如果需要,使用PBS清洗组织。将胚胎置于含有冷PBS(约10ml)的无菌100mm培养皿中。
    4. 解剖所有胚胎的大脑,小心地转移到含有1毫升HBBS的35毫米培养皿中。将菜肴放在冰上以确保培养基充分冷却。&nbsp;
    5. 通过首先解剖嗅球和小脑然后分裂半球,非常仔细地将两个海马体解剖出大脑。对于每个半球,去除丘脑,并在刮刀的帮助下非常轻柔地将海马体拉出大脑。
    注意:要制备含有约1 x 10 6 细胞的NSPC悬浮液,至少取5到6个胚胎(10到十二个海马,处于发育阶段E16-E17)。

  3. 隔离NSPC
    1. 将0.5 ml 0.25%胰蛋白酶-EDTA加入15 ml Falcon试管中,并在CO 2 培养箱中加热(35°C,20-30分钟)。&nbsp;
    2. 海马细胞的酶促解离 将分离的海马转移到温暖的0.25%胰蛋白酶-EDTA中。在CO 2 培养箱中孵育5-7分钟。孵育后,通过加入1ml体积的Neurobasal培养基(室温)停止酶消化。轻轻一甩含有纸巾的管子,将它混合起来。
      注意:可以使用无菌手术刀将每个海马体切成几个部分,以促进组织的酶促消化。此外,我们建议在组织培养期间摇动管(每分钟2-3次)。
    3. 细胞的机械解离
      使用火焰抛光玻璃巴斯德吸管上下移取海马组织。首先使用具有最大尺寸内部尖端直径的移液管吸取组织,然后使用中等尺寸的移液管。最后,使用具有最小尺寸尖端的移液管吸移解离的细胞。所有步骤均在室温下进行。
      注意:使用每个移液器进行上下移液不超过10次,以便在细胞机械解离时增加细胞活力。轻轻施加非常小的压力,以最小化细胞损伤,同时破坏细胞聚集体。在细胞分离的任何步骤都要避免气泡。
    4. 取5ml Neurobasal培养基,加入得到的细胞悬浮液中,轻轻混合,同时加入培养基。
    5. 让Neurobasal培养基中的分离细胞的混合物通过40-μm细胞过滤器进入50ml离心管。
    6. 将得到的悬浮液在200℃下离心10分钟(室温)。
    7. 吸出上清液,使细胞沉淀完整。向沉淀中加入10ml Neurobasal培养基并轻轻重悬。
    8. 如前所述,以200 x g 离心细胞10分钟(图2)。
    9. 吸出上清液,使细胞沉淀完整。向沉淀中加入1ml PBS并轻轻重悬细胞沉淀。
    10. 以密度梯度离心
      1. 取1 ml获得的细胞悬浮液,转移到22%Percoll溶液(总体积:10 ml)的表面。&nbsp;
      2. 将混合物在450 x g 下离心10分钟(室温)。去除上清液,保持细胞沉淀完整(图2)。&nbsp;
      3. 向沉淀中加入10ml Neurobasal培养基并轻轻重悬。&nbsp;
    11. 将获得的NSPC在200 x g 离心10分钟(室温)。除去上清液,在沉淀中加入1 ml生长培养基,轻轻重悬。
      注意:使用1 ml移液器混合并重新悬浮细胞悬液。这可以最大限度地减少细胞损伤并提高孤立NSPC的可行性。


      图2.通过控制酶消化和随后的细胞机械解离从胎鼠小鼠海马中分离NSPC的主要步骤的示意图,然后进行多次离心/再悬浮步骤

  4. 测试分离的干细胞的可行性
    1. 台盼蓝染色
      一旦获得NSPC的悬浮液,使用台盼蓝进行细胞活力测试。为此,取20μl细胞悬浮液,并向液滴中加入20μl0.4%台盼蓝溶液。将样品充分混合。将混合物非常温和地装入玻璃血细胞计数器中。让细胞通过毛细管作用填充血细胞计数器的腔室。计算活细胞与受损细胞的比较(考虑那些用台盼蓝染色的细胞)。建议活细胞的比例应至少超过75%。
      注意:为了评估细胞活力,可以使用另一种染料。我们探测了碘化丙锭(一种常用的红色荧光DNA复染剂)来标记人群中的死细胞。该方法需要用于检测碘化丙啶介导的荧光的设备。
    2. 基于FACS的分析
      取所得NSPC(约5×10 5个细胞)悬浮液,并将其重悬于100μlBD细胞洗涤缓冲液中。将混合物转移至流式细胞仪并加入7-AAD(5μl)。仔细混合。在室温下孵育细胞10分钟。孵育后,进行分析(我们使用BD FACSDiva TM 6.1.2软件)。通常,活细胞计数高达~88%(参见图3)。这证实了使用所述方案从小鼠海马获得的胚胎NSPC的高活力。


      图3.基于FACS的胚胎NSPCs分析,从胎鼠海马新鲜分离。 A.点图与7-AAD阳性无活力(红色)细胞的门显示比例隔离后死细胞。 B.在点图FSC对SSC上通过形态学分布活的(绿色)和非活的(红色)细胞。使用BD FACSDiva TM 软件分析每个样品的至少5×10 4个细胞。

  5. 在2D培养中培育胚胎NSPCs
    1. 取得获得的NSPC悬浮液(1×10 5 细胞/ ml)并向细胞中加入250μlFGF-2(20ng / ml)。我们通过稀释混合物使总体积为50ml,在24孔板上将细胞接种在Matrigel涂层的盖玻片上。
      注意:总体积取决于要增长的所需细胞密度(盖玻片数量)。
    2. 为了在2D培养物中接种NSPC,取0.5ml混合物并将细胞转移到24孔板上的Matrigel涂覆的盖玻片上。
      注意:对于2D培养,通过在盖玻片表面均匀分布细胞来种子NSPC,因为细胞聚集显着影响单层细胞培养物中NSPCs的生长和随后的分化。
    3. 将平板置于CO 2 培养箱(35°C,5%CO 2 )中并孵育24小时。&nbsp;
    4. 第二天,使用倒置显微镜检查细胞存活率。此时,通过从含有盖玻片的每个孔中轻轻吸出整个培养基来改变生长培养基(0.5ml /孔)(参见配方)。
    5. 每两天更换一次生长培养基。在进行变化时,目测评估细胞生长的培养物。我们在2D培养物中接种后不久呈现胚胎干细胞的实例(参见图4A)。未分化的细胞可以通过巢蛋白的免疫染色来确认,巢蛋白是未分化的神经干细胞的特异性标记物,在2D培养物中通常显示高达97%的巢蛋白阳性染色(参见图4B)。


      图4. 2D培养中胚胎NSPCs的示例图像。 A.在贴壁2D培养物中铺板后不久从胎鼠海马分离的未分化NSPC的透射光图像(第1天快照体外)。 B.免疫染色的NSPC对巢蛋白(绿色)和Hoechst 33342(蓝色)染色的共聚焦图像。比例尺,30μm。

  6. 在2D文化中传递NSPC
    1. NSPC达到约80%融合后,将细胞传代。&nbsp;
    2. 从24孔板中含有细胞的每个孔中吸出所有培养基。用温PBS轻轻洗涤附着的细胞(参见食谱)。&nbsp;
    3. 取200μlActutase(1x溶液,在室温下预先解冻)并将其加入细胞(板的每个孔)中。将细胞与Accutase在CO 2 培养箱(35°C)中孵育5分钟。
    4. 在显微镜下检查细胞是否脱落,然后向细胞中加入1ml生长培养基(稀释/灭活Accutase)。
      注意:如果细胞在与Accutase孵育5分钟后未分离,则将24孔板放回CO 2 培养箱中(最多5分钟),如果细胞分离,每分钟或每两分钟检查一次。
    5. 将分离的细胞收集到1.5ml Eppendorf管中,并将悬浮液在200μL离心下离心10分钟(室温)。弃去上清液,将沉淀重悬于1 ml生长培养基中。
      注意:检查每个获得的通道的NSPC活力(我们使用台盼蓝染色,但也可以使用另一种方法)。此外,在接种细胞之前估计所得悬浮液的密度。如果密度高于1 x 10 5 细胞/ ml,则相应地稀释悬浮液(通常为1:2)。&nbsp;
    6. 取0.5ml所得悬浮液,将细胞转移到24孔板中的每个Matrigel包被的盖玻片上,接种下一代NSPCs。
      注意:我们建议在2D培养基中以每个盖玻片至少3 x 10 4 细胞培养NSPC,因为我们有通过实验确定。

  7. 通过胚胎NSPCs产生3D神经球体
    1. 形成贴壁神经球体(神经球)
      为了形成3D神经球,取NSPC悬浮液(每个盖玻片8×10×4×/ sup)细胞,并将细胞重悬于培养基中(不补充FGF-2)。正如我们常规观察到的那样,从培养基中去除FGF-2可促进胚胎NSPCs的自发分化,并促进贴壁神经球体的形成。&nbsp;
    2. 将细胞转移到24孔板上的Matrigel涂覆的盖玻片上,每个盖玻片的体积为0.5ml。将平板置于CO 2 培养箱中(35°C,5%CO 2 )。
      注意:对于贴壁神经球体的形成,盖玻片需要涂有Matrigel。如果实验设计包括浮动神经球体的形成,则不需要涂覆盖玻片。
    3. 在接种细胞后的最初24-48小时内,神经球将形成。在这段时间内不需要改变培养基,以免破坏形成。神经球在3D培养中逐渐增长。在3D培养中6-7天后,胚胎NSPCs产生稳定的神经球体,其发育可达150-200μm(见图5)。


      图5.胚胎小鼠NSPCs在贴壁3D培养中5天后产生的典型神经球体。比例尺:50μm。

    注意:我们建议不要在3D培养物中培养胚胎NSPC超过9-10天,因为3D神经球促进自发细胞分化。在那个时间点,3D神经球体可以达到300微米以上,这可以促进地层核心内的细胞死亡。因此,强烈建议在3D神经球体到达过度生长之前传代NSPC。

  8. 在2D或3D培养中免疫染色NSPCs
    1. 从胚胎NSPCs的2D或3D培养物中吸取所有培养基。用PBS洗涤细胞(预热至室温)以除去死细胞和任何碎片。
    2. 在含有细胞的每个孔中加入500μl4%PFA的0.1M磷酸盐缓冲液。在室温下孵育30分钟。&nbsp;
    3. 用PBS清洗至少2次。
      注意:我们建议使用~0.5 ml的缓冲液以确保充分洗涤。在该步骤中,可以储存细胞直至进行免疫染色(长达一周时间)。在这种情况下,用封口膜密封含有盖玻片的盖板,以避免干燥并保持在4°C。
    4. 为了阻断非特异性染色,将细胞(或神经球体)在含有0.3%Triton X-100和0.5%BSA(参见配方)的封闭溶液中在室温下孵育1.5小时。
    5. 与含有0.3%Triton X-100和0.5%BSA的抗体溶液中的一抗孵育(参见配方),在4℃下过夜。我们每孔使用200μl抗体溶液。
      注意:在封闭溶液中孵育或使用抗体促进治疗时,以缓慢的速度摇动平板是有用的(但不是必需的)。
    6. 在PBS中洗涤细胞/神经球3次,每次15分钟。
    7. 与含有0.5%BSA(不含Triton X-100)的抗体溶液中的第二抗体在室温下在黑暗中孵育1小时。我们每孔使用200μl溶液。&nbsp;
    8. 在PBS中洗涤3次,持续15分钟。&nbsp;
    9. 在室温下避光孵育Hoechst 33342(PBS中1:5,000)3-5分钟。&nbsp;
    10. 在PBS中洗涤3次。然后在ddH 2 O中洗涤以冲洗掉所有盐。细胞/球状体已准备好进行成像。
    11. 成像
      我们在载玻片上进行免疫染色细胞的成像。为此,使用封固剂(ImmunoHistoMount)将含有2D培养物(或3D神经球体)的盖玻片安装到SuperFrost ®玻璃载玻片上。然后让载玻片在室温下风干过夜。将样品保持在4°C。通过这种方法,可以根据需要储存免疫染色细胞制剂。我们提供了i)胚胎NSPCs产生的贴壁神经球体用于巢蛋白和β-微管蛋白III染色的免疫染色的实例图像(见图6)和ii)胚胎NSPCs的贴壁单层胚胎,用于巢蛋白和Hoechst染色。 2D文化(见图4B)。其他蛋白质的染色也可以使用这种方法进行。


      图6. 3D培养中小鼠胚胎NSPCs产生的神经球体的免疫荧光染色。 A和B.胚胎NSPCs产生的神经球体的共聚焦图像合并为巢蛋白(绿色)和β-微管蛋白在第3天在3D培养物中进行III(红色)染色。比例尺,50μm(A)和40μm(B)。

  9. 小鼠胚胎NSPCs植入器官型海马组织
    1. 海马切片的制备
      根据贵研究所批准的程序,在P8-P9处牺牲FVB小鼠的幼仔。如上所述和之前所述(Rybachuk et al。,2017),将动物斩首,解剖大脑并将海马体移除到冷的解剖培养基中(参见食谱)。将每个海马放置在组织切碎盘上,安装在组织切碎机(McIlwain)上,并开始切碎以将组织切割成350μm厚的横切片。将海马切片放入含有冷解剖培养基的培养皿中,并将切片翻转以使其内侧朝上。只收集那些具有公认的海马样形态的海马切片。
      注意:为了“好”切割组织,我们建议在切碎器盘上方放一张滤纸,并将海马体置于其上,以避免刀片滑动和摆动组织。此外,在开始切碎之前,去除组织周围的多余培养基。&nbsp;
    2. 器官型海马组织培养物
      使用玻璃巴斯德吸管将火焰抛光的大尺寸尖端(内径接近3mm)转移到置于6孔板上的0.4-μm膜插入物的表面上。我们通常在0.4-μm膜插入物上铺板四片。使用玻璃巴斯德吸管(小直径(内径约0.8 mm))取出备用培养基。向每个含有膜插入物上的切片的孔中加入1ml培养基(参见食谱)。将板置于CO 2 培养箱(35℃,5%CO 2 )中。接种后第2天更换培养基。之后,每两天更换一次媒体。
      注意:许多膜插入物(组织培养板上的孔)取决于切割“好”的切片量,可以从6孔板调整到24孔板。&nbsp; >
    3. 胚胎NSPCs的植入
      取50μl新鲜分离的NSPCs悬浮液,用标准移液管将细胞置于器官型海马组织表面(Eppendorf,Research ® Plus)。我们在接种后7天将胚胎NSPCs移植到海马组织上,但是可以使用任何时间点。任何时间都需要根据实验设计进行调整。通过非常小心地将细胞均匀地分布在组织表面上而不形成细胞聚集体来转移NSPC。此外,确保NSPC仅在组织表面沉降。不要急剧移动平板以防止新植入的细胞落在膜插入物上。在植入后第2天,更换培养基以清洗未附着于宿主组织的NSPC。
      注意:我们建议每片切片密度为0.25 x 10 5 细胞移植NSPC。来自2D或3D培养的NSPC也可以在仔细解离细胞后移植,以确保在宿主组织表面均匀分布。
    4. 胚胎干细胞在宿主器官组织内的生长。
      在CO 2 培养箱(35°C,5%CO 2 )中用NSPC移植物维持器官型海马组织直至使用。每两天更换一次培养基。
      注意:我们建议通过在膜插入物下吸出来更换培养基。
    5. 检查宿主组织内分化NSPCs的生长情况。由于组织内GFP标记的NSPC的简单追踪,这可以在任何时间点进行。 3天后,移植的NSPC开始掺入宿主组织中(参见图7)。器官型海马组织中的免疫染色与上文和我们之前的工作中针对细胞培养所描述的程序类似地进行(Rybachuk 等人,2017; Kopach 等人,2018)。 。


      图7.胚胎小鼠NSPC移植到宿主海马组织上的实例。 A和B.免疫染色的器官型海马组织与GFP标记的NSPC(绿色,A)的共聚焦图像和GFP标记的NSPC(绿色)和GFAP(红色)染色(B)在植入后第3天的合并图像。比例尺,100微米。

数据分析

获得的胚胎NSPC悬浮液可用于在体外<或>在宿主组织的内源环境中生长和/或分化细胞。

  1. 在2D培养中,可以通过视觉监测细胞的生长,并通过巢蛋白(红色)和Hoechst 33342(蓝色)的免疫染色来确认,如图4所示。参见Kopach 等人( 2018),图1B,用于在1d 体外免疫染色NSPC级分的图像以及图6A用于在不同实验条件下对巢蛋白阳性NSPC(未分化祖细胞)的比例进行统计分析。
  2. 在3D文化中,NSPC可以遵循强大的多谱系差异。从胎鼠海马中分离的NSPC可以产生神经球体,这种形成可以增强细胞生长和分化,并有助于维持分化祖细胞的高生存力。为了评估在3D培养中是否从胚胎NSPC产生神经元或神经胶质,需要使用细胞类型特异性标记物。为了观察神经球体内的表型分布,我们对星形胶质细胞标记GFAP的分化祖细胞以及神经元标记物NeuN和少突胶质细胞特异性标记物olig-2进行了免疫染色。所示的实例是在3D培养的第5天采集的胚胎NSPC的表型分化的图像(参见图8)。


    图8.胚胎小鼠NSPCs产生的3D神经球体的免疫荧光染色。 A.胚胎海马NSPCs免疫染色的神经球体对GFAP(青色)和β-微管蛋白III(红色)染色的共聚焦图像在3D文化的第5天。 B.由胚胎NSPC产生的GFAP(红色)和olig-2(青色)染色产生的神经球体的共聚焦图像。 C.用于Hoechst 33342(蓝色)和NeuN(红色)染色的产生NSPC的神经球体的共聚焦图像。比例尺,50μm(A)和20μm(B和C)。

  3. 在宿主脑组织中,器官型海马切片,其中形态层结构和信号传导途径组件在所需的一段时间内保持(组织维持数周),胚胎NSPCs显示出迅速的多谱系神经发生。使用器官型海马组织,我们通过电生理学方法监测神经元兴奋性,从而能够追踪NSPC衍生的海马神经元的时间依赖性成熟。特别地,我们记录了来自分化祖细胞的被动膜特性[见Kopach 等(2018),图1],神经元放电[见Kopach 等人(2018) ),图3]和在NSPC衍生的神经元中自发诱发的突触事件[参见Kopach 等人(2018),图2]。器官型海马组织代表了一种有用的工具,用于在移植后的不同时间点可行地评估在内源宿主环境中分化NSPC的神经生理学特性的成熟。此外,可以实现成熟祖细胞的功能特性与内源性主要神经元之间的直接定量比较。可以使用针对细胞类型特异性标记物的免疫染色进一步证实分化NSPC的胶质谱系(参见Kopach 等 [2018]和图6F中的方案的描述,用于NSPC衍生的图像)。移植后2或3周时少突胶质细胞或星形胶质细胞。

食谱

  1. 0.2 M磷酸盐缓冲液(总体积250 ml; pH 7.4)
    1. 取1.56g磷酸二氢钠(NaH 2 PO 4 )并加入50ml ddH 2 O&lt;
    2. 取5.68g磷酸二钠(Na 2 HPO 4 )并加入200ml ddH 2 O&lt;
    3. 将200ml稀释的Na 2 HPO 4 与50ml稀释的NaH 2 PO 4 混合。
    4. 在4°C下储存0.2 M磷酸盐缓冲液(pH 7.4)
  2. 在0.1M磷酸盐缓冲液中的4%多聚甲醛(PFA)(总体积100ml)
    1. 取4g PFA并将其加入50ml ddH 2 O中,同时在60℃下搅拌
    2. 为了促进在ddH 2 O中稀释PFA,在搅拌下向混合物中加入20μlNaOH。
    3. 搅拌混合物约30分钟
    4. 向混合物中加入50ml 0.2M磷酸盐缓冲液
    5. 使用0.45μm过滤器过滤混合物&nbsp;
    6. 如果在接下来的几天内不使用PFA,请将其保存在-20°C
  3. 生长培养基(总量50毫升)
    1. 取48.5 ml Neurobasal培养基,加入50 ml Falcon试管中。
    2. 加入1毫升B-27 ®(50x)&nbsp;
    3. 加0.5 ml GlutaMAX TM (100x)
    4. 加入50μlN-乙酰基-L-半胱氨酸(NAC)和0.25ml P / S.
    5. 如果不立即使用,将介质保存在4°C&nbsp;
    6. 使用前将20 ng / ml FGF-2加入培养基中
  4. 组织解剖培养基(总体积100 ml; pH 7.3)
    1. 取50毫升MEM,将其加入Falcon管中。
    2. 加入25毫升HBSS
    3. 加入60μgTris使其终浓度为5 mM
    4. 加入17.5μgNaHCO 3 (终浓度2 mM)
    5. 加入1.26毫升HEPES(终浓度12.5 mM)
    6. 加入276.5μg葡萄糖(终浓度15 mM)
    7. 加入1毫升P / S&nbsp;
    8. 在ddH 2 O中稀释,得到总体积为100ml
    9. 使用0.45-μm无菌过滤器过滤制备的培养基
    10. 将介质保存在4°C(如果在接下来的几天内不使用,则冻结)
  5. 组织培养基(总体积100ml; pH7.2)
    1. 取50毫升MEM,将其加入Falcon管中。
    2. 加入25毫升HBSS
    3. 加入30μgTris(终浓度2.5 mM)
    4. 加入17.5μgNaHCO 3 (2 mM)
    5. 加入1.26毫升HEPES(12.5 mM)
    6. 加入276.5μg葡萄糖(15 mM)
    7. 加入1毫升P / S&nbsp;
    8. 通过0.45-μm无菌过滤器过滤培养基并将其储存在4°C(如果不使用则冷冻)
    9. 在使用之前,向培养基中加入250μl/ ml马血清。
    10. 在使用之前,向培养基中加入20μl/ ml B-27 ®补充剂(50x)。
  6. 抗体解决方案
    在使用之前将BSA(0.5%)溶解在PBS中
  7. 阻止解决方案
    使用前将BSA(0.5%)和Triton X-100(0.3%)溶解于PBS中

致谢

这项工作得到了乌克兰国家科学院的资助。

利益争夺

作者声明没有利益冲突或竞争利益。

伦理

所有程序均按照Bogomoletz生理学研究所和国家遗传与再生医学研究所(乌克兰基辅)动物护理和使用委员会批准的方案使用,并符合欧盟委员会指令(86/609 / EEC)指南。

参考

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  4. Kempermann,G.,Gage,FH,Aigner,L.,Song,H.,Curtis,MA,Thuret,S.,Kuhn,HG,Jessberger,S.,Frankland,PW,Cameron,HA,Gould,E。, Hen,R.,Abrous,DN,Toni,N.,Schinder,AF,Zhao,X.,Lucassen,PJ和Frisen,J。(2018)。 人类成人神经发生:证据及其余问题。 细胞干细胞 23(1):25-30。
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  6. Kopach,O。和Pivneva,T。(2018)。 基于细胞的治疗中风相关神经变性神经替代策略的方法:干细胞祖细胞神经发生的神经生理学见解在主机环境中。 Neural Regen Res 13(8):1350-1351。
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  9. Pagano,SF,Impagnatiello,F.,Girelli,M.,Cova,L.,Grioni,E.,Onofri,M.,Cavallaro,M.,Etteri,S.,Vitello,F.,Giombini,S.,Solero ,CL和Parati,EA(2000)。 从成人嗅球中分离和鉴定神经干细胞。 干细胞 18(4):295-300。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rybachuk, O., Kopach, O., Pivneva, T. and Kyryk, V. (2019). Isolation of Neural Stem Cells from the Embryonic Mouse Hippocampus for in vitro Growth or Engraftment into a Host Tissue. Bio-protocol 9(4): e3165. DOI: 10.21769/BioProtoc.3165.
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