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Aug 2019

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Integration of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons into Rat Brain
人诱导多能干细胞(hiPSC)源神经元与大鼠脑回路的整合   

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Abstract

Human neuron transplantation offers novel opportunities for modeling human neurologic diseases and potentially replacement therapies. However, the complex structure of the human cerebral cortex, which is organized in six layers with tightly interconnected excitatory and inhibitory neuronal networks, presents significant challenges for in vivo transplantation techniques to obtain a balanced, functional and homeostatically stable neuronal network. Here, we present a protocol to introduce human induced pluripotent stem cell (hiPSC)-derived neural progenitors to rat brains. Using this approach, hiPSC-derived neurons structurally integrate into the rat forebrain, exhibit electrophysiological characteristics, including firing, excitatory and inhibitory synaptic activity, and establish neuronal connectivity with the host circuitry.

Keywords: Human neuron transplantation (人神经元移植), Excitatory and inhibitory neuronal networks (兴奋性和抑制性神经网络), hiPSC-derived neurons (hiPSC源神经元), Electrophysiological characteristics (电生理特征), Neuronal connectivity (神经元连接)

Background

The human cerebral cortex is a complex cellular mosaic containing diversified neuronal subtypes in distinct cortical layers (I-VI) that establish specific patterns of axonal output and dendritic input, providing the essential substrate of cortical circuitry (Rakic, 2009; Lodato et al., 2011; Lui et al., 2011). In particular, a balance of excitatory and inhibitory neurotransmission is necessary for proper brain function (Turrigiano and Nelson, 2004). Human induced pluripotent stem cells (hiPSCs) allow modeling human neurological diseases in a human genetic context (Dolmetsch and Geschwind, 2011; Brennand et al., 2015; Vera and Studer, 2015). Considerable advances have been made in establishing in vitro systems to differentiate hiPSCs into neurons, including generating excitatory (glutamatergic) projection neurons or inhibitory (GABAergic) interneurons (Pasca et al., 2011; Shi et al., 2012; Liu et al., 2013; Maroof et al., 2013; Nicholas et al., 2013; Espuny-Camacho et al., 2017). In vivo studies also addressed that hiPSC-derived neurons exhibit neuronal morphology and possess synaptic activity after grafting to the rodent brain (Weick et al., 2011; Nicholas et al., 2013; Espuny-Camacho et al., 2017). However, development of a balanced network of both excitatory and inhibitory neurons representative of the complex constitution of human cortex had not been achieved until our recent reports (Xu et al., 2016; Yin et al., 2019).

The protocol described here generates an in vivo model which provides a more accurate representation of the human cortex, allowing the study of the interplay between excitatory and inhibitory networks in both normal and pathological conditions. In addition, in this system, transplanted human neurons display functional neuron behaviors and show mature electrophysiological profiles. Together, using this method, hiPSC-derived neural progenitors develop into functional neurons after grafting in the rat neonatal brain, with physiological properties including excitatory and inhibitory postsynaptic currents, which are indicative of the copresence of excitatory and inhibitory networks. This model enables the study of human cortical development and diseases such as autism, schizophrenia, and Alzheimer’s disease, which are thought to involve imbalances in excitatory and inhibitory neural transmission.

Materials and Reagents

  1. Animals
    NIH nude rat pups (Charles River Laboratories, RRID: RGD_2312499) (postnatal Day 1, P1) are used for transplant recipients. These athymic nude rats are T-cell deficient and show depleted cell populations in thymus-dependent areas of peripheral lymphoid organs. Animals are housed and maintained in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committees.

  2. Culture products
    1. Regular 6-well plates (Thermo Fisher Scientific, catalog number: 140675 )
    2. Ultra-Low attachment 6-well plates (Corning, catalog number: 3471 )
    3. Laminin/poly-D-lysine coated plates (Corning, catalog number: 354595 )
    4. hiPSC dsRED-SC1014 cell line (Johns Hopkins Medical Institutions) (SC1014 cell line is a characterized human iPSC line which was reprogrammed by non-integrating Sendai virus and lentivirus. The stable dsRED-SC1014 cell line was established by nucleofection with piggybac-dsRED transposon and piggyback transposase)
    5. Mouse embryonic fibroblasts (MEFs, Gibco, catalog number: S1520-100 )
    6. Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, catalog number: 10569010 )
    7. Fetal Bovine Serum (FBS), ESC-qualified (Thermo Fisher Scientific, catalog number: 10439024 )
    8. Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12, Thermo Fisher Scientific, catalog number: 11320033 )
    9. Knockout serum replacement (KSR, Thermo Fisher Scientific, catalog number: 10828028 )
    10. Fibroblast growth factor 2 (FGF2; 4 ng/ml; PeproTech, catalog number: 100-18B )
    11. 1 mM Glutamax (Thermo Fisher Scientific, catalog number: 35050061 )
    12. 100 μM non-essential amino acids (Thermo Fisher Scientific, catalog number: 11140050 )
    13. 100 μM 2-mercaptoethanol (Thermo Fisher Scientific, catalog number: 21985023 )
    14. Noggin (50 ng/ml, R&D system, catalog number: 6057-NG-025 )
    15. Dorsomorphin (1 μM, Tocris, catalog number: 3093 )
    16. SB431542 (10 μM, Tocris, catalog number: 1614 )
    17. 1% N2 supplement (Thermo Fisher Scientific, catalog number: 17502001 )
    18. Heparin (2 μg/ml, Millipore Sigma, catalog number: H3149 )
    19. Neurobasal medium (Thermo Fisher Scientific, catalog number: 21103049 )
    20. B27 minus VitA (Thermo Fisher Scientific, catalog number: 12587001 )
    21. Retinoic acid (RA, Millipore Sigma, catalog number: R2625 )
    22. Sonic Hedgehog (SHH, 50 ng/ml, R&D system, catalog number: 1845-SH-025 )
    23. Purmorphamine (2 μM, R&D system, catalog number: 4551/10 )
    24. Brain-derived neurotrophic factor (BDNF, 20 ng/ml, PeproTech, catalog number: 450-02 )
    25. Glial cell line-derived neurotrophic factor (GDNF, 20 ng/ml, PeproTech, catalog number: 450-10 )
    26. Ascorbic acid (0.2 mM, Millipore Sigma, catalog number: 1043003 )
    27. Dibutyryl cAMP (0.5 mM, Millipore Sigma, catalog number: D0260 )
    28. Collagenase (1 mg/ml, Millipore Sigma, catalog number: C9722 )
    29. Matrigel (Corning, catalog number: 354230 )
    30. Accutase (Corning, catalog number: 25-058-Cl )
    31. Dulbecco’s PBS (DPBS) without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190144 )
    32. Human embryonic stem cell (ESC) medium (see Recipes)
    33. Mouse embryonic fibroblast (MEF) medium (see Recipes)
    34. N2-induction medium (NIM) (see Recipes)
    35. Neural differentiation medium (see Recipes)

  3. Immunostaining
    1. Ketamine (Henry Schein, catalog number: 1049007 )
    2. Xylazine (Rompun, catalog number: 321350RX )
    3. Tissue-Tek OCT Compound (SAKURA, catalog number: 4583 )
    4. 2-methylbutane (Fisher Scientific, catalog number: 03551-4 )
    5. Donkey serum (Abcam, catalog number: ab7475 )
    6. Triton X-100 (Millipore Sigma, catalog number: T8787 )
    7. Anti-human-specific NES (Millipore, MAB5326, RRID: AB_11211837)
    8. Anti-TBR1 (Abcam, catalog number: ab31940 , RRID: AB_2200219)
    9. Anti-CTIP2 (Abcam, catalog number: ab18465 , RRID: AB_2064130)
    10. Anti-BRN2 (Santa Cruz Biotechnology, catalog number: sc-6029 , RRID: AB_2167385)
    11. Anti-SATB2 (Abcam, catalog number: ab51502 , RRID: AB_882455)
    12. Anti-PROX1 (Abcam, catalog number: ab37128 , RRID: AB_882189)
    13. Anti-TUJ1 (Millipore, catalog number: MAB1637 , RRID: AB_2210524)
    14. Anti-MAP2 (Millipore Sigma, catalog number: M2320 , RRID: AB_609904; Millipore, catalog number: AB5622 , RRID: AB_91939)
    15. Phosphate-buffered saline (PBS), 1x, pH 7.4 (see Recipes)
    16. 4% paraformaldehyde (PFA) (see Recipes)
    17. Ketamine/xylazine solution (see Recipes)

  4. Electrophysiological Recordings
    1. Isoflurane (Henry Schein, catalog number: 1182097 )
    2. NaCl (Millipore Sigma, catalog number: 71380 )
    3. KCl (Millipore Sigma, catalog number: 31248 )
    4. MgSO4 (Millipore Sigma, catalog number: 63138 )
    5. NaH2PO4 (Millipore Sigma, catalog number: S5011 )
    6. NaHCO3 (Millipore Sigma, catalog number: S5761 )
    7. CaCl2 (Millipore Sigma, catalog number: C7902 )
    8. D-glucose (Millipore Sigma, catalog number: G6152 )
    9. HEPES (Millipore Sigma, catalog number: H6147 )
    10. EGTA (Millipore Sigma, catalog number: E8145 )
    11. MgATP (Millipore Sigma, catalog number: A9187 )
    12. Na3GTP (Millipore Sigma, catalog number: G8877 )
    13. K-gluconate (Millipore Sigma, catalog number: G4500 )
    14. CsCl (Millipore Sigma, catalog number: 203025 )
    15. Tetrodoxin (TTX, 1 μM, Tocris, catalog number: 10-691 )
    16. Tetraethylammonium (TEA, 10 mM, Tocris, catalog number: 30-685-0 )
    17. Picrotoxin (10 μM, Tocris, catalog number: 11-281 )
    18. Bicuculline (Bic, 10 μM, Tocris, catalog number: 25-031-0 )
    19. 6-Cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 20 μM, Tocris, catalog number: 10-451-0 )
    20. D-2-amino-5-phosphonovaleric acid (D-AP5, 50 μM, Tocris, catalog number: 01-061-0 )
    21. ACSF (see Recipes)
    22. Internal solution (see Recipes)

Equipment

  1. CO2 incubator (Eppendorf, Hauppauge, NY, USA)
  2. 37 °C water bath
  3. Centrifuge (Thermo Fisher Scientific, Waltham, MA, USA)
  4. Auto-nanoliter injector (Drummond, model: Nanoinject II )
  5. Cryostat (Leica, model: CM3050 )
  6. LSM880 confocal laser-scanning microscope (Carl Zeiss, Germany)
  7. Vibratome (Leica, model: VT1200S )
  8. Flaming-Brown micropipette puller (Sutter Instruments, model: P-1000 )
  9. HEKA EPC10 amplifier (HEKA Elektronik, Lambrech, Germany)

Software

  1. ZEN lite (Carl Zeiss, https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html)
  2. PatchMaster (HEKA Elektronik, https://www.heka.com/downloads/downloads_main.html)
  3. Clampfit (Molecular devices, https://www.moleculardevices.com/products/axon-patch-clamp-system/acquisition-and-analysis-software/pclamp-software-suite#gref)
  4. MiniAnalysis (Synaptosoft, http://www.synaptosoft.com/MiniAnalysis/)

Procedure

  1. hiPSCs culture and passage
    1. Culture hiPSC dsRED-SC1014 cells on inactivated MEFs in human embryonic stem cell (ESC) medium (see Recipes) in regular 6-well plates. Change medium every day.
    2. Passage cells at a ratio 1:6 to 1:12 when cells reach 80-90% confluency.
    3. Prepare MEF plates following Steps A4-A6 one or two days before passaging hiPSCs culture.
    4. Cover the whole surface of culture vessels with 2% Matrigel (in DMEM/F12) and incubate the vessels at room temperature for at least 1 hour. 
    5. Aspirate Matrigel and plate 30,000 cells/cm2 of mitotically inactivated MEFs in the vessels in MEF medium (see Recipes).
    6. Keep MEF plates in a 37 °C, 5% CO2 incubator.
    7. Aspirate the MEF medium from a plate containing inactivated MEFs and add pre-warmed human ESC medium 3-4 h before plating hiPSCs.
    8. Remove hiPSC colonies which need to be passaged under a dissection microscope.
    9. Add collagenase (1 mg/ml in DMEM/F12) solution to the plate containing hiPSCs, and incubate the plate in a 37 °C, 5% CO2 incubator for 30-60 min.
    10. Stop the incubation when the edges of the colonies start to pull away from the plate.
    11. Remove the collagenase carefully to avoid disturbing the attached cell layer.
    12. Add human ESC medium to the plate, gently blow hiPSCs off, and collect hiPSCs in a new conical tube. Make sure to minimize foaming.
    13. Centrifuge at 200 x g for 5 min, and then remove the supernatant from the hiPSC pellet.
    14. Resuspend the pellet with human ESC medium.
    15. Add appropriate volume of cell suspension to each MEF plate, and return the plate to the incubator. Replace medium daily.

  2. Neural differentiation of hiPSCs
    1. Differentiate hiPSCs to neurons based on rosette neural aggregates (termed RONAs) method (Xu et al., 2016) as below.
    2. Incubate hiPSC colonies with collagenase (1 mg/ml in DMEM/F12) in the incubator for about 5-10 min, and then gently wash off the collagenase with medium.
    3. Grow detached hiPSC colonies in suspension as embyroid bodies (EBs) (100-200 μm in diameter, ~10 EBs/ml medium) in human ESC medium without FGF2 for 2 days in Ultra-Low attachment 6-well plates (Corning).
    4. From day 2 to day 6, add Noggin (50 ng/ml, R&D system) or Dorsomorphin (1 μm, Tocris) and SB431542 (10 μM, Tocris) in human ESC medium (without FGF2).
    5. On day 7, transfer free-floating EBs to Matrigel precoated culture plates (as described above) to allow the complete attachment of EB aggregates with the supplement of N2-induction medium (NIM, see Recipes).
    6. Continuously feed cultures with N2-medium every other day from days 7 to 12.
    7. Change N2-induction medium every day from day 12 onward. 
    8. Attached EB aggregates break down to form a monolayer colony on day 8 to 9 with typical neural-specific rosette formation. With the extension of neural induction, highly compact three-dimensional column-like neural aggregates RONAs form in the center of attached colonies. Microisolate RONAs manually, taking special care to minimize contaminating the peripheral monolayer of flat cells and cells underneath RONAs.
    9. At day 17, collect and maintain RONA clusters as neurospheres in Neurobasal medium (Thermo Fisher Scientific) containing B27 minus VitA (Thermo Fisher Scientific), 1 mM Glutamax (Thermo Fisher Scientific) for one day. A typical RONA is shown in Figure 1.


      Figure 1. Typical RONAs on day 17. Immunostaining of PAX6 and NKX2.1 showing cortical patterning related transcriptional factors in neural progenitors (reproduced from Xu et al., 2016).

    10. At day 18, dissociate neurospheres into single cells following Steps B11 to B19.
    11. Remove neurosphere suspension from culture plate and transfer to a 15 ml conical tube.
    12. Let neurospheres settle down in the tube 2-5 min, and then remove the medium carefully leaving ~100 μl medium remaining with the neurospheres.
    13. Resuspend neurospheres in 5 ml DPBS (Thermo Fisher Scientific).
    14. Let neurospheres settle down in the tube 2-5 min, and remove DPBS carefully leaving ~100 μl DPBS remaining with the neurospheres.
    15. Add 1 ml Accutase (Corning) to the neurospheres and incubate for 10 min at room temperature. 
    16. Pipet up and down until all the neurospheres are in a single cell suspension.
    17. Add 4 ml neural differentiation medium (see Recipes) to the tube, and centrifuge the cells at 200 x g for 5 min.
    18. Remove the supernatant gently. 
    19. Resuspend the cells in neural differentiation medium to desired cell density, and plate the cells on Laminin/poly-D-lysine coated plates (Corning).
    20. From days 24 to 30, apply RA (2 μM, Millipore Sigma), SHH (50 ng/ml, R&D system), and Purmorphamine (2 μM, R&D system) into neural differentiation medium to maintain the identity of neural progenitors, which provides an appropriate percentage of excitatory (~80-85%) and inhibitory (~15-20%) neurons representative of the human cerebral cortex.
    21. hiPSC-derived neural progenitors at days 31-32 are injected into neonatal rat cortices as described below (Procedure C), and characterization are performed at 10 weeks after transplantation.
    22. In culture conditions, hiPSC-derived neurons show mature human cortical identity after 10 weeks (Figure 2).


      Figure 2. hiPSC-derived neurons show human cortical identity in culture after 10 weeks of differentiation. A. Cultures express the cortical layer-specific markers TBR1, CTIP2, BRN2, and STAB2. B. Some cells express the hippocampal marker PROX1 (reproduced from Yin et al., 2019).

  3. Animal transplantation
    1. The whole procedure from the cell culture to transplantation is shown in Figure 3.


      Figure 3. An outline showing transplantation of forebrain progenitors from hiPSCs (reproduced from Yin et al., 2019).

    2. Trypsinize hiPSC-derived neural progenitors at days 31-32 in culture and thoroughly dissociate with a pipette. Then, centrifuge the cells at 300 x g for 3 min.
    3. Carefully remove the supernatant, and resuspend the pellet in ice cold 1x PBS with a final density of 2 x 106 cells/μl.
    4. Fill 1.0 μl of cells into a pulled glass micropipette (0.5 mm I.D., 1 mm O.D.) by suction, and then fit glass micropipette to an auto-nanoliter injector ( Nanoinject II , Drummond). Using nanoinjector allows to minimize tissue damage and inject small volume at a relatively slow rate.
    5. Gently and deeply anesthesize neonatal rat pups.
    6. Inject the cells into the right cortex (2.0 mm posterior and 1.9 mm lateral to bregma, 2.6 mm below the dura) over 1 min. Keep the micropipette tip in place for a further 2 min before withdrawal.

  4. Brain sections immunostaining
    1. Anesthetize rats (10-week-old, also 10 weeks after transplantation) with ketamine/xylazine solution (0.5 ml/kg, see Recipes).
    2. Perfuse rats through the left ventricle of the heart with 1x PBS until the liver is bleached.
    3. Continue perfusion with freshly prepared 4% PFA.
    4. Dissect the brains and post-fix them in 4% PFA for 2-3 days at 4 °C.
    5. Transfer the brains to 30% sucrose until they sink to the bottom when they get saturated with sucrose solution.
    6. Remove the brains from sucrose solution and put them in 2-methylbutane (Fisher Scientific) in a container already chilled in liquid nitrogen for 30 s to 1 min.
    7. Drop some OCT compound (SAKURA) on the cryostat tissue holder already placed on dry ice.
    8. Place the brain tissue in the center of the holder and add more OCT compound surrounding the brain until it becomes solidified. 
    9. Store the OCT brain blocks at -80 °C until being processed for serial sections.
    10. Prepare brain coronal sections (25 μm) using a cryostat ( CM3050 , Leica).
    11. Antigen retrieval step is optional and should be standardized depending upon the guidelines for each antibody. For the antibodies used in the present study, antigen retrieval is not necessary. 
    12. Proceed sections for blocking with 10% (v/v) donkey serum and 0.2% (v/v) Triton X-100 in 1x PBS for one hour at room temperature.
    13. Incubate sections overnight at 4 °C with primary antibodies of interest.
    14. Wash sections with PBS three times of 5 min each, followed by incubations with appropriate, fluorescently labeled secondary antibodies at room temperature for one hour.
    15. After incubation for one hour at room temperature, wash sections with PBS three times for 5 min each and mount them on a clean slide. Dry the slide completely, and then proceed for imaging with a confocal microscope (Figure 4 and Video 1).
    16. Video 1 is produced using Z-Stack images. Set the first and last positions for the Z-Stack, i.e., the depth of the section. 1.01 µm is used for the Interval in the current study. Alternatively, choosing interval can be done by clicking on Optimal to set the number of slices.


      Figure 4. Human neurons expressing RFP colocalize with the mature neuronal marker MAP2, suggesting hiPSC-derived neurons integrate into the rat brain. A. A low magnification image showing RFP positive human cells in rat brain. B. Brain sections immunostained with MAP2 and DAPI (reproduced from Yin et al., 2019).

      Video 1. hiPSC-derived neurons in rat brain as shown by RFP/MAP2 double positive cells (reproduced from Yin et al., 2019)

  5. Electrophysiological recordings of acute brain slices
    1. Anesthetize rat (10-week-old, also 10 weeks following transplantation) with isoflurane (300 μl in a 500 ml container for drop jar anesthesia) and quickly remove the brain.
    2. Prepare transverse brain slices of 350 μm thickness using a vibratome (Leica VT1200S ).
    3. Incubate slices in ACSF solution (see Recipe 4) continuously bubbled with 95% O2 and 5% CO2, first at 34 °C for 30 min, and then at room temperature.
    4. Transfer a single slice into a submerged recording chamber constantly perfused with carbogen-equilibrated ACSF.
    5. Visualize injected human neurons expressing RFP under a 40x water immersion objective by fluorescence and DIC optics (Carl Zeiss).
    6. Perform whole-cell patch clamp recordings using a glass pipettes (3-5 MΩ) filled with internal solution (see Recipes). Randomly select RFP-positive human neurons for recording 300 μm to 1,000 μm from graft site.
    7. Induce action potentials by a series of hyperpolarizing and depolarizing step currents.
    8. Evoke sodium and potassium currents by a series of voltage steps (from –100 mV to +60 mV in 20-mV steps).
    9. Record spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) in voltage-clamp configuration at –70 mV. Obtain sEPSCs in the presence of picrotoxin (100 μM), and sIPSCs in the presence of CNQX (20 μM) and D-AP5 (50 μM).
    10. The recording results show that grafting hiPSC-derived neurons functionally integrate to the rat brain, and they are able to fire and display excitatory and inhibitory synaptic activity (Figure 5).


      Figure 5. hiPSC-derived neurons functionally integrate into the synaptic circuitry of the rat brain. A. AP firing patterns of hiPSC-derived neurons. B. Representative traces of whole-cell Na+ (inwards) and K+ (outwards) currents recorded from grafted cells. C. Representative traces of sEPSCs and sIPSCs (reproduced from Yin et al., 2019).

Data analysis

The hiPSC cultures and human neurons obtained using these protocols were investigated and compared for their human cortical identity using a set of cortical-specific markers through a series of assays (Xu et al., 2016; Yin et al., 2019). Electrophysiological recording data were analyzed using Clampfit 10.5 software (Molecular devices) and MiniAnalysis software (Synaptosoft) as published (Yin et al., 2019). Therefore, the data analysis has not been discussed here.

Notes

  1. At Step A4, the collagenase should be gently washed off the plate with growth medium after the colony borders begin to peel away from the plate while the colony center remains attached, then the colonies will be selectively detached undisturbed.
  2. At Step A8, please note that attached aggregates usually break down to form a monolayer colony on day 8 to 9 with typical neural specific rosette formation. With the extension of neural induction, highly compact 3-dimensional column-like neural aggregates RONAs will form in the center of attached colonies.
  3. At Step A10, when collect the RONAs, the isolation should be done under microscope, taking special care to minimize contaminating the peripheral monolayer of flat cells and cells underneath RONAs.
  4. When preparing acute brain slices, the brain should be removed as soon as possible.
  5. ACSF solution should be prepared freshly every day before experiment. Also, it is very important to keep the slices in ACSF solution continuously bubbled with 95% O2 and 5% CO2 during recording. These will help maintain the brain slices in a healthily physiological condition.

Recipes

  1. 1x PBS, pH 7.4
    NaCl 8 g (0.137 M)
    KCl 200 mg (0.0027 M)
    Na2HPO4 1.44 g (0.01 M)
    KH2PO4 240 mg (0.0018 M)
    Make up the volume to 1 L with MilliQ water, adjust the pH to 7.4
  2. 4% PFA
    Dissolve 2 g PFA in 50 ml 1x PBS
  3. Human embryonic stem cell (ESC) medium
    DMEM/F12 (Thermo Fisher Scientific)
    20% knockout serum replacement (KSR, Thermo Fisher Scientific)
    4 ng/ml FGF2 (PeproTech)
    1 mM Glutamax (Thermo Fisher Scientific)
    100 μM non-essential amino acids (Thermo Fisher Scientific)
    100 μM 2-mercaptoethanol (Thermo Fisher Scientific) 
  4. Mouse embryonic fibroblast (MEF) medium
    DMEM (Thermo Fisher Scientific)
    10% FBS, ESC-qualified (Thermo Fisher Scientific)
    100 μM non-essential amino acid solution (Thermo Fisher Scientific)
    100 μM 2-mercaptoethanol (Thermo Fisher Scientific)
  5. N2-induction medium (NIM)
    DMEM/F12
    1% N2 supplement
    100 μM MEM non-essential amino acids solution
    1 mM Glutamax
    2 μg/ml Heparin
  6. Neural differentiation medium
    Neurobasal/B27 (Thermo Fisher Scientific)
    BDNF (20 ng/ml, PeproTech)
    GDNF (20 ng/ml, PeproTech)
    Ascorbic acid (0.2 mM, Millipore Sigma)
    Dibutyryl cAMP (0.5 mM, Millipore Sigma)
  7. Ketamine/xylazine solution
    Ketamine (100 mg/ml)
    Xylazine (20 mg/ml)
    Mix them in sterile saline (0.9% NaCl) for anesthesia
  8. ACSF
    NaCl 125 mM
    KCl 2.5 mM
    MgSO4 1 mM
    NaH2PO4 1.2 mM
    NaHCO3 26 mM
    CaCl2 2 mM
    D-glucose 10 mM
  9. Internal solution
    K-gluconate 126 mM
    KCl 8 mM
    HEPES 20 mM
    EGTA 0.2 mM
    NaCl 2 mM
    MgATP 3 mM
    Na3GTP 0.5 mM
    Adjust the pH to 7.3, and the osmolality to 290-300 mOsm

Acknowledgments

The protocols were developed with the grants from MSCRF and NIH. We thank Dr. Jin-Chong Xu, Dr. Gun-sik Cho and Dr. Chulan Kown for helping to standardize the protocols. The protocols were used in two separate studies published in 2016 and 2019 (Xu et al., 2016; Yin et al., 2019).

Competing interests

The authors declare no competing financial or non-financial interests.

Ethics

All experiments using hiPSCs were conducted in accordance with the policy of the Johns Hopkins University (JHU) School of Medicine (SOM) that research involving hiPSCs being conducted by JHU faculty, staff or students or involving the use of JHU facilities or resources shall be subject to oversight by the JHU Institutional Stem Cell Research Oversight (ISCRO) Committee.
  All the animal procedures used in establishing the protocols were in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committees. The approval ID of the animal experiment in this protocol was M017M288, which is validated from 10/1/2017 to 9/30/2020.

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  8. Nicholas, C. R., Chen, J., Tang, Y., Southwell, D. G., Chalmers, N., Vogt, D., Arnold, C. M., Chen, Y. J., Stanley, E. G., Elefanty, A. G., Sasai, Y., Alvarez-Buylla, A., Rubenstein, J. L. and Kriegstein, A. R. (2013). Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development. Cell Stem Cell 12(5): 573-586. 
  9. Pasca, S. P., Portmann, T., Voineagu, I., Yazawa, M., Shcheglovitov, A., Pasca, A. M., Cord, B., Palmer, T. D., Chikahisa, S., Nishino, S., Bernstein, J. A., Hallmayer, J., Geschwind, D. H. and Dolmetsch, R. E. (2011). Using iPSC-derived neurons to uncover cellular phenotypes associated with Timothy syndrome. Nat Med 17(12): 1657-1662.
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  13. Vera, E. and Studer, L. (2015). When rejuvenation is a problem: challenges of modeling late-onset neurodegenerative disease. Development 142(18): 3085-3089. 
  14. Weick, J. P., Liu, Y. and Zhang, S. C. (2011). Human embryonic stem cell-derived neurons adopt and regulate the activity of an established neural network. Proc Natl Acad Sci U S A 108(50): 20189-20194. 
  15. Xu, J. C., Fan, J., Wang, X., Eacker, S. M., Kam, T. I., Chen, L., Yin, X., Zhu, J., Chi, Z., Jiang, H., Chen, R., Dawson, T. M. and Dawson, V. L. (2016). Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity. Sci Transl Med 8(333): 333ra348. 
  16. Yin, X., Xu, J. C., Cho, G. S., Kwon, C., Dawson, T. M. and Dawson, V. L. (2019). Neurons derived from human induced pluripotent stem cells integrate into rat brain circuits and maintain both excitatory and inhibitory synaptic activities. eNeuro 6(4).

简介

[摘要] 人类神经元移植为建模人类神经系统疾病和潜在的替代疗法提供了新的机会。然而,人脑皮层的复杂结构分为六层,具有紧密互连的兴奋性和抑制性神经元网络,这对体内移植技术获得平衡,功能稳定和稳态稳定的神经元网络提出了重大挑战。在这里,我们提出了一项协议,将人类诱导的多能干细胞(hiPSC )衍生的神经祖细胞引入大鼠脑。使用这种方法,hiPSC 衍生的神经元在结构上整合到大鼠前脑中,表现出电生理特性,包括放电,兴奋性和抑制性突触活性,并与宿主电路建立神经元连通性。


[背景] 人类大脑皮层是一个复杂的细胞镶嵌体,在不同的皮质层(I-VI)中包含多样化的神经元亚型,可建立轴突输出和树突状输入的特定模式,提供了皮质电路的基本底物(Rakic,2009; Lodato 等等人,2011; Lui 等人,2011)。特别地,兴奋性和抑制性神经传递的平衡对于适当的脑功能是必需的(Turrigiano和Nelson,2004)。人类诱导的多能干细胞(hiPSC )可以在人类遗传背景下对人类神经系统疾病进行建模(Dolmetsch和Geschwind,2011; Brennand 等,2015; Vera和Studer,2015)。建立体外系统以将hiPSCs 分化为神经元方面已经取得了重大进展,包括生成兴奋性(谷氨酸能的)投射神经元或抑制性(GABA能的)中间神经元(Pasca 等,2011; Shi 等,2012; Liu 等。,2013; Maroof 等,2013; Nicholas 等,2013; Espuny-Camacho 等,2017)。体内研究还指出,hiPSC 衍生的神经元移植到啮齿动物大脑后表现出神经元形态并具有突触活性(Weick 等人,2011; Nicholas 等人,2013; Espuny-Camacho 等人,2017)。然而,直到我们最近的报道(Xu 等人,2016; Yin 等人,2019),才实现了代表人类皮质复杂结构的兴奋性和抑制性神经元的平衡网络的开发。

这里描述的协议生成了一个体内模型,该模型提供了人类皮层的更准确表示,从而可以研究正常和病理条件下兴奋性和抑制性网络之间的相互作用。另外,在该系统中,移植的人类神经元显示出功能性神经元行为并显示出成熟的电生理特征。在一起使用这种方法,hiPSC 衍生的神经祖细胞移植到大鼠新生脑后便发展为功能神经元,其生理特性包括兴奋性和抑制性突触后电流,这表明兴奋性和抑制性网络并存。该模型可以研究人类皮质发育和疾病,例如自闭症,精神分裂症和阿尔茨海默氏病,这些疾病被认为与兴奋性和抑制性神经传递失衡有关。

关键字:人神经元移植, 兴奋性和抑制性神经网络, hiPSC源神经元, 电生理特征, 神经元连接

材料和试剂


 


动物
NIH裸鼠幼崽(Charles River Laboratories,RRID:RGD_2312499 )(出生后第1天,P1)用于移植对象。这些无胸腺裸鼠是T细胞缺陷的,并且在外周淋巴器官的胸腺依赖性区域中显示出耗尽的细胞群。根据美国国家卫生研究院(NIH)的《实验动物的护理和使用指南》以及机构动物护理和使用委员会的规定,对动物进行饲养和维护。


 


文化产品
常规6孔板(Thermo Fisher Scientific,目录号:140675)
超低附件6孔板(Corning,目录号:3471)
层粘连蛋白/聚-D-赖氨酸涂层板(Corning,目录号:354595)
的hiPSC 的DsRed-SC1014细胞系(约翰霍普金斯杂志升机构)(SC1014细胞系是表征的人iPSC系,其通过非整合型仙台病毒和慢病毒重新编程。稳定的DsRed-SC1014细胞系通过用核转染建立的piggyBac转-dsRED 转座子和背back 转座酶)
小鼠胚胎成纤维细胞(MEF中,GIBCO,Ç atalog编号:S1520-100)
Dulbecco氏改进的Eagle培养基(DMEM,赛默飞世尔科技,Ç atalog号:10569010)
胎牛血清(FBS),ESC-合格(赛默飞世尔科技,Ç atalog号:10439024)
Dulbecco改良的Eagle培养基/营养混合物F-12(DMEM / F12,Thermo Fisher Scientific,目录号:11320033)
剔除血清替代品(KSR,Thermo Fisher Scientific,目录号:10828028 )
成纤维细胞生长因子2(FGF2; 4 ng / ml; PeproTech ,目录号:100-18B)
1 mM Glutamax (Thermo Fisher Scientific,目录号:35050061)
100 μM 非必需氨基酸(赛默飞世尔科技,产品目录号:11140050)
100 μM 2-巯基乙醇(赛默飞世尔科技,产品目录号:21985023)
头胶(50 ng / ml,R&D系统,货号:6057-NG-025)
Dorsomorphin(1μM ,Tocris ,目录号:3093)
SB431542(10 μ 中号,Tocris ,目录号:1614)
1%的N2补充(Thermo Fisher Scientific,目录号:17502001)
肝素(2μg / ml,Millipore Sigma,目录号:H3149 )
神经基础培养基(Thermo Fisher Scientific,目录号:21103049)
B27减去VitA (Thermo Fisher Scientific,目录号:12587001)
维甲酸(RA,Millipore Sigma,目录号:R2625 )
声波刺猬(SHH,50 ng / ml,R&D系统,目录号:1845-SH-025)
紫吗啡(2μM ,研究与开发系统,目录号:4551/10)
脑源性神经营养因子(BDNF,20 ng / ml,PeproTech ,目录号:450-02)
胶质细胞系衍生的神经营养因子(GDNF,20 ng / ml,PeproTech ,目录号:450-10)
抗坏血酸(0.2 mM,Millipore Sigma,目录号:1043003 )
二丁酰cAMP(0.5 mM,Millipore Sigma,目录号:D0260)
胶原酶(1 mg / ml,Millipore Sigma,目录号:C9722)
Matrigel(Corning,目录号:354230)
Accutase (Corning,目录号:25-058-Cl)
不含钙和镁的Dulbecco PBS(DPBS)(Thermo Fisher Scientific,目录号:14190144)
人胚胎干细胞(ESC)培养基(请参阅食谱)
小鼠胚胎成纤维细胞(MEF)培养基(请参阅食谱)
N2感应介质(NIM)(请参阅食谱)
神经分化培养基(请参阅食谱)
 


免疫染色
氯胺酮(Henry Schein,目录号:1049007)
赛拉嗪(Rompun ,目录号:321350RX)
Tissue-Tek OCT化合物(樱花,目录号:4583)
2-甲基丁烷(Fisher Scientific,目录号:03551-4)
驴血清(Abcam,目录号:ab7475)
Triton X-100(Millipore Sigma,目录号:T8787)
抗人特异性NES(Millipore,MAB532 6 ,RRID:AB_11211837)
抗TBR1(Abcam,目录号:ab31940,RRID:AB_2200219)
抗CTIP2(Abcam,目录号:ab18465 ,RRID:AB_2064130)
抗BRN2(圣克鲁斯生物技术,目录号:sc-6029,RRID:AB_2167385)
抗SATB2(Abcam,目录号:ab51502,RRID:AB_882455)
Anti-PROX1(Abcam,目录号:ab37128 ,RRID:AB_882189)
Anti-TUJ1(Millipore ,目录号:MAB1637 ,RRID:AB_2210524)
抗MAP2(Millipore Sigma,目录号:M2320 ,RRID:AB_609904; Millipore ,目录号:AB5 622,RRID:AB_91939)
PHO sphate缓冲盐水(PBS),1 X,pH为7.4(参见食谱)
4%多聚甲醛(PFA)(请参阅食谱)
氯胺酮/甲苯噻嗪溶液(请参见食谱)
 


电生理记录
异氟烷(Henry Schein,目录号:1182097)
NaCl(Millipore Sigma,目录号:71380)
氯化钾(Millipore Sigma,目录号:31248)
MgSO 4 (Millipore Sigma,目录号:63138)
NaH 2 PO 4 (Millipore Sigma,目录号:S5011)
NaHCO 3 (Millipore Sigma,目录号:S5761)
CaCl 2 (Millipore Sigma,目录号:C7902)
D-葡萄糖(Millipore Sigma,目录号:G6152)
HEPES(Millipore Sigma,目录号:H6147)
EGTA(Millipore Sigma,目录号:E8145)
MgATP (Millipore Sigma,目录号:A9187)
Na 3 GTP(Millipore Sigma,目录号:G8877)
葡萄糖酸K(Millipore Sigma,目录号:G4500)
CsCl (Millipore Sigma,目录号:203025)
河豚毒素(TTX,1μM ,Tocris ,目录号:10-691)
四乙铵(TEA,10 mM,Tocris ,目录号:30-685-0)
微小毒素(10μM ,Tocris ,目录号:11-281)
Bicuculline(Bic,10μM ,Tocris ,目录号:25-031-0)
6-Cyano-7-nitroquinoxaline-2,3-dione(CNQX,20μM ,Tocris ,目录号:10-451-0)
d-2-氨基-5-膦酰基戊酸(d-AP5,50 μM ,Tocris ,目录号:01-061-0)
ACSF(请参阅食谱)
内部解决方案(请参阅食谱)
 


设备


 


CO 2 培养箱(美国纽约州豪珀格,埃彭多夫)
37 °C水浴
离心机(Thermo Fisher Scientific,美国马萨诸塞州沃尔瑟姆)
自动纳升注射器(德拉蒙德,型号:Nanoinject II )
低温恒温器(Leica ,型号:CM3050 )
LSM880共聚焦激光扫描显微镜(德国卡尔蔡司)
振动刀(Leica ,型号:VT1200S )
火焰棕色微量移液器(萨特仪器,型号:P -1000 )
HEKA EPC10放大器(HEKA Elektronik ,Lambrech ,德国)
 


软件


 


ZEN lite(卡尔·蔡司,https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html)
PatchMaster (HEKA Elektronik ,https ://www.heka.com/downloads/downloads_main.html )
Clampfit (分子设备,https://www.moleculardevices.com/products/axon-patch-clamp-system/acquisition-and-analysis-software/pclamp-software-suite#gref)
MiniAnalysis (Synaptosoft ,http://www.synaptosoft.com/MiniAnalysis/)
 


程序


 


hiPSC的文化和传承
培养的hiPSC 的DsRed-SC1014细胞对人胚胎干细胞(ESC)培养基(见配方)灭活的MEF中定期6孔板中。每天更换培养基。
当细胞达到80-90%融合时,以1:6至1:12的比例传代细胞。
制备MEF平板以下小号TEP 小号甲4- 甲6一个或传代前两天的hiPSC 培养。
用2%Matrigel(在DMEM / F12中)覆盖培养皿的整个表面,并将其在室温下孵育至少1小时。
抽吸基质胶,并在MEF培养基中的血管中接种30,000个细胞/ cm 2 的有丝分裂失活的MEF(请参见食谱)。
将MEF板保存在37 °C ,5%CO 2的培养箱中。
从含有灭活的MEF的板中吸出MEF培养基,并在铺板hiPSC 之前3-4 h加入预热的人类ESC培养基。
去除需要在解剖显微镜下传代的hiPSC 菌落。
将胶原酶(在DMEM / F12中为1 mg / ml)溶液添加到含有hiPSC的培养板中,然后在37 °C ,5%CO 2的培养箱中孵育30-60分钟。
当菌落的边缘开始从板中拉开时,停止孵育。
小心去除胶原酶,以免干扰附着的细胞层。
将人类ESC培养基添加到板中,轻轻吹出hiPSC ,然后将hiPSC 收集在新的锥形管中。确保减少泡沫。
以200 x g 离心5分钟,然后从hiPSC 沉淀中去除上清液。
用人类ESC培养基重悬沉淀。
向每个MEF板中添加适当体积的细胞悬液,然后将板返回培养箱。每天更换培养基。
hiPSC的神经分化
如下所示,基于玫瑰花形神经聚集体(称为RONA)方法将hiPSC 与神经元区分开(Xu et al。,2016)。
在培养箱中用胶原酶(在DMEM / F12中为1 mg / ml)将hiPSC 集落孵育约5-10分钟,然后用培养基轻轻冲洗掉胶原酶。
生长分离的hiPSC 菌落悬浮液embyroid 体(EB)(100-200 微米直径,10〜的EB / ml的培养基)在人类ESC培养基中没有FGF2用于超低附件6孔板2天(Corning)中。
从第2天到第6天,在人ESC培养基(不含FGF2)中添加Nog gin(50 ng / ml,R&D系统)或Dorsomorphin(1μm ,Tocris )和SB431542(10μM ,Tocris )。
在第7天,将自由漂浮的EB转移至预先涂有Matrigel的培养板中(如上所述),以使EB聚集体与N2诱导培养基(NIM,请参见食谱)的完全结合。
连续地供给培养物用N 2培养基每隔一天从d AY 小号7至12。
从第12天起每天更换N2诱导培养基。
附着的EB聚集体在第8至9天分解形成单层菌落,并形成典型的神经特异性玫瑰花结。随着神经诱导的扩展,高度紧密的三维柱状神经聚集体RONAs在附着菌落的中心形成。手动微隔离RONAs,要特别小心以最大程度地减少对扁平细胞和RONAs下方细胞的外围单层污染。
在第17天,在含有B27减去VitA (Thermo Fisher Scientific ),1 mM Glutamax (Thermo Fisher Scientific )的神经基础培养基(Thermo Fisher Scientific )中收集和维持RONA簇作为神经球,持续一天。典型的RONA如图1所示。
 


D:\ Reformatting \ 2020-7-1 \ 1903012--1517西泠印837248 \图jpg \图1.jpg


图1.第17天的典型RONA。PAX6 和NKX2.1的免疫染色显示了神经祖细胞中与皮层模式相关的转录因子(转自Xu 等人,2016)。


 


在第18天,在S tep s B 11至B 19 之后,将神经球解离为单个细胞。
从培养板上取下神经球悬液,并转移到15 ml锥形管中。
让神经球在试管中静置2-5分钟,然后小心地除去培养基,使〜100μl的培养基留在神经球中。
在5 ml DPBS(Thermo Fisher Scientific)中重悬神经球。
让神经球在管2-5定下来分钟,并除去DPBS仔细离去〜100个微升与剩余的DPBS 神经球。
向神经球中加入1 ml Accutase (Corning),并在室温下孵育10分钟。
上下吸移,直到所有神经球都在单个细胞悬液中。
向试管中加入4 ml神经分化培养基(请参阅食谱),并以200 x g 离心5分钟。
轻轻去除上清液。
将细胞在神经分化培养基中重悬至所需的细胞密度,然后将细胞铺在层粘连蛋白/聚-D-赖氨酸包被的平板上(Corning)。
从第24 天到第30 天,将RA(2μM ,Millipore Sigma),SHH(50 ng / ml,R&D系统)和Purmorphamine (2μM ,R&D系统)应用于神经分化培养基中,以维持神经祖细胞的身份。提供适当百分比的代表人类大脑皮层的兴奋性神经元(〜80-85%)和抑制性神经元(〜15-20%)。
的hiPSC 衍生的在d神经祖细胞AY 小号31-32注入如下(方法C)中所述,和表征是10周移植后进行新生大鼠皮质。
在培养条件下,hiPSC 衍生的神经元在10周后显示出成熟的人类皮质身份(图2)。
 


D:\ Reformatting \ 2020-7-1 \ 1903012--1517西陵银837248 \图jpg \图2.jpg


图2。hiPSC 衍生的神经元在分化10周后在培养物中显示出人类皮质身份。A.培养物表达皮质层特异性标记物TBR1,CTIP2,BRN2和STAB2。B.一些细胞表达海马标记物PROX1 (从Yin 等人复制,2019)。


 


动物移植
从细胞培养到移植的整个过程如图3所示。
 


D:\ Reformatting \ 2020-7-1 \ 1903012--1517西陵银837248 \图jpg \图3.jpg


图3.轮廓显示了从hiPSCs 移植fo rebrain祖细胞(转载自Yin 等人,2019)。


 


胰蛋白酶化的hiPSC 衍生的神经祖细胞在DA ý 小号在培养31-32,并用移液管充分解离。然后,将细胞以300 x g 离心3分钟。
小心除去上清液,然后将沉淀重悬于冰冷的1x PBS中,终密度为2 x 10 6 细胞/ μl 。
填1.0 微升细胞的成拉玻璃微量移液管(0.5毫米内径1毫米OD)通过抽吸,然后贴合玻璃微量给自动纳升注射器(Nanoinject II,德拉蒙德)。使用纳米注射器可以最大程度地减少组织损伤,并以相对较慢的速率注射少量。
轻轻和深度麻醉新生大鼠幼崽。
在1分钟内将细胞注入右皮质(后2.0毫米,前reg外侧1.9毫米,硬脑膜以下2.6毫米)。退出前,将微量移液器吸头再固定2分钟。
 


脑切片免疫染色
用氯胺酮/甲苯噻嗪溶液(0.5 ml / kg,请参见食谱)麻醉大鼠(10周大,移植后也要10周)。
用1x PBS灌注大鼠的心脏左心室直到肝脏变白。
继续用新鲜制备的4%PFA灌注。
解剖大脑和后固定它们在4%PFA 2-3天,在4 ℃下。
将大脑转移到30%的蔗糖中,直到当蔗糖溶液饱和时它们沉入底部。
从蔗糖溶液中取出大脑,然后将它们放在2-甲基丁烷(Fisher Scientific)中,该容器已在液氮中冷却30 s至1分钟。
将一些OCT化合物(SAKURA)放在已经放在干冰上的低温恒温器纸架上。
将脑组织放在支架的中央,并在脑周围添加更多的OCT化合物,直到其固化。
将OCT脑块保存在-80 °C,直到进行连续切片处理。
使用低温恒温器(CM3050,Leica)准备脑冠状切片(25μm )。
抗原回收步骤是可选的,应根据每种抗体的指南进行标准化。对于本研究中使用的抗体,抗原修复不是必需的。
在室温下,继续用1%PBS中的10%(v / v)驴血清和0.2%(v / v)Triton X-100 封闭1 小时。
将切片与感兴趣的一抗在4 °C下孵育过夜。
用PBS清洗切片,每次3次,每次5分钟,然后在室温下与适当的荧光标记的二抗孵育1小时。
在室温下孵育1小时后,用PBS清洗切片3次,每次5分钟,然后将它们安装在干净的载玻片上。完全干燥玻片,然后用共聚焦显微镜成像(图4和视频1)。
视频1是使用Z-Stack图像生成的。设置在Z-Stack的,第一个和最后一个位置,即,该部分的深度。当前研究中的间隔使用1.01 µm。另外,选择间隔可以通过单击最佳设置切片数来完成。
 


D:\ Reformatting \ 2020-7-1 \ 1903012--1517西泠印837248 \图jpg \图4.jpg


图4.表达RFP 与成熟神经元标记MAP2 共同定位的人类神经元,表明hiPSC 衍生的神经元整合到大鼠大脑中。A.低倍放大图像显示大鼠脑中RFP阳性人类细胞。B.脑切片我mmunostained 与MAP2和DAPI (从阴再现等人,2019) 。


D:\ Reformatting \ 2020-7-1 \ 1903012--1517西泠印837248 \ video1.jpg


V 记意1. 的hiPSC 衍生的神经元在大鼠脑中如图RFP / MAP2双阳性细胞。(摘自Yin 等人,2019)


 


急性脑切片的电生理记录
麻醉大鼠(10周龄,也10周移植后)用异氟烷(300 微升在滴罐麻醉的500ml容器)的d迅速取出大脑。
制备350层的横向脑切片微米使用振动机(Leica VT1200S)的厚度。
将切片在ACSF溶液中孵育切片(请参见第4章),该切片应先在34°C下持续30分钟,然后在室温下连续通入95%O 2 和5%CO 2 。
将单个切片转移到不断充满经碳原平衡的ACSF的浸没式记录室中。
通过荧光和DIC光学镜(Carl Zeiss),在40倍水浸物镜下可视化表达RFP的注射人类神经元。
使用装有内部溶液的玻璃移液器(3-5MΩ)进行全细胞膜片钳记录(请参见食谱)。随机选择RFP阳性的人神经元,用于记录300 微米到1000 微米,从移植位点。
通过一系列超极化和去极化阶跃电流来感应动作电位。
以一系列电压步长(从–100 mV到+60 mV以20 mV的步长)激发钠和钾电流。
以–70 mV的电压钳位记录自发性兴奋性突触后电流(sEPSCs )和自发性抑制性突触后电流(sIPSCs )。获得sEPSCs 在印防己毒素存在(100 μM ),并sIPSCs 在CNQX(20的存在μM )和d-AP5(50 μM )。
记录结果表明,嫁接hiPSC的神经元在功能上与大鼠大脑整合,并且能够激发并显示出兴奋性和抑制性突触活性(图5)。
 


D:\ Reformatting \ 2020-7-1 \ 1903012--1517西陵银837248 \图jpg \图5.jpg


图5. hiPSC 衍生的神经元在功能上整合到大鼠脑的突触电路中。A. hiPSC 衍生的神经元的AP激发模式。B. 从移植细胞记录的全细胞Na + (向内)和K + (向外)流的代表性迹线。C.sEPSC 和sIPSC的代表性痕迹(从Yin 等人复制,2019)。


 


数据分析


 


研究了使用这些方案获得的hiPSC 培养物和人类神经元,并通过一系列测定使用一系列皮质特异性标记物比较了它们的人类皮质身份(Xu 等人,2016; Yin 等人,2019)。使用公布的Clampfit 10.5软件(Molecular devices)和MiniAnalysis 软件(Synaptosoft )(Yin et al。,2019)分析电生理记录数据。因此,这里不讨论数据分析。


 


笔记


 


在步骤A4中,在菌落边界开始从培养皿上剥落而菌落中心仍保持附着状态后,应使用生长培养基轻轻地将胶原酶从培养皿中冲洗掉,然后将菌落选择性地分离而不会受到干扰。
在步骤A8,请注意,附着的聚集体通常在第8到9天分解形成单层菌落,并形成典型的神经特异性玫瑰花结。随着神经诱导的扩展,高度紧凑的3维列状神经聚集体RONAs将在附着菌落的中心形成。
在步骤A10中,在收集RONAs时,应在显微镜下进行隔离,并要特别小心以最大程度地减少对扁平细胞和RONAs下方细胞的外围单层污染。
准备急性脑切片时,应尽快摘除大脑。
实验前每天应新鲜配制ACSF溶液。同样,在记录过程中,使切片在ACSF溶液中连续充满95%O 2 和5%CO 2 也是非常重要的。这些将有助于保持脑片处于健康的生理状态。
 


菜谱


 


1x PBS,pH 7.4
氯化钠8克(0.137 M)


氯化钾200 mg(0.0027 M)


Na 2 HPO 4 1.44 g(0.01 M)


KH 2 PO 4 240毫克(0.0018 M)


用M illiQ 水补足至1 L ,将pH调节至7.4


PFA 4%
将2 g PFA溶于50 ml 1x PBS


人类胚胎干细胞(ESC)培养基
DMEM / F12(赛默飞世尔科技)


淘汰20%的血清替代品(KSR,Thermo Fisher Scientific )


4 ng / ml FGF2(PeproTech )


1 mM Glutamax (Thermo Fisher Scientific )


100 μM 非必需氨基酸(赛默飞世尔科技)


100 μM 2-巯基乙醇(赛默飞世尔科技)


小鼠胚胎成纤维细胞(MEF)培养基
DMEM(赛默飞世尔科技)


10%FBS,ESC合格(Thermo Fisher Scientific )


100 μM 非必需氨基酸溶液(赛默飞世尔科技)


100 μM 2-巯基乙醇(赛默飞世尔科技)


N2感应培养基(NIM)
DMEM / F12


1%N2补充


100 μM MEM非必需氨基酸溶液


1 mM的Glutamax


2 微克/ ml肝素


神经分化培养基
Neurobasal / B27(赛默飞世尔科技)


BDNF(20 ng / ml,PeproTech )


GDNF(20 ng / ml,PeproTech )


抗坏血酸(0.2 mM,Millipore Sigma)


二丁酰cAMP(0.5 mM,Millipore Sigma)


氯胺酮/甲苯噻嗪溶液
氯胺酮(100毫克/毫升)


赛拉嗪(20毫克/毫升)


将它们混合在无菌盐水(0.9%NaCl)中进行麻醉


联合会
氯化钠125 mM


氯化钾2.5 mM


硫酸镁4 1 mM


NaH 2 PO 4 1.2 毫米


碳酸氢钠3 26 mM


氯化钙2 2 mM


D-葡萄糖10 mM


内部解决方案
葡萄糖酸钾126 mM


氯化钾8 毫米


HEPES 20 毫米


EGTA 0.2 毫米


氯化钠2 mM


镁ATP 3 毫米


Na 3 GTP 0.5 毫米


调节pH值至7.3,重量克分子渗透压浓度为290 -300 mOsm


 


致谢


 


该协议是在MSCRF和NIH 的资助下开发的。我们感谢博士金-Chong许,Gun-博士植Cho和博士朱兰Kown 帮助规范的协议。该方案在2016年和2019年发表的两项独立研究中使用(Xu 等人,2016; Yin 等人,2019)。


 


利益争夺


 


作者声明没有任何竞争性的金融或非金融利益。


 


伦理


 


所有使用hiPSC的实验均根据约翰·霍普金斯大学(JHU)医学院(SOM)的政策进行,该研究涉及JHU教职员工或学生进行的涉及hiPSC的研究,或涉及JHU设施或资源的使用。由JHU机构干细胞研究监督(ISCRO)委员会进行监督。


  建立方案时使用的所有动物程序均符合美国国立卫生研究院(NIH)的《实验动物的护理和使用指南》以及机构动物护理和使用委员会的指导。此协议中动物实验的批准ID为M017M288,已从10/1/2017到9/30/2020进行验证。


 


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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yin, X., Dawson, T. M. and Dawson, V. L. (2020). Integration of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons into Rat Brain . Bio-protocol 10(17): e3746. DOI: 10.21769/BioProtoc.3746.
  2. Yin, X., Xu, J. C., Cho, G. S., Kwon, C., Dawson, T. M. and Dawson, V. L. (2019). Neurons derived from human induced pluripotent stem cells integrate into rat brain circuits and maintain both excitatory and inhibitory synaptic activities. eNeuro 6(4).
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