Differentiation of Human Induced Pluripotent Stem Cells (iPSCs) into an Effective Model of Forebrain Neural Progenitor Cells and Mature Neurons
人诱导多能干细胞 (iPSCs) 分化为前脑神经祖细胞和成熟神经元的有效模型   

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Stem Cell Reports
Jul 2018

 

Abstract

Induced Pluripotent Stem Cells (iPSCs) are pluripotent stem cells that can be generated from somatic cells, and provide a way to model the development of neural tissues in vitro. One particularly interesting application of iPSCs is the development of neurons analogous to those found in the human forebrain. Forebrain neurons play a central role in cognition and sensory processing, and deficits in forebrain neuronal activity contributes to a host of conditions, including epilepsy, Alzheimer’s disease, and schizophrenia. Here, we present our protocol for differentiating iPSCs into forebrain neural progenitor cells (NPCs) and neurons, whereby neural rosettes are generated from stem cells without dissociation and NPCs purified from rosettes based on their adhesion, resulting in a more rapid generation of pure NPC cultures. Neural progenitor cells can be maintained as long-term cultures, or differentiated into forebrain neurons. This protocol provides a simplified and fast methodology of generating forebrain NPCs and neurons, and enables researchers to generate effective in vitro models to study forebrain disease and neurodevelopment. This protocol can also be easily adapted to generate other neural lineages.

Keywords: iPSC (iPSC细胞), Forebrain (前脑), Cortical (皮质), NSC (NSC), NPC (NPC), Neuron (神经元)

Background

Induced pluripotent stem cells (iPSCs) are stem cells produced from non-pluripotent source cells and tissues (Shi et al., 2017). Due to their ability to differentiate into a wide range of cell types, they are a promising avenue for improving our understanding of human development and treatment of degenerative diseases (Marchetto et al., 2011). Of particular interest are iPSC-derived models of human forebrain neurons, as these cells are known to mediate higher order brain functions, including consciousness (Baxter and Chiba, 1999), emotion (Morgane et al., 2005), and sleep (Schwartz and Roth, 2008). As a result, deficits in these cells can cause a wide range of neurological disorders, including neurodegenerative diseases like Alzheimer’s (Auld et al., 2002) and Huntington’s (McColgan and Tabrizi, 2018), as well as neurodevelopmental diseases such as autism (Donovan and Basson, 2017) and epilepsy (Heath, 1976). As there are few effective forebrain models for humans, the discovery of iPSCs spurred a rapid push to develop effective protocols to differentiate iPSCs to forebrain neurons (Srikanth and Young-Pearse, 2014). The first protocols that were developed drew upon previous work using embryonic stem cells (ESCs), which relied upon feeder cell cultures. This complicated the procedure and raised concerns about clinical applications. Later protocols were able to generate forebrain neurons without using feeder cells (Bell et al., 2017), with some eliminating all animal generated products entirely (Yuan et al., 2015). It can be difficult to make an all-encompassing statement about the protocols currently used to generate forebrain NPCs, due to the multitude of labs currently generating forebrain neurons and the many variables that can be changed and optimized. However, many of the recently most cited published protocols for the generation of forebrain NPCs and neurons can be divided into two kinds, monolayer and embryoid bodies (EBs) protocols. In an EB based protocol, iPSCs are dissociated and plated in suspension in a neural induction media to allow them to form EBs, which gradually aggregate over 5-7 days (Pasca et al., 2011). These EBs are then transferred to a plate that supports cell attachment, enabling the embryoid bodies to attach to the bottom of the plate and spread out into a neural rosette. From this rosette, neural stem cells (NSCs) arise, which can be passaged to form relatively stable neural progenitors cells (NPCs) (Shi et al., 2012). NPCs can then be plated in a neuronal induction media to give rise to mature neurons (Bell et al., 2017). Monolayer based protocols chiefly differ in that iPSC colonies are maintained as a monolayer during neural induction, and develop directly into rosettes without aggregation (Chandrasekaran et al., 2017). Using either approach, generation of NPCs from iPSCs is typically reported to require 21-30 days, with electrically active neurons requiring an additional 30+ days of differentiation from NPCs, for a total time of 50+ days to generate forebrain neurons from iPSCs (Yuan et al., 2015).

This protocol describes a methodology for generating forebrain neurons from iPSCs, where iPSC colonies are induced to form neural rosettes without mechanical dissociation, and neural progenitor cells are purified from immature clusters of neural cells, known as neural rosettes based on differential adhesion. Neural progenitor cells will not attach to non-adherent plates and aggregate together in a floating mass, while other cells types either adhere or float but do not aggregate with NPCs (Bell et al., 2017). This allows rapid purification of NPCs, has the potential for automation and enables the generation of NPC cultures within 14 days of initiation of differentiation. This modification does not appear to negatively influence the fate of the cells, as we observe uniform staining for key neural progenitor cells markers (Zhang et al., 2010; Venere et al., 2012; Zhang and Jiao, 2015). Indeed, we have found that we are capable of recording electrical activity from neurons consistently in as little as five days of differentiation from NPCs. This protocol can be used to generate forebrain neurons simply and effectively for use in investigating neurodevelopment, the etiology of diseases that affect the forebrain, and drug testing.

Materials and Reagents

  1. For Cell Culture
    1. 6-well plate (SARSTEDT, catalog number: 83.3920)
    2. 35-mm dish (SARSTEDT, catalog number: 83.3900)
    3. 60-mm dish (SARSTEDT, catalog number: 83.3901.300)
    4. 100-mm dish (SARSTEDT, catalog number: 83.3902.300)
    5. Petri dishes (Fisher Scientific, catalog number: FB0875713)
    6. Coverslips (Fisher, catalog number: 12-545-80)
    7. Liquid nitrogen (PRAXAIR, catalog number: 7727-37-9)
    8. iPSCs, either derived from somatic cells or thawed from a frozen aliquot
    9. TeSRTM-E8TM Media (Stem Cell Technologies, catalog number: 05990)
    10. BrainPhysTM Neuronal Medium (Stem Cell Technologies, catalog number: 05790)
    11. Matrigel® (Corning, catalog number: 354277) 
    12. KnockOutTM DMEM/F-12 (Thermo Fisher, catalog number: 12660012)
    13. DMSO (Sigma-Aldrich, catalog number: C6164)
    14. StemPro NSC SFM (Thermo Fisher, catalog number: A1050901)
    15. SM1 Neuronal Supplement (Stem Cell Technologies, catalog number: 05711)
    16. N2 Supplement-A (Stem Cell Technologies, catalog number: 07152)
    17. BSA (Gibco, catalog number: 16140071)
    18. Non-Essential Amino Acid (NEAA) (Gibco, catalog number: 11140050)
    19. SB431542 (Stem Cell Technologies, catalog number: SB431542)
    20. Noggin (Gibco, catalog number: PHC1506)
    21. Laminin (Sigma-Aldrich, catalog number: L2020)
    22. Gentle Cell Dissociation Reagent (Stem Cell Technologies, catalog number:07174)
    23. Epidermal growth factor (EGF) (Sigma, catalog number: E9644)
    24. Fibroblast growth factor (FGF) (Sigma, catalog number: F0291)
    25. Brain-Derived Neurotrophic Factor (GenScript, catalog number: Z03208-25)
    26. Glial-Derived Neurotrophic Factor (GenScript, catalog number: Z02927-50)
    27. Accutase (Sigma-Aldrich, catalog number: SCR005)
    28. DPBS without CaCl2 and MgCl2 (Sigma-Aldrich, catalog number: D8537)
    29. Neural Induction Medium 1 (see Recipes)
    30. Neural Induction Medium 2 (see Recipes)
    31. Neural Progenitor Media (see Recipes)
    32. Neuronal Media (see Recipes)
    33. Culture Dish Coating with Matrigel® (see Recipes)

  2. For Immunocytochemistry (ICC)
    1. Glass coverslips (Fisher, catalog number: 12-545-81)
    2. Microscope slides (Fisher, catalog number: 12-552-3)
    3. Pipette tips, 1 ml (SARSTEDT,70.1186) 
    4. Pipette tips, 200 µl (SARSTEDT,70.1186)
    5. Pipette tips, 20 µl (SARSTEDT,70.1186)
    6. 15-ml conical tube (SARSTEDT, 62.554.002)
    7. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 252549)
    8. BSA (Sigma-Aldrich, catalog number: A2058)
    9. Triton X-100
    10. DAPI (Thermo Fisher, catalog number: 62248)
    11. Vectashield® (Vector Labs, catalog number: H-1000)
    12. Nail Polish (Sally Hansen Insta-Dri Fast-Dry Clear Nail Color)
    13. Antibodies
      Antibodies for iPSCs:
      1. TRA-1-60 (Embryonic Stem Cell Marker Panel, Abcam, catalog number: ab109884)
      2. SSEA (Embryonic Stem Cell Marker Panel, Abcam, catalog number: ab109884)
      3. Nanog (Embryonic Stem Cell Marker Panel, Abcam, catalog number: ab109884)
      4. OCT4 (Stemcell Technologies, catalog number: 60093)
      5. PAX6 (Stemcell Technologies, catalog number: 60094)
      Antibodies for NPCs:
      1. SOX1 (Stemcell Technologies, catalog number: 60095)
      2. Nanog (Embryonic Stem Cell Marker Panel, Abcam, catalog number: ab109884)
      3. Nestin (Stemcell Technologies, catalog number: 60091)
      4. PAX6 (Stemcell Technologies, catalog number: 60094)
      Antibodies for Neurons:
      1. Tuj1 (Abcam, catalog number: ab14545)
      2. S100B (Abcam, catalog number: ab52642)
      3. VGLUT1 (Abcam, catalog number: ab77822)
      4. GABA (Abcam, catalog number: ab86186)
      5. GFAP (Abcam, catalog number: ab7260)
      Secondary Antibodies:
      1. ALEXA 488 anti-mouse (Invitrogen, catalog number: A-11008)
      2. ALEXA 555 anti-rabbit (Invitrogen, catalog number: A-21422)
    14. Coating glass coverslips with Poly-ornithine and laminin (see Recipes)

  3. For Electrophysiology
    1. Borosilicate pipettes with resistances of 3-6 MΩ (World Precision Instruments, catalog number: 1B150-4)
    2. Cell strainer (40 µm) (Sigma, catalog number: CLS431750)
    3. Tetrodotoxin (TTX) (Alomone labs, catalog number: T-550)
    4. BrainPhysTM Without Phenol Red (Stem Cell Technologies, catalog number: 05791)
    5. HEPES (Sigma-Aldrich, catalog number: H3375)
    6. KCl (Sigma-Aldrich, catalog number: 793590)
    7. Potassium Gluconate (Sigma-Aldrich, catalog number: G4500)
    8. EGTA (Sigma-Aldrich, catalog number: 324626)
    9. Mg-ATP (Sigma-Aldrich, catalog number: A9187)
    10. Creatine phosphate (Sigma-Aldrich, catalog number: CRPHO-RO)
    11. Guanosine triphosphate (Sigma-Aldrich, catalog number: G8877)
    12. NMDA (Alomone labs, catalog number: N-170)
    13. Magnesium Chloride hexahydrate (Sigma, catalog number: M2393)
    14. Internal pipette solution (see Recipes)

    Equipment

    1. Pipettes (Fisher, catalog number: 4680100)
    2. Pipette puller (Sutter Instrument, model: P-1000)
    3. Osmometer (Advanced Instruments, model: 3320)
    4. Bead bath (Lab Armor, model: M706)
    5. Fluorescent microscope (Olympus, model: 1X73)
    6. Recording chamber with six-channel valve controller (Warner Instruments)
    7. Automatic temperature controller (Warner Instruments, model: TC-324C)
    8. Micromanipulator (Sutter Instrument, model: MP-225)
    9. Microelectrode amplifier Multiclamp 770B (Molecular Devices)
    10. Acquisition system Axon digidata 1550A (Molecular Devices)
    11. Biological Safety Cabinet Class 2 (Nuaire, Model: NU440400)
    12. Incubator (Thermo Fisher, catalog number: 51030287)
    13. Centrifuge (Allegra, model: X-12)

    Software

    1. Clampex 10.5 (Molecular Devices, www.moleculardevices.com)
    2. GraphPad Prism 7 (GraphPad, www.graphpad.com)
    3. Excel 2016 (Microsoft, https://products.office.com/en-ca/excel)

    Procedure

    1. Differentiation from iPSCs to Forebrain NSCs (Neural induction)
      Note: The following steps are described assuming high-quality iPSCs (see Figure 1) are plated on a 60-mm tissue culture dish that is coated with Matrigel® (for more details, see Recipes and Table 2).


      Figure 1. Sample ICC staining for high-quality iPSC cultures. In order for differentiation to proceed effectively, ensure that you begin differentiation with high-quality iPSC cultures. IPSCs should uniformly express pluripotency markers SSEA, OCT4, TRA-1-60, and NANOG (A) when assessed using ICC. Additional pluripotency markers DNMT3b, EST2, and ZFP42 can also be assessed using a qPCR assay (B). IPSCs should be free of karyotypic abnormalities (C), possess the ability to differentiate into all three germ lineages and express characteristic markers of each lineage (D), and test negative for mycoplasma contamination (E). Scale bars represent 100 μm.

      1. Working in a class II biological safety cabinet, use appropriately sized pipettes to plate iPSCs in E8 medium in a 60-mm dish. If starting from a frozen aliquot of iPSC, we recommend plating at least 300,000 cells. This is considered Day 0 of iPSC culture.
      2. On Day 1 of iPSC culture (15%-25% confluency), aspirate the spent medium to remove non-attached cells, and check the size of colonies. If colonies are approximately 100-200 µm in diameter, they are an appropriate size to begin differentiation. Add 3 ml of complete Neural Induction Medium 1, pre-warmed in a bead bath to each plate using a 5 ml pipette. Return the plates to an incubator maintained at 37 °C, 5% CO2 and atmospheric (~20%) Oxygen. If plates do not contain colonies of sufficient size, add 3 ml of E8 media and check daily until colonies reach the appropriate size.
      3. On Day 2 (about 48 h after switching to Neural Induction Medium), change the medium by aspirating old medium from each well. Add 3 ml of pre-warmed complete Neural Induction Medium 1 to each plate.
      4. On Day 4 of neural induction, cells will be reaching confluency. If necessary, mark any colonies with non-neural differentiation. Remove these unwanted colonies with a 200 µl pipette tip. Aspirate the spent medium from each well. Add 3 ml of pre-warmed complete Neural Induction Medium 1 to each plate.
        Note: Due to high cell density in the culture from Day 4 onwards, doubling the volume of Neural Induction Medium is very critical for cell nutrition. Also, minimal cell death should be observed from Days 4 to 7 after neural induction. If the color of cells turns yellowish with many floating cells during Days 4 to 7 of neural induction, it indicates that the starting density of iPSCs was too high. In this case, change the Neural Induction Medium every day, remove some colonies and double the volume per well/plate. Ideally, work with these variables to ensure that the media does not continue to turn yellow.
      5. On Day 6 of neural induction, cells should be near maximal confluence. Remove any non-neural differentiated cells that can be observed and add 3 ml of complete Neural Induction Medium into each plate.
      6. On Day 7 of neural induction, the medium should be switched into Neural Induction Medium 2. Add 3 ml of complete Neural Induction Medium 2 to each plate. The medium should be changed every day for 5 days. For example morphologies, see Figure 2.


      Figure 2. Morphology of iPSCs differentiating into forebrain NPCs. A. Day 0: Showing a single iPSC colony of appropriate size immediately prior to addition of Neural Induction Media 1. B. Day 2: The iPSC colony, which has been treated with Neural Induction Media 1 for 2 days, begins to change cellular morphology and some cells extend processes. C. Day 5: Increased expansion of the colony with some differentiation of outer cells. D. Day 12: Appearance of rosettes in the colony become visible. NSCs are present in high confluence in the middle of these structures. It is at this point that colonies are detached and re-plated on non-adherent plates at D13 for two days. E. D13 immediately after plating on adherent plates. This image shows floating rosette colonies that will continue to proliferate and differentiate in a floating mass. Non-rosette cells either remained on the dish at D12 after chemical release or float as single cells on the non-adherent plates shown in (E). F. At D15, rosette clusters expand in size and are moved to adherent plates. Cell aggregates here are 3-dimensional, but are attached to the plate. Note the purity of the clusters at this point (F). Scale bars represent 130 µm.


    2. Harvest and expansion of NPCs
      Note: On Day 12 of neural induction, NPCs are ready to be harvested and expanded.
      1. Aspirate the spent Neural Induction Medium from each plate to be passaged.
      2. Gently add DPBS without CaCl2 and MgCl2 to each plate twice to rinse the cells.
      3. Add 1.5 ml of pre-warmed Gentle Cell Dissociation Reagent to each plate and incubate for 5 min at 37 °C until most cells detach from the surface of the culture vessels. Tap plates gently to dislodge cells still attached.
      4. Use a pipette to gently rinse the surface of the plates with the Gentle Cell Dissociation Reagent already in the plates to detach any remaining cells.
      5. Using a pipette, transfer the cell suspension to a 15-ml conical tube.
      6. Add 1 ml of DPBS to each plate to collect residual cells and transfer the cell suspension to the conical tube.
      7. Gently pipet the cell suspension up and down 3 times with a 5-ml or 10-ml pipette to break up the cell clumps.
      8. Centrifuge the cells at 300 x g for 5 min.
      9. Aspirate the supernatant and re-suspend the cells in pre-warmed Neural Progenitor Cell (NPC) Medium (i.e., 10 ml for all cells from each plate).
      10. Plate the cells suspended in NPC Medium onto a 10 cm Petri dish.
      11. Culture the cells in a CO2 incubator for 2 days. During this time, NPCs will form aggregations while floating in NPC medium.
      12. Once aggregates have reached an appropriate size of approximately 70-200 µm, prepare a 10 cm tissue culture dish coated with 5 ml of Matrigel® for at least 1 h. If few aggregates have formed, plate cells in a 60 mm dish instead.
      13. Using a 5-ml or 10-ml pipette, take up and pass the NPC medium through a cell strainer to collect NPC aggregations. 
      14. Reverse the strainer and pass 10 ml of fresh pre-warmed NPC medium through strainer where the cell aggregates are bound so that they are transferred onto the Matrigel®-coated 10 cm plate.
      15. Culture the cells in a CO2 incubator to allow for NPC aggregates to attach to the coated dish and migrate and proliferate.
      16. Change medium every 2-3 days until cells reach confluence and are ready for passaging or cryopreservation. Dissociate using warm accutase at 37 °C for five minutes.
      17. To assess the purity of NPC culture, fix cells and check for NPC markers using ICC (see Figure 3).
      18. To cryopreserve NPCs, freeze in an 80/20 mix of FBS/DMSO. Store at -80 °C for use within a few months, or in liquid nitrogen for long-term storage.


        Figure 3. Sample ICC staining for high-quality NPC cultures. In order for differentiation to proceed effectively, ensure that iPSC cultures uniformly express Nestin, SOX1, and PAX6. NPC cultures should have no expression of the pluripotent marker OCT4 (DAPI shown in blue in merge of PAX6 and OCT4; all cells express PAX6, i.e., 100% purity of the culture). Scale bars represent 100 μm.

    3. Differentiation of forebrain NPCs into neurons
      1. Plate NPCs on a tissue culture dish that is coated with Matrigel®. Wait until cells have achieved 70%-95% confluency before beginning differentiation.
      2. Once NPCs have reached desired confluency, aspirate media and replace with an equal volume of Neuronal Media.
      3. Every 2-3 days, aspirate half of the media in the plate and replace with fresh Neuronal Media.
        Note: As some media will be lost to evaporation, you may need to add a little more media than you remove from the plate in order to keep the media volume stable over time.
      4. Continue to change the media until neurons reach the desired stage of development. For example morphologies of developing forebrain neurons, consult Figure 4. For example ICC characterization of forebrain neurons, consult Figure 5.
        Notes:
        1. The purity of your line will be very easily detected during this stage of development. Cell lines that contain a high percentage of NPCs will rapidly polarize and form neuronal projections (usually around Days 2-5), whereas lines that contain a high percentage of non NPC cells (Astrocytes, neural crest cells, etc.) will not.
        2. There is variability in how long neurons in a particular plate will take to reach a certain stage of development depending on line, clone, passage number etc. However, we have found that neurons in good quality cultures consistently achieve the following landmarks by the following number of days into differentiation.

        Day 5 = Cells are post-mitotic
        Day 15 = Cells have clearly polarized axons and dendrites, and have clearly detectable electrophysiological properties, such as action potentials


        Figure 4. Example morphology for forebrain NPCs differentiating into neurons. Morphology of a forebrain NPC culture differentiating into neurons. Images taken at D0 (A), D5 (B), D15 (C), D30 (D). Scale bars represent 130 µm.

      5. To assess the purity of your neuronal culture, fix cells and check for forebrain markers using ICC. To assess the quality of the neurons produced, ensure that the cells display proper electrophysiological properties (see Data analysis).


        Figure 5. Sample ICC staining for high-quality forebrain neuronal. Representative ICC of forebrain neuronal culture following 30 days of differentiation (D30) from NPCs, demonstrating the relative abundance of glutamatergic, GABAergic, and astrocytic markers in the population. These cultures are approximately 65% glutamatergic, 30% GABAergic, and 5%-10% astroglial. Scale bars represent 50 μm.

    4. Assessment of culture purity using immunocytochemistry
      Note: The following steps are described assuming cells are plated on glass coverslips coated with poly-ornithine and laminin (For more details, see Recipes).
      1. Fix samples in 4% paraformaldehyde (PFA) diluted in PBS. Incubate at room temperature for 15 min.
      2. Wash samples with PBS (3 x 15 min) at room temperature.
      3. Permeabilize samples by incubation in PBS + 0.1% Triton X-100 at room temperature for 10 min. 
      4. Aspirate permeabilization buffer and replace with 5% BSA diluted in PBS to block samples. Incubate at room temperature for 60 min. 
      5. Prepare working stocks of primary antibodies by diluting in blocking 5% BSA-PBS. See Table 1 for recommended working dilutions and antibodies for different cells types. Coat coverslips in primary antibody solution and incubate overnight at 2-8 °C.

        Table 1. Antibodies used in immunocytochemistry


      6. Wash samples with PBS (3 x 15 min) at room temperature.
      7. Aspirate PBS and add secondary antibodies diluted in 5% BSA-PBS. See Table 1 for recommended secondary antibodies and dilutions. Incubate coverslips at room temperature, away from light for one hour.
      8. Wash samples with PBS (3 x 15 min) at room temperature.
      9. If desired, add DAPI diluted in PBS, incubate for 5 min in room temperature.
      10. Add a drop of Vectashield® to a glass slide. Carefully use a needle and forceps to transfer the coverslip, cell-side down, to the slide. Seal using nail polish.

    5. Assessment of neuronal quality using Electrophysiology
      1. Pull pipettes from glass capillaries. Their resistance should range from 3 to 6 MΩ when filled with the internal pipette solution.
      2. Transfer individual coverslips containing differentiated human iPSC-derived neurons into a heated recording chamber and continuously perfused (1 ml/min) with BrainPhys Neuronal Medium without phenol bubbled with a mixture of CO2 (5%) and O2 (95%) and maintained at 35 °C using an automatic temperature controller.
      3. Choose the cells that you will record from.
      4. Fill the pipette with the internal pipette solution and place it in the electrode holder. Lower the pipette to place it into the external solution. After compensating offsets, approach the pipette to the chosen cell with the help of the remote micromanipulator to form a high resistance cell-attached seal. 
      5. Once the seal is formed and the whole cell configuration is established, compensate series resistance at 80%-90%. 
      6. Wait for 5 to 10 min before starting to record. This allows the cell content to equilibrate with the internal pipette solution.
      7. For acquisition, set your filter at 2 kHz and your sampling rate at 20 kHz.
      8. Once whole-cell recording had been established, recordings of fundamental neuronal properties, including rheobase, resting membrane potential, action potential parameters and spontaneous postsynaptic currents can be performed. Add NMDA via pipette. Assessed neurons should be hold neurons in voltage clamp at -70 mV except when examining changes in the resting membrane potential and rheobase, which should be performed in current clamp. Clampex and GraphPad Prism 7 are recommended software to use to display data. For example electrophysiological recordings obtained from cortical neurons, consult Figure 6.


        Figure 6. Electrophysiological properties of high-quality forebrain neurons. A. Differential image contrast of a glass microelectrode recording from a single neuron in the whole-cell configuration. Scale bars represent 20 μm. B. A hyperpolarizing pulse showing a depolarizing sag followed by multiple rebound action potentials. C. Left: Representative traces of voltage clamp recordings showing inward Na+ currents; Right: Sodium current traces disappear after tetrodotoxin (TTX) 1 μM application. D. Representative current-clamp recording from a spontaneously active neuron with resting membrane potential -40 mV. E. Representative recording showing action potentials fired by forebrain neurons during a current ramp protocol. F. Representative voltage-clamp recording from a neuron with spontaneous synaptic input. All electrophysiological data was obtained from D14 neurons.

    Data analysis

    Electrophysiological data should be processed with currents should be filtered at 2 kHz and digitized at 20 kHz. Values should be reported correcting for the nominal membrane potential in voltage- and current-clamp recordings for the calculated 10-mV liquid junction potential.

    Recipes

    1. Media
      Note: All media as given as recipes for 50 ml as it is possible to prepare this volume in a single 50 ml tube, suitable for warming in a bead or water bath.

      1. Neural Induction Medium 1


      2. Neural Induction Medium 2


        Note: Complete Neural Induction Medium 1 and 2 can be stored at 2-8 °C in the dark for up to 2 weeks. Warm the Neural Induction Medium in a 37 °C water bath for 5-10 min before using. Do not warm the Neural Induction Medium in a 37 °C water bath for longer than 10 min, as this may cause degradation of the medium.

      3. Neural Progenitor Media


      4. Neuronal Media


    2. Buffers and solutions
      1. Internal pipette solution
        5 mM HEPES
        2 mM KCl
        136 mM potassium gluconate
        5 mM EGTA
        5 mM ATP-Mg2+
        8 mM creatine phosphate
        0.35 mM guanosine triphosphate
        The pH is adjusted to 7.2 with KOH and the osmolality is adjusted with distilled water or concentrated potassium gluconate if needed to between 295 and 298 mOsm.
        Note: The difference in osmolality between Internal and external solutions should be near 5%.
      2. Culture Dish Coating with Matrigel®
        1. Thaw a frozen aliquot of Matrigel® (250 μl) from -80 °C by placing it in a 4 °C fridge for 1 h
        2. To create a working solution, dilute the thawed Matrigel® in 25 ml of cold PBS or media
          Note: Diluting Matrigel® in DMEM or other media with a strong coloration will make it easier to determine that the whole dish is evenly covered.
        3. Quickly cover the whole surface of each culture vessel with the appropriate amount matrix solution (Table 2)
        4. Incubate the culture vessels in a 37 °C, 5% CO2 incubator for at least 1 h
        5. The culture vessels are now ready for use. Just before use, aspirate the diluted Matrigel® solution from the culture vessels. Cells can be plated directly onto the Matrigel®-coated culture vessels without rinsing
          Note: Coated culture vessels can also be stored at 2-8 °C for up to one week. When storing, seal culture vessels with Parafilm® laboratory film to prevent drying. Before using, warm up the coated culture vessels stored at 2-8°C at room temperature for 30 min.

          Table 2. Required volume of Matrigel® matrix solution for coating different culture vessels


      3. Coating glass coverslips with Poly-ornithine and laminin
        1. Place glass coverslips in a Petri dish or suspension plate
        2. Sterilize coverslip by exposing plates to UV radiation for 20 min
        3. Coat coverslips in 100 μl of 50 μg/ml polyorinithine in PBS. Wait two hours
        4. Aspirate solutions. Wash once using PBS, then coat coverslip in 10 μg/ml laminin diluted in PBS
        5. Incubate at 37 °C for two hours
        6. Aspirate solution, coat coverslips in 10% FBS DMEM
        7. The plates are now ready to use. For best results, use within 24 h of preparation. Before plating cells, wash plates once with PBS, as FBS may influence differentiation

    Acknowledgments

    This work was supported by grants from the Canada Research Chairs program and the Canadian Institute of Health Research to CE. Scott Bell is funded by the (Fonds de la recherche en santé du Québec) FRQS, Nuwan Hettige is funded by FRQS, Malvin Jefri is supported by the Government of Malaysia, and Lilit Antonyan is supported by CONACYT (Mexico). This protocol was adapted from previous work (Bell et al., 2017 and 2018).

    Competing interests

    Carl Ernst is president of ManuStem.com.

    Ethics

    All work was approved by the Research Ethics Board of the Douglas Hospital.

    References

    1. Auld, D. S., Kornecook, T. J., Bastianetto, S. and Quirion, R. (2002). Alzheimer's disease and the basal forebrain cholinergic system: relations to β-amyloid peptides, cognition, and treatment strategies. Prog Neurobiol 68(3): 209-245.
    2. Baxter, M. G. and Chiba, A. A. (1999). Cognitive functions of the basal forebrain. Curr Opin Neurobiol 9(2): 178-183.
    3. Bell, S., Maussion, G., Jefri, M., Peng, H., Theroux, J. F., Silveira, H., Soubannier, V., Wu, H., Hu, P., Galat, E., Torres-Platas, S. G., Boudreau-Pinsonneault, C., O'Leary, L. A., Galat, V., Turecki, G., Durcan, T. M., Fon, E. A., Mechawar, N. and Ernst, C. (2018). Disruption of GRIN2B impairs differentiation in human neurons. Stem Cell Reports 11(1): 183-196.
    4. Bell, S., Peng, H., Crapper, L., Kolobova, I., Maussion, G., Vasuta, C., Yerko, V., Wong, T. P. and Ernst, C. (2017). A rapid pipeline to model rare neurodevelopmental disorders with simultaneous CRISPR/Cas9 gene editing. Stem Cells Transl Med 6(3): 886-896.
    5. Chandrasekaran, A., Avci, H. X., Ochalek, A., Rosingh, L. N., Molnar, K., Laszlo, L., Bellak, T., Teglasi, A., Pesti, K., Mike, A., Phanthong, P., Biro, O., Hall, V., Kitiyanant, N., Krause, K. H., Kobolak, J. and Dinnyes, A. (2017). Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells. Stem Cell Res 25: 139-151.
    6. Donovan, A. P. and Basson, M. A. (2017). The neuroanatomy of autism - a developmental perspective. J Anat 230(1): 4-15.
    7. Heath, R. G. (1976). Brain function in epilepsy: midbrain, medullary, and cerebellar interaction with the rostral forebrain. J Neurol Neurosurg Psychiatry 39(11): 1037-1051.
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    12. Schwartz, J. R. and Roth, T. (2008). Neurophysiology of sleep and wakefulness: basic science and clinical implications. Curr Neuropharmacol 6(4): 367-378.
    13. Shi, Y., Inoue, H., Wu, J. C. and Yamanaka, S. (2017). Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov 16(2): 115-130.
    14. Shi, Y., Kirwan, P. and Livesey, F. J. (2012). Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc 7(10): 1836-1846.
    15. Srikanth, P. and Young-Pearse, T. L. (2014). Stem cells on the brain: modeling neurodevelopmental and neurodegenerative diseases using human induced pluripotent stem cells. J Neurogenet 28(1-2): 5-29.
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简介

诱导多能干细胞(iPSCs)是可以从体细胞产生的多能干细胞,并且提供了一种模拟体外神经组织发育的方法。 iPSC的一个特别有意义的应用是类似于人前脑中发现的神经元的发育。前脑神经元在认知和感觉处理中发挥核心作用,前脑神经元活动缺陷导致许多疾病,包括癫痫,阿尔茨海默病和精神分裂症。在这里,我们提出了将iPSCs分化为前脑神经祖细胞(NPC)和神经元的方案,其中神经玫瑰花结从干细胞产生而不会解离,并且基于它们的粘附从玫瑰花结中纯化NPC,从而更快速地产生纯NPC培养物。神经祖细胞可以作为长期培养物维持,或分化为前脑神经元。该协议提供了一种简化且快速的方法来生成前脑NPC和神经元,并使研究人员能够生成有效的体外模型来研究前脑疾病和神经发育。该协议也可以很容易地适应生成其他神经谱系。
【背景】诱导的多能干细胞(iPSC)是由非多能源细胞和组织产生的干细胞(Shi 等,,2017)。由于它们能够分化成广泛的细胞类型,它们是一种有前途的途径,可以提高我们对人类发育和退行性疾病治疗的理解(Marchetto et al。,2011)。特别感兴趣的是iPSC衍生的人类前脑神经元模型,因为已知这些细胞介导更高级的脑功能,包括意识(Baxter和Chiba,1999),情绪(Morgane et al。,2005) ),睡觉(Schwartz和Roth,2008)。因此,这些细胞的缺陷可导致广泛的神经系统疾病,包括阿尔茨海默氏症(Auld et al。,2002)和亨廷顿氏症(McColgan和Tabrizi,2018)等神经退行性疾病,以及神经发育疾病,如自闭症(Donovan和Basson,2017)和癫痫(Heath,1976)。由于人类很少有效的前脑模型,iPSCs的发现促使人们迅速推动开发有效的方案来区分iPSC和前脑神经元(Srikanth和Young-Pearse,2014)。开发的第一个方案利用了先前使用胚胎干细胞(ESCs)的工作,胚胎干细胞依赖于饲养细胞培养。这使程序复杂化并引起对临床应用的担忧。后来的协议能够在不使用饲养细胞的情况下产生前脑神经元(Bell et al。,2017),其中一些完全消除了所有动物产生的产物(Yuan et al。,2015 )。由于当前生成前脑神经元的众多实验室以及可以改变和优化的许多变量,因此很难对目前用于生成前脑NPC的协议做出全面的陈述。然而,最近引用的许多已发表的用于产生前脑NPC和神经元的方案可分为两种类型,单层和胚状体(EB)方案。在基于EB的方案中,将iPSC解离并在神经诱导培养基中悬浮铺板以使它们形成EB,其在5-7天内逐渐聚集(Pasca 等人,2011)。然后将这些EB转移到支持细胞附着的平板上,使胚状体附着在平板的底部并扩散到神经莲座丛中。从该花环中,出现神经干细胞(NSCs),其可以传代以形成相对稳定的神经祖细胞(NPC)(Shi 等人,,2012)。然后可以将NPC铺在神经元诱导培养基中以产生成熟神经元(Bell et al。,2017)。基于单层的方案主要不同之处在于iPSC集落在神经诱导期间保持为单层,并直接发育成玫瑰花结而没有聚集(Chandrasekaran et al。,2017)。使用任何一种方法,通常报告从iPSC产生NPC需要21-30天,电活性神经元需要额外30天以上从NPC分化,总共50天以上从iPSC产生前脑神经元(元) et al。,2015)。

该协议描述了从iPSC产生前脑神经元的方法,其中诱导iPSC集落形成神经玫瑰花结而没有机械解离,并且神经祖细胞从神经细胞的未成熟簇中纯化,称为基于差异粘附的神经玫瑰花结。神经祖细胞不会附着在非粘附板上并在浮动块中聚集在一起,而其他细胞类型要么粘附或漂浮,要么不与NPC聚集(Bell et al。,2017)。这允许NPC的快速纯化,具有自动化的潜力并且能够在分化开始后14天内产生NPC培养物。这种修饰似乎不会对细胞的命运产生负面影响,因为我们观察到关键神经祖细胞标记物的均匀染色(Zhang et al。,2010; Venere et al。,2012; Zhang and Jiao,2015)。实际上,我们发现我们能够在与NPC分化的短短五天内始终记录神经元的电活动。该协议可用于简单有效地生成前脑神经元,用于研究神经发育,影响前脑的疾病的病因学和药物测试。

关键字:iPSC细胞, 前脑, 皮质, NSC, NPC, 神经元

材料和试剂

  1. 对于细胞培养
    1. 6孔板(SARSTEDT,目录号:83.3920)
    2. 35毫米碟(SARSTEDT,目录号:83.3900)
    3. 60毫米碟(SARSTEDT,目录号:83.3901.300)
    4. 100毫米盘(SARSTEDT,目录号:83.3902.300)
    5. 培养皿(Fisher Scientific,目录号:FB0875713)
    6. 盖弗斯(Fisher,目录号:12-545-80)
    7. 液氮(PRAXAIR,目录号:7727-37-9)
    8. iPSC,来源于体细胞或从冷冻等分试样中解冻
    9. TeSR TM -E8 TM Media(Stem Cell Technologies,目录号:05990)
    10. BrainPhys TM 神经元培养基(干细胞技术,目录号:05790)
    11. Matrigel ®(康宁,目录号:354277) 
    12. KnockOut TM DMEM / F-12(Thermo Fisher,目录号:12660012)
    13. DMSO(Sigma-Aldrich,目录号:C6164)
    14. StemPro NSC SFM(Thermo Fisher,目录号:A1050901)
    15. SM1神经元补充剂(干细胞技术,目录号:05711)
    16. N2 Supplement-A(干细胞技术,目录号:07152)
    17. BSA(Gibco,目录号:16140071)
    18. 非必需氨基酸(NEAA)(Gibco,目录号:11140050)
    19. SB431542(干细胞技术,目录号:SB431542)
    20. Noggin(Gibco,目录号:PHC1506)
    21. 层粘连蛋白(Sigma-Aldrich,目录号:L2020)
    22. Gentle Cell Dissociation Reagent(干细胞技术,目录号:07174)
    23. 表皮生长因子(EGF)(西格玛,目录号:E9644)
    24. 成纤维细胞生长因子(FGF)(西格玛,目录号:F0291)
    25. 脑源性神经营养因子(GenScript,目录号:Z03208-25)
    26. 胶质衍生的神经营养因子(GenScript,目录号:Z02927-50)
    27. Accutase(Sigma-Aldrich,目录号:SCR005)
    28. 不含CaCl 2 和MgCl 2 的DPBS(Sigma-Aldrich,目录号:D8537)
    29. 神经诱导培养基1(见食谱)
    30. 神经诱导培养基2(见食谱)
    31. 神经祖细胞培养基(见食谱)
    32. 神经元媒体(见食谱)
    33. 用Matrigel ®培养皿涂层(见食谱)

  2. 用于免疫细胞化学(ICC)
    1. 玻璃盖玻片(Fisher,目录号:12-545-81)
    2. 显微镜载玻片(Fisher,目录号:12-552-3)
    3. 移液器吸头,1毫升(SARSTEDT,70.1186) 
    4. 移液器吸头,200μl(SARSTEDT,70.1186)
    5. 移液器吸头,20μl(SARSTEDT,70.1186)
    6. 15毫升锥形管(SARSTEDT,62.554.002)
    7. 多聚甲醛(PFA)(西格玛奥德里奇,目录号:252549)
    8. BSA(Sigma-Aldrich,目录号:A2058)
    9. Triton X-100
    10. DAPI(Thermo Fisher,目录号:62248)
    11. Vectashield ®(Vector Labs,目录号:H-1000)
    12. 指甲油(Sally Hansen Insta-Dri快干透明指甲油)
    13. 抗体
      用于iPSC的抗体:
      1. TRA-1-60(胚胎干细胞标记板,Abcam,目录号:ab109884)
      2. SSEA(胚胎干细胞标记板,Abcam,目录号:ab109884)
      3. Nanog(胚胎干细胞标记板,Abcam,目录号:ab109884)
      4. OCT4(Stemcell Technologies,目录号:60093)
      5. PAX6(Stemcell Technologies,目录号:60094)
      NPC抗体:
      1. SOX1(Stemcell Technologies,目录号:60095)
      2. Nanog(胚胎干细胞标记板,Abcam,目录号:ab109884)
      3. Nestin(Stemcell Technologies,目录号:60091)
      4. PAX6(Stemcell Technologies,目录号:60094)
      神经元抗体:
      1. Tuj1(艾博抗(Abcam),目录号:ab14545)
      2. S100B(Abcam,目录号:ab52642)
      3. VGLUT1(艾博抗(Abcam),目录号:ab77822)
      4. GABA(Abcam,目录号:ab86186)
      5. GFAP(Abcam,目录号:ab7260)
      二抗:
      1. ALEXA 488抗小鼠(Invitrogen,目录号:A-11008)
      2. ALEXA 555抗兔(Invitrogen,目录号:A-21422)
    14. 用聚鸟氨酸和层粘连蛋白涂覆玻璃盖玻片(见食谱)

  3. 对于电生理学
  1. 电阻为3-6MΩ的硼硅酸盐移液器(World Precision Instruments,目录号:1B150-4)
  2. 细胞过滤器(40μm)(Sigma,目录号:CLS431750)
  3. 河豚毒素(TTX)(Alomone labs,目录号:T-550)
  4. 没有酚红的BrainPhys TM (干细胞技术,目录号:05791)
  5. HEPES(西格玛奥德里奇,目录号:H3375)
  6. KCl(Sigma-Aldrich,目录号:793590)
  7. 葡萄糖酸钾(Sigma-Aldrich,目录号:G4500)
  8. EGTA(Sigma-Aldrich,目录号:324626)
  9. Mg-ATP(Sigma-Aldrich,目录号:A9187)
  10. 磷酸肌酸(Sigma-Aldrich,目录号:CRPHO-RO)
  11. 三磷酸鸟苷(Sigma-Aldrich,目录号:G8877)
  12. NMDA(Alomone labs,目录号:N-170)
  13. 氯化镁六水合物(Sigma,目录号:M2393)
  14. 内部移液器解决方案(见食谱)

设备

  1. 移液器(Fisher,目录号:4680100)
  2. 移液器拉拔器(Sutter Instrument,型号:P-1000)
  3. 渗透压计(Advanced Instruments,型号:3320)
  4. 珠浴(Lab Armor,型号:M706)
  5. 荧光显微镜(奥林巴斯,型号:1X73)
  6. 带六通道阀门控制器的录音室(华纳仪器)
  7. 自动温度控制器(华纳仪器,型号:TC-324C)
  8. 微操纵器(Sutter Instrument,型号:MP-225)
  9. 微电极放大器Multiclamp 770B(Molecular Devices)
  10. 采集系统Axon digidata 1550A(Molecular Devices)
  11. 生物安全柜2级(Nuaire,型号:NU440400)
  12. 孵化器(Thermo Fisher,目录号:51030287)
  13. 离心机(Allegra,型号:X-12)

软件

  1. Clampex 10.5(Molecular Devices, www.moleculardevices.com
  2. GraphPad Prism 7(GraphPad, www.graphpad.com
  3. Excel 2016(Microsoft, https://products.office.com/en-ca/excel

程序

  1. 从iPSC到Forebrain NSCs的分化(神经诱导)
    注意:*假设将高质量的iPSC(见图1)涂在涂有基质胶 ® (有关详细信息,请参阅食谱和表2)。


    图1.高质量iPSC培养物的ICC染色样本。为了有效地进行分化,确保您开始使用高质量的iPSC培养物进行分化。当使用ICC评估时,IPSCs应均匀表达多能性标志物SSEA,OCT4,TRA-1-60和NANOG(A)。还可以使用qPCR测定法评估额外的多能性标志物DNMT3b,EST2和ZFP42(B)。 IPSCs应该没有核型异常(C),具有分化成所有三个种系的能力并表达每个谱系的特征标记(D),并且对支原体污染测试为阴性(E)。比例尺代表100微米。

    1. 在II级生物安全柜中工作,使用适当尺寸的移液器在60mm培养皿中的E8培养基中培养iPSC。如果从冷冻的iPSC等分试样开始,我们建议至少接种300,000个细胞。这被认为是iPSC培养的第0天。
    2. 在iPSC培养的第1天(15%-25%汇合),吸出用过的培养基以除去未附着的细胞,并检查菌落的大小。如果菌落直径约为100-200μm,则它们是适合开始分化的大小。加入3ml完全神经诱导培养基1,用5ml移液管在珠子浴中预热至每个平板。将平板放回保持在37℃,5%CO 2 和大气(~20%)氧气的培养箱中。如果平板不含足够大小的菌落,则每天加入3毫升E8培养基并检查,直到菌落达到合适的大小。
    3. 在第2天(切换至神经诱导培养基后约48小时),通过从每个孔中吸出旧培养基来更换培养基。向每个板中加入3ml预热的完全神经诱导培养基1。
    4. 在神经诱导的第4天,细胞将达到融合。 如有必要,请标记任何具有非神经分化的菌落。用200μl移液管尖端去除这些不需要的菌落。从每个孔中吸出用过的培养基。在每个平板上加入3毫升预热的完全神经诱导培养基1。
      5.在神经诱导的第6天,细胞应该接近最大汇合。去除任何可以观察到的非神经分化细胞,并在每个平板中加入3ml完全神经诱导培养基。
    5. 在神经诱导的第7天,应将培养基转换为神经诱导培养基2.向每个平板中加入3ml完全神经诱导培养基2。介质应每天更换5天。例如形态学,见图2.

    注意:由于第4天以后培养物中的细胞密度很高,神经诱导培养基的体积加倍对于细胞营养非常关键。此外,在神经诱导后第4天至第7天应观察到最小的细胞死亡。如果在神经诱导的第4天至第7天期间细胞的颜色变为带有许多漂浮细胞的黄色,则表明iPSC的起始密度太高。在这种情况下,每天更换神经诱导培养基,去除一些菌落,每孔/板的体积加倍。理想情况下,使用这些变量以确保介质不会继续变黄。


    图2.分化为前脑NPC的iPSC的形态学。 A.第0天:在加入神经诱导培养基之前立即显示适当大小的单个iPSC集落.B。第2天:iPSC集落,已经用神经诱导培养基1处理2天,开始改变细胞形态,并且一些细胞延伸过程。 C.第5天:随着外细胞的一些分化,集落的扩增增加。 D.第12天:菌落中玫瑰花结的外观变得清晰可见。 NSCs在这些结构的中间以高汇合存在。此时,将菌落分离并在D13的非粘附平板上重新铺板两天。 E.在粘附板上铺板后立即进行D13。该图像显示浮动莲座丛,其将继续增殖并在浮动块中分化。化学释放后,非玫瑰花结细胞在D12处保留在培养皿上,或者作为单细胞漂浮在(E)中所示的非粘附平板上。 F.在D15处,莲座丛的尺寸扩大并移动到粘附板上。这里的细胞聚集体是三维的,但是附着在板上。注意此时簇的纯度(F)。比例尺代表130微米。


  2. 收获和扩大NPC
    注意:在神经诱导的第12天,NPC已准备好收获和扩大。
    1. 从每个板上吸出用过的神经诱导培养基进行传代。
    2. 轻轻地将不含CaCl 2 2 和MgCl 2 的DPBS加入每个板中两次以冲洗细胞。
    3. 向每个板中加入1.5ml预热的温和细胞解离试剂,并在37℃下孵育5分钟,直到大多数细胞从培养皿表面脱离。轻轻敲打板以移除仍附着的细胞。
    4. 使用移液管轻轻冲洗板表面,使用板中已有的Gentle Cell Dissociation Reagent,以分离剩余的细胞。
    5. 使用移液管将细胞悬浮液转移至15ml锥形管中。
    6. 向每个板中加入1ml DPBS以收集残留的细胞并将细胞悬浮液转移到锥形管中。
    7. 用5毫升或10毫升移液管轻轻吸取细胞悬液3次,以打碎细胞团块。
    8. 将细胞在300 x g 下离心5分钟。
    9. 吸出上清液并将细胞重新悬浮在预热的神经祖细胞(NPC)培养基中(即,对于来自每个平板的所有细胞,10ml)。
    10. 将悬浮在NPC培养基中的细胞铺板到10cm培养皿上。
    11. 在CO 2 培养箱中培养细胞2天。在此期间,NPC将在NPC媒体中浮动时形成聚合。
    12. 一旦聚集体达到约70-200μm的合适尺寸,制备涂有5ml Matrigel 的10cm组织培养皿至少1小时。如果形成的聚集体很少,则在60mm培养皿中培养细胞。
    13. 使用5毫升或10毫升移液器,将NPC培养基吸收并通过细胞过滤器以收集NPC聚集体。 
    14. 翻转过滤器并通过过滤器通过10ml新鲜预热的NPC培养基,其中细胞聚集体被结合,使得它们被转移到涂有Matrigel 涂层的10cm板上。
    15. 在CO 2 培养箱中培养细胞以使NPC聚集体附着到包被的培养皿上并迁移和增殖。
    16. 每2-3天更换一次培养基,直至细胞达到融合,并准备进行传代或冷冻保存。使用温热的accutase在37°C下解离5分钟。
    17. 为了评估NPC培养物的纯度,使用ICC修复细胞并检查NPC标记物(参见图3)。
    18. 为冷冻保存NPC,冷冻80/20混合的FBS / DMSO。储存在-80°C,可在几个月内使用,或在液氮中储存,以便长期储存。


      图3.高质量NPC培养物的ICC染色样本为了有效地进行分化,确保iPSC培养物均匀表达Nestin,SOX1和PAX6。 NPC培养物不应表达多能标记物OCT4(DAPI在PAX6和OCT4的合并中以蓝色显示;所有细胞均表达PAX6,即,100%纯度的培养物)。比例尺代表100微米。

  3. 前脑NPC分化为神经元
    1. 将板状NPC置于涂有Matrigel ®的组织培养皿上。等到细胞在开始分化之前达到70%-95%汇合。
    2. 一旦NPC达到所需的汇合度,吸出培养基并用等量的Neuronal Media替换。
    3. 每隔2-3天,将一半的培养基吸入培养皿中,换上新鲜的Neuronal培养基。
      注意:由于某些介质会因蒸发而丢失,因此您可能需要添加比从印版中移除更多的介质,以便随着时间的推移保持介质体积稳定。
    4. 继续改变培养基,直到神经元达到理想的发育阶段。例如,发展前脑神经元的形态,参见图4.例如前脑神经元的ICC表征,参见图5.
      注意:
      1. 在这个开发阶段,您的生产线的纯度将很容易被发现。含有高百分比NPC的细胞系将迅速极化并形成神经元投射(通常在第2-5天左右),而含有高百分比的非NPC细胞(星形胶质细胞,神经嵴细胞等)的细胞系则不会。 / EM>
      2. 特定平板中的神经元需要多长时间才能达到某一发育阶段,这取决于品系,克隆,传代数等。但是,我们发现优质培养物中的神经元始终通过以下方式获得以下标志:以下几天分化。

      第5天=细胞是有丝分裂后的 第15天=细胞具有明显极化的轴突和树突,并具有明显可检测的电生理特性,如动作电位


      图4.前脑NPC分化为神经元的示例形态。前脑NPC培养物分化为神经元的形态学。在D0(A),D5(B),D15(C),D30(D)拍摄的图像。比例尺代表130微米。

    5. 要评估您的神经元培养物的纯度,修复细胞并使用ICC检查前脑标记物。为了评估产生的神经元的质量,确保细胞显示出适当的电生理特性(参见数据分析)。

  4. 使用免疫细胞化学评估培养物纯度
    注意:假设细胞涂在涂有聚鸟氨酸和层粘连蛋白的玻璃盖玻片上,将描述以下步骤(更多细节见食谱)。
    1. 将样品固定在用PBS稀释的4%多聚甲醛(PFA)中。在室温下孵育15分钟。
    2. 在室温下用PBS(3×15分钟)洗涤样品。
    3. 通过在室温下在PBS + 0.1%Triton X-100中孵育10分钟来使样品透化。 
    4. 吸出透化缓冲液并用PBS中稀释的5%BSA代替以阻断样品。在室温下孵育60分钟。 
    5. 通过在阻断5%BSA-PBS中稀释来制备初级抗体的工作原种。有关不同细胞类型的推荐工作稀释液和抗体,请参见表1。在一抗溶液中涂上盖玻片,在2-8°C下孵育过夜。

      表1.免疫细胞化学中使用的抗体


    6. 在室温下用PBS(3×15分钟)洗涤样品。
    7. 吸出PBS并加入用5%BSA-PBS稀释的二抗。推荐的二抗和稀释度见表1。在室温下孵育盖玻片,远离光照一小时。
    8. 在室温下用PBS(3×15分钟)洗涤样品。
    9. 如果需要,加入用PBS稀释的DAPI,在室温下孵育5分钟。
    10. 将一滴Vectashield ®添加到载玻片上。小心地使用针头和镊子将盖玻片(细胞侧朝下)转移到载玻片上。密封使用指甲油。


      图5.高质量前脑神经元的ICC染色样本。 NPC分化30天(D30)后前脑神经元培养的代表性ICC,证明了谷氨酸能,GABA能和星形胶质细胞标志物的相对丰度。人口。这些培养物约为65%谷氨酸能,30%GABA能,和5%-10%星形胶质细胞。比例尺代表50微米。

  5. 使用电生理学评估神经元质量
    1. 从玻璃毛细管中拉出移液器。当用内部移液器溶液填充时,它们的电阻应在3到6MΩ的范围内。
    2. 将含有分化的人类iPSC衍生神经元的个别盖玻片转移到加热的记录室中,用BrainPhys神经元培养基连续灌注(1 ml / min),不用苯酚鼓泡,用CO 2 (5%)和O的混合物 2 (95%)并使用自动温度控制器维持在35°C。
    3. 选择要录制的单元格。
    4. 用内部移液器溶液填充移液管并将其放入电极支架中。放下移液器,将其放入外部溶液中。在补偿偏移后,在远程微操纵器的帮助下将移液管接近所选择的细胞,以形成高阻细胞附着的密封。 
    5. 一旦形成密封并建立整个电池配置,将串联电阻补偿在80%-90%。 
    6. 在开始录制之前等待5到10分钟。这允许细胞内容物与内部移液管溶液平衡。
    7. 对于采集,将滤波器设置为2 kHz,采样率设置为20 kHz。
    8. 一旦建立了全细胞记录,就可以进行基本神经元特性的记录,包括rheobase,静息膜电位,动作电位参数和自发突触后电流。通过移液器添加NMDA。评估神经元应该在电压钳中保持神经元在-70 mV,除非检查静息膜电位和rheobase的变化,这应该在电流钳中进行。 Clampex和GraphPad Prism 7是推荐用于显示数据的软件。例如从皮质神经元获得的电生理记录,参见图6.


      图6.高质量前脑神经元的电生理特性。 A.全细胞构型中单个神经元记录的玻璃微电极的差分图像对比度。比例尺代表20微米。 B.超极化脉冲,显示去极化下垂,随后是多个反弹动作电位。 C.左:电压钳记录的代表性迹线显示向内Na + 电流;右图:在施用1μM的河豚毒素(TTX)后,钠电流痕迹消失。 D.具有静息膜电位-40 mV的自发活跃神经元的代表性电流钳记录。 E.代表性记录显示在当前斜坡方案期间由前脑神经元发射的动作电位。 F.来自具有自发突触输入的神经元的代表性电压钳记录。所有电生理数据均来自D14神经元。

数据分析

应处理电生理数据,电流应以2 kHz过滤,并以20 kHz数字化。应报告值,校正计算出的10 mV液体结电势的电压和电流钳记录中的标称膜电位。

食谱

  1. 媒体
    注意:所有培养基均为50 ml的配方,因为可以在一个50 ml的管中准备这个体积,适合在珠子或水浴中加热。

    1. 神经诱导培养基1


    2. 神经诱导培养基2


      注意:完整的神经诱导培养基1和2可以在2-8°C的黑暗中储存长达2周。在使用前,将神经诱导培养基在37°C水浴中加热5-10分钟。不要将神经诱导培养基在37°C水浴中加热超过10分钟,因为这可能会导致培养基降解。

    3. 神经祖先媒体


    4. 神经元媒体


  2. 缓冲和解决方案
    1. 内部移液器解决方案
      5 mM HEPES
      2 mM KCl
      136 mM葡萄糖酸钾
      5 mM EGTA
      5mM ATP-Mg 2 +
      8 mM肌酸磷酸盐
      0.35 mM鸟苷三磷酸
      用KOH将pH调节至7.2,并且如果需要,用蒸馏水或浓缩的葡萄糖酸钾调节重量摩尔渗透压浓度至295至298mOsm之间。
      注意:内部和外部溶液的渗透压差异应接近5%。
    2. 用Matrigel ®培养皿涂层
      1. 从-80°C解冻冷冻的Matrigel ®(250μl)等分试样,将其置于4°C冰箱中1小时
      2. 要制作工作溶液,将解冻的Matrigel ®稀释在25 ml冷PBS或培养基中
        注意:在DMEM或其他具有强烈着色的介质中稀释基质胶 ® 可以更容易地确定整个培养皿是均匀的覆盖。
      3. 用适量的基质溶液快速覆盖每个培养皿的整个表面(表2)
      4. 将培养皿在37°C,5%CO 2 培养箱中孵育至少1 h
      5. 培养皿现在可以使用了。在使用前,从培养皿中吸出稀释的Matrigel ®溶液。细胞可直接接种到Matrigel ®涂层培养皿上,无需冲洗。
        注意:涂层培养皿也可以在2-8°C下储存长达一周。储存时,用Parafilm ® 实验室薄膜密封培养皿以防止干燥。使用前,将涂在2~8°C的培养皿在室温下预热30分钟。

        表2.用于涂覆不同培养皿的基质胶所需体积 ® 基质溶液


    3. 用聚鸟氨酸和层粘连蛋白涂覆玻璃盖玻片
      1. 将玻璃盖玻片放入培养皿或悬浮板中
      2. 通过将板暴露于UV辐射20分钟来消毒盖玻片
      3. 用PBS中的100μl50μg/ ml聚鸟氨酸涂覆盖玻片。等两个小时
      4. 吸气解决方案。用PBS洗涤一次,然后用PBS稀释的10μg/ ml层粘连蛋白包被盖玻片
      5. 在37°C孵育2小时
      6. 吸出溶液,在10%FBS DMEM中涂覆盖玻片
      7. 这些板现在可以使用了。为获得最佳效果,请在准备24小时内使用。在铺板细胞之前,用PBS洗涤板一次,因为FBS可能影响分化

致谢

这项工作得到了加拿大研究教席计划和加拿大卫生研究院对CE的资助。斯科特•贝尔(Scott Bell)由魁北克省资助会(FRQS)资助,Nuwan Hettige由FRQS资助,Malvin Jefri由马来西亚政府资助,Lilit Antonyan由CONACYT(墨西哥)资助。该协议改编自先前的工作(Bell et al。,2017和2018)。

利益争夺

Carl Ernst是ManuStem.com的总裁。

伦理

所有工作均经道格拉斯医院研究伦理委员会批准。

参考

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引用:Bell, S., Hettige, N. C., Silveira, H., Peng, H., Wu, H., Jefri, M., Antonyan, L., Zhang, Y., Zhang, X. and Ernst, C. (2019). Differentiation of Human Induced Pluripotent Stem Cells (iPSCs) into an Effective Model of Forebrain Neural Progenitor Cells and Mature Neurons. Bio-protocol 9(5): e3188. DOI: 10.21769/BioProtoc.3188.
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