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May 2017

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Generation of Human iPSC-derived Neural Progenitor Cells (NPCs) as Drug Discovery Model for Neurological and Mitochondrial Disorders
人来源iPSC获得的神经祖细胞(NPCs)的产生作为神经和线粒体疾病的药物发现模型   

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Abstract

The high attrition rate in drug development processes calls for additional human-based model systems. However, in the context of brain disorders, sampling live neuronal cells for compound testing is not applicable. The use of human induced pluripotent stem cells (iPSCs) has revolutionized the field of neuronal disease modeling and drug discovery. Thanks to the development of iPSC-based neuronal differentiation protocols, including tridimensional cerebral organoids, it is now possible to molecularly dissect human neuronal development and human brain disease pathogenesis in a dish. These approaches may allow dissecting patient-specific treatment efficacy in a disease-relevant cellular context. For drug discovery approaches, however, a highly reproducible and cost-effective cell model is desirable. Here, we describe a step-by-step process for generating robust and expandable neural progenitor cells (NPCs) from human iPSCs. NPCs generated with this protocol are homogeneous and highly proliferative. These features make NPCs suitable for the development of high-throughput compound screenings for drug discovery. Human iPSC-derived NPCs show a metabolism dependent on mitochondrial activity and can therefore be used also to investigate neurological disorders in which mitochondrial function is affected. The protocol covers all steps necessary for the preparation, culture, and characterization of human iPSC-derived NPCs.


Graphic abstract:



Schematic of the protocol for the generation of human iPSC-derived NPCs


Keywords: Human iPSCs (人的诱导性万能干细胞), Neural progenitor cells (神经前体细胞), Drug discovery (药物发现), Stem cell differentiation (干细胞分化), Neuronal disease modeling (神经疾病建模), Mitochondrial disorders (线粒体疾病)

Background

In recent years, the downsides of target-centered drug discovery have become evident, in particular for programs addressing neurological diseases (Paul et al., 2010). The challenges of target-based drug discovery led to the revival of phenotypic drug discovery. Phenotypic screenings require the identification of a disease-specific trait (phenotype) that can be modulated within the physiological environment of a cell or organism (Khurana et al., 2015). This approach has the advantage of identifying compounds that show an effect within complex biological environments. At the same time, phenotypic drug discovery requires the presence of a robust disease-relevant phenotype that can be efficiently modulated using a reliable high-throughput detection method. Current drug discovery pipelines strongly rely on cancer-derived immortalized cell lines that lack the functional properties of brain cells. Moreover, immortalized cell lines are mostly glycolytic and less sensitive to mitochondrial impairment than neuronal cells (Bénit et al., 2016). Therefore, immortalized cells are not an effective drug discovery model for neurological diseases and diseases in which mitochondrial oxidative phosphorylation (OXPHOS) is dysfunctional, such as mitochondrial disorders and neurodegeneration (Cunnane et al., 2020).


An important breakthrough in the field of neuronal disease modeling and drug discovery is represented by cellular reprogramming technologies. Cellular reprogramming allows the conversion of the identity of a given cell, thereby enabling the generation of brain cells from patient-specific somatic cells. Cellular conversion can be accomplished through the derivation of induced pluripotent stem cells (iPSCs) (Takahashi et al., 2007) or through the process of direct conversion, which can reprogram skin cells directly into post-mitotic induced neurons (iNs) (Vierbuchen et al., 2010). iPSCs can also be used to derive three-dimensional (3D) cerebral organoids that may recapitulate the organization of the developing human brain (Lancaster et al., 2013).


In order to perform meaningful phenotypic screens, it is necessary to use disease-relevant cellular models. Among reprogramming-derived cell types, patient-specific neural progenitor cells (NPCs) appear particularly well suited for phenotypic drug discovery of neurological diseases, given their homogeneous features, cost-effective culture conditions, and mild proliferative state. Indeed, iPSC-derived NPCs have been successfully employed for drug discovery in the context of neurological and neuropsychiatric disorders (Lorenz et al., 2017; Readhead et al., 2018; Walter et al., 2019).


During the process of neurogenesis, which is characterized by a metabolic shift from glycolysis to OXPHOS (Zheng et al., 2016), NPCs represent the first cell type to depend on mitochondrial respiration (Beckervordersandforth, 2017; Lorenz and Prigione, 2017). Since NPCs show sensitivity to conditions that impair OXPHOS, they can be used as an effective cell model to carry out high-throughput screenings for neurological and mitochondrial diseases. Accordingly, iPSC-derived NPCs from patients affected by mitochondrial diseases exhibited meaningful phenotypes that could be harnessed to perform phenotypic compound screenings (Lorenz et al., 2017). Altogether, iPSC-derived NPCs might be instrumental for identifying novel treatments in the context of neurological and mitochondrial diseases that are hard to model using conventional drug discovery systems (Inak et al., 2017).


Here, we describe in detail the generation and maintenance of human iPSC-derived NPCs. Different protocols can be used to obtain these cells. Some protocols give rise to NPC populations with limited proliferative features (Elkabetz et al., 2008), while others may depend on the addition of human LIF, which makes culturing the cells very cost-intensive (Li et al., 2011). However, most useful approaches rely solely on small molecule to produce highly proliferative NPC populations (Reinhardt et al., 2013). A key characteristic of these NPCs is their capability of robust and homogenous expansion. At the same time, NPCs should be able to efficiently differentiate into neuronal and glial cell types (Reinhardt et al., 2013; Lorenz et al., 2017).


Our approach is based on the protocol of Reinhardt et al. ( 2013) and was published in Lorenz et al. (2017) for drug discovery applications in the context of neurological mitochondrial diseases. The protocol allows the generation of NPCs that maintain their characteristics under chemically defined conditions over several passages and can be readily differentiated into the desired brain cell types. The protocol is highly reproducible and leads to homogenous cell populations. It is similarly effective starting from human embryonic stem cells, healthy control iPSCs, and patient-specific iPSCs (Lorenz et al., 2017).


To initiate neural induction, bone morphogenic protein (BMP) inhibitor dorsomorphin (DM), and transforming growth factorβ (TGFβ)-signaling inhibitor SB43152 (SB) are supplemented to the basic NPC media (BM1). Purmorphamine (PU) and CHIR99021 (CHIR) are added to stimulate the sonic hedgehog (SHH) signaling pathway, inhibit glycogen synthase kinase-3 (GSK3), and activate canonical WNT signaling (Chambers et al., 2009; Kim et al., 2010). After the first days in the induction medium, embryoid bodies (EBs) form neuroepithelial structures (Figure 1A). After 6 days, the EBs are gently dissociated and seeded on Matrigel-coated plates for purification. Neural progenitor morphology becomes homogenous after 4-6 passages post neural induction (Figure 1A). NPCs should express typical NPC markers such as NESTIN, PAX6, SOX1, SOX2, and DACH1. Spontaneous differentiation of cells expressing the pan-neuronal marker TUJ1 is always present (Figure 1C-1D). Conversely, pluripotency markers like OCT4, NANOG, DNMT3B, and DPPA4 should be downregulated compared to the parental iPSC lines. NPCs retain their multipotent identity over multiple passages (> 30) as well as after freezing and thawing without changes in cell morphology.


The following step-by-step protocol allows the experimenter to obtain human iPSC-derived NPCs with a high degree of homogeneity within 4-8 weeks, and to culture and characterize the cells for subsequent use in disease modeling or drug discovery applications.


Materials and Reagents

The following list provides examples of the materials and equipment that we routinely use in our laboratory. Nevertheless, reagents and equipment with similar specifications will work as well.

  1. Cellstar cell culture multiwell plates (6-24 well) (Greiner Bio-One, catalog numbers: 657 160 [6-well; 665 180 [12-well]; 662 160 [24-well])

  2. Corning 60 mm Ultra-low attachment culture dish (Corning, catalog number: 3261)

  3. Cell spatula (TPP, catalog number: 99010)

  4. Serological pipettes (Greiner Bio-One, catalog numbers: 760180 [2 ml]; 606180 [5 ml]; 607180 [10 ml]; 760107 [25 ml]; 768160 [50 ml])

  5. BemisTM Curwood ParafilmTM M Laboratory Film (Fisher Scientific, product code HS234526C, catalog number: 10018130)

  6. Greiner centrifuge tubes (50 ml and 15 ml) (Sigma-Aldrich, manufacturer: Greiner; catalog number for 15 ml: T1818, for 50 ml: T2318)

  7. Liquid nitrogen

  8. Corning ® Matrigel ® Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenolred-free, LDEV-free (Corning, CAS number: 356231, format: 10 ml, store at -20 °C and follow instructions from supplier for preparation)

  9. CHIR 99021 (Caymen Chemical, CAS number: 252917-06-9, catalog number: 13122; product format: 2.5 mg/ml in DMSO; storage at -20 °C as 6 mM stock; stability: ≥ 2 years)

  10. SB431542 in solution (MACS Miltenyi, CAS number: 130-106-543, catalog number: 130-106-543; product format: 10 mM in DMSO; storage at -20 °C as 10 mM stock, protected from light; stability: 6 months)

  11. Dorsomorphin (StemCell; CAS number: 866405-64-3, catalog number: 72102; product format 10 mg solid; storage at -20 °C as 5 mM stock in DMSO (heat applied) or ethanol; stability: 12 months)

  12. Purmorphamin (MACS Miltenyi, CAS number: 483367-10-8; catalog number: 130-104-465 product format: 5 mg solid; storage at -20 °C as 10mM stock in DMSO, protected from light)

  13. (+)-Sodium L-ascorbate (Vitamin C) (Merck, Sigma-Aldrich, catalog number: A4034; product format solid powder; store as 200 µM stock in water at -20 °C)

  14. N-2 Supplement (100×) (Thermo Fisher Scientific, Gibco, catalog number: 17502-048; product format: 5 ml liquid; store at -20 °C, protected from light; stability: 18 months)

  15. B27 without Vitamin A (50×) (Thermo Fisher Scientific, Gibco, catalog number: 12587010; product format: 10 ml liquid; store at -20 °C protected from light)

  16. Neurobasal® Medium (Thermo Fisher Scientific, Gibco, catalog number: 21103049; product format: 500 ml liquid; store at 2-8 °C, protected from light; shelf life: 12 months)

  17. DMEM/F12, HEPES (Thermo Fisher Scientific, Gibco, catalog number: 31330038; product format: 500 ml liquid; store at 2-8 °C, protected from light; shelf life: 12 months)

  18. KnockOut DMEM (Thermo Fisher Scientific, Gibco, catalog number: 10829018; product format: 500 ml liquid; store at 2-8 °C protected from light

  19. KnockOut Serum replacement (Thermo Fisher Scientific, Gibco, catalog number: 10828010; product format: 500 ml liquid; store at -20 °C protected from light

  20. StemMACSTM iPS-Brew XF, human (MACS Miltenyi Biotec, catalog number: 130-104-368)

  21. mTeSRTM1 (STEMCELL Technologies, catalog number for 500 ml kit: 85850)

  22. Penicilin-Streptomycin (P/S; 10,000 U/ml Penicilin, 10,000 µg/ml Streptomycin) (Thermo Fisher Scientific, Gibco, catalog number 15140122; product format: 100 ml liquid; store at -20 °C; shelf life: 12 months)

  23. L-Glutamine (200 mM) (Thermo Fisher Scientific, Gibco, Catalog number: 25030081; product format: 100 ml liquid; store at -20 °C, protected from light; shelf life: 24 months)

  24. Non-essential amino acids (NEAA, 100×) (Thermo Fisher Scientific, Gibco, catalog number: 11140035, product format: 100 ml, store at 2-8 °C; shelf life: 12 months)

  25. Sodium pyruvate (100 mM) (Thermo Fisher Scientific, Gibco, catalog number: 11360070; product format: 100 ml; store at 2-8 °C protected from light; shelf life: 12 months)

  26. MycoZap Plus-CL (Lonza, catalog number VZA-2012; product format: 20 ml liquid; store at -20 °C, protected from light)

  27. StemProTM AccutaseTM Cell Dissociation Reagent (Thermo Fisher Scientific, Gibco/StemPro, catalog number: A1110501; product format: 100 ml liquid, store at -20 °C, protected from light)

  28. DPBS (without calcium and magnesium) (Thermo Fisher Scientific, Gibco, catalog number: 14190250; product format: 500 ml liquid; store at 15-30 °C; shelf life: 36 months)

  29. ROCK inhibitor (RI), Y-27632, dihydrochloride (Enzo, CAS number: 129830-38-2; catalog number: ALX-270-333-M005; product format 5 mg powder; store at -20 °C; soluble in DMSO as 25 mg/ml; stability: stock solution stable for 1 month at -20 °C)

  30. UltraPureTM 0.5 M EDTA (Invitrogen, catalog number: 15575020)

  31. Bambanker (NIPPON Genetics, catalog number: BB03)

  32. ShandonTM Immu-MountTM (Thermo ScientificTM, catalog number: 77-86-1; reference number 9990402; product formal: 20 ml)

  33. Anti-Nestin Antibody, clone 10C2 (Millipore, catalog number: MAB5326; Brand family: Chemicon®)

  34. Anti-Pax-6 Antibody (Covance, catalog number: 901301)

  35. SOX2 antibody (R&D Systems, catalog number: AF2018)

  36. DACH1 antibody (ProteinTech, catalog number: 10914-1-AP)

  37. Anti-β-Tubulin III (TUJ-1) (Sigma-Aldrich, catalog number: T8578)

  38. MAP2 (Synaptic system, catalog number: 188004)

  39. Hoechst 33342 (Hoechst) (InvitrogenTM, catalog number: H3570)

  40. 16% Paraformaldehyde Aqueous Solution (PFA) (Electron Microscopy Sciences; catalog number: 50-980-487)

  41. Tween-20 (Sigma-Aldrich, catalog number: P9416)

  42. TritonTM-X100 (Sigma-Aldrich, catalog number: T8532)

  43. Alexa FluorTM 488 donkey anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific, catalog number: A-21206)

  44. Alexa FluorTM 647 donkey anti-mouse IgG (H+L) secondary antibody (Thermo Fisher Scientific, catalog number: A-31571)

  45. Donkey anti-goat Cy 3 (Sigma-Aldrich, catalog number: AP180C)

  46. Donkey anti-guinea pig Cy 3 (Sigma-Aldrich, catalog number: AP193C)

  47. SYBRTM Green PCR Master Mix (Thermo Fisher Scientific, catalog number: 4309155)

  48. M-MLV Reverse Transkriptase (Invitrogen, catalog number: 28025013)

  49. RNeasy Mini Kit (50) (Qiagen, catalog number: 74104)

  50. Rnase-Free Dnase Set (50) (Qiagen, catalog number: 79254)

  51. Oligo(dT) 12-18 Primer (Invitrogen, catalog number: 18418012)

  52. dNTP-Mix (10 mM) (Invitrogen, catalog number: 18427088)

  53. MicroAmpTM Optical 348-well-reaction plate with barcode (Thermo Fisher Scientific, catalog number: 4343814)

  54. Basic medium 1 (BM1) (see Recipes, Table 1)

  55. BM2 (sm-) medium (see Recipes, Table 1)

  56. M1 medium (see Recipes, Table 2)

  57. M2 medium (see Recipes, Table 2)

  58. M3 (sm+) medium (see Recipes, Table 2)

Equipment

  1. Centrifuge (Eppendorf, model: 5810R)

  2. Water bath (Thermo Fisher Scientific, FisherbrandTM IsotempTM Digital-Control Water Baths: Model 220)

  3. CO2 incubator for cell culture (Thermo Fisher Scientific, model: Heraeus BBD 6220)

  4. Hypoxia incubator for iPSC culture (Binder GmbH, model: Binder BCB 160)

  5. qPCR machine (Thermo Fisher Scientific, model: ViiA 7)

  6. Confocal Microscope system (Zeiss, model: Axio Imager Z1)

  7. Freezing container (Nalgene, model: Mr. Frosty; Corning CoolCellTM)

  8. -80°C Freezer (New Brunswick, model: Innova 4725)

  9. Laminar flow hood (BDK, Luft und Reinraumtechnik GMBH)

  10. Sterile scissor (for example: Surgical scissors, sharp, Sigma-Aldrich, catalog number: Z265977)

  11. Shaker (Rocky, LTF Labortechnik)

Procedure

In general:

  1. Work under sterile conditions, e.g., under a laminar flow hood.

  2. Disinfect reagents and consumables before transferring under the hood.

  3. All media should be prepared fresh and used within one week.

  4. Do not thaw N-2 Supplement and B-27 Supplement in the water bath. Instead thaw them at room temperature (RT, 20-22 °C) for 2-4 h or in the fridge (4 °C) over night.

  5. All media should be pre-warmed before applying to the cells. Do not keep the media at 37 °C for long periods but rather pre-warm at RT.

  6. Culture the NPCs and iPSCs in a controlled environment with 37 °C and 5% CO2.

    1. Optional: If possible, culture the iPSCs under hypoxic conditions (37 °C, 5% CO2, 5% O2), which more closely mimics in vivo conditions.

    2. We culture iPSCs under feeder-free conditions in either StemMACSTM iPS-Brew XF, or mTeSRTM1 on Matrigel-coated 6-well plates.

    3. We passage iPSCs using 0.5 mM EDTA/PBS in ratios ranging from 1:4-1:12. To increase cell survival, we recommend adding ROCK inhibitor (RI) after each splitting in a concentration of 10 µM.


  1. Step-by-step protocol for NPC generation

    Day 0 (Monday): Harvesting of the iPSCs and transfer on Corning Ultra-Low Attachment 60mm/15mm dishes.

    1. Prepare the basic medium 1 (BM1) (Table 1).

    2. Prepare M1 medium (Table 2).

    3. Choose one to two wells of iPSCs (~80% confluent).

    4. Remove the media and rinse the wells twice with DPBS to remove dead cells and debris.

    5. Detach cells from the plate using Accutase (1 mg/ml). Add 0.5-1 ml/well and incubate for 2-5 min at 37 °C, check visually in between.

    6. Continue when colonies are mainly detaching from the plate.

    7. Add 4-10 ml PBS or medium to reduce Accutase activity.

    8. Optional: Mechanically detach cells using a cell spatula if cells are not completely detached by Accutase activity.

    9. Transfer the cell suspension to a 15 ml Falcon tube (pool both wells), let the cells settle to the bottom (10-15 min) in water bath.

    10. Optional: Centrifuge at 120 × g for 5 min (potentially more damaging but faster and results in a more solid pellet).

    11. Remove as much medium as possible from the sedimented cells by pipetting (for example with a 5 ml serological pipette). Make sure not to disperse or suck up the cells.

    12. Add 5 ml of M1 to the cells, transfer the cells to a non-treated Corning Ultra-Low Attachment 60 mm/15 mm dish (1:1; or X number of wells in case of high confluence).

    13. Incubate at 37 °C, 5% CO2 for 2 days.

    14. Incubation with M1 will form embryoid bodies (EBs) floating in the media. If they attach at day 1 to the bottom of the Corning Ultra-Low attachment 60 mm/15 mm dish, detach them by gently pipetting using a 1 ml pipette (cut off the tip with sterile scissors!). Make sure not to shred the floating EBs. Alternatively, use a cell spatula. Keep the media in the well and gently scrape throughout the well using a cell spatula to detach EBs.


    Day 2 (Wednesday): 50% media exchange to M2

    1. Prepare BM2 (sm-) medium (Table 1).

    2. Prepare M2 medium (Table 2).

    3. Cut off the tip of a 1 ml pipet tip using a sterile scissor.

    4. Transfer EBs to a 15 ml Falcon tube using a 1 ml pipette tip (cut off the tip!).

    5. Optional: If some EBs attached, detach EBs using a cell spatula.

    6. Let EBs settle for 15 min in the water bath at 37 °C.

    7. Remove 50% of M1 and add 50% of M2 instead.

      Optional: Remove all M1 and add 100% M2 (but potentially more damaging for the cells).

    8. Transfer the EBs back to the same Corning Ultra-Low Attachment 60 mm/15 mm dish using a 1 ml pipette tip (cut off the tip using a sterile scissor!).


    Day 3 (Thursday): 100% media exchange to M2

    1. Prepare M2 medium (Table 2).

    2. Cut off the tip of a 1 ml pipet tip using a sterile scissor.

    3. Transfer EBs to a 15 ml Falcon tube using a 1 ml pipette tip (cut off the tip!).

    4. Optional: If necessary, detach EBs using a cell spatula as described before.

    5. Let EBs settle for 15 min in the water bath at 37 °C.

    6. Exchange the medium completely to 100% of M2 medium (5-6 ml final volume).

    7. Transfer EBs back to the same Corning Ultra-Low Attachment 60 mm/15 mm dish using a 1 ml pipette tip (cut off the tip using a sterile scissor!).


    Day 4 (Friday): 100% media exchange to M3 (sm+) and preparation of Matrigel-coated plates
    1. Prepare M3 (sm+) medium (Table 2).

    2. Cut off the tip of a 1 ml pipet tip using a sterile scissor.

    3. Transfer EBs to a 15 ml Falcon tube using a 1 ml pipette tip (cut off the tip!).

    4. Optional: If necessary, detach EBs using a cell spatula as described before.

    5. Let EBs settle for 15 min in the water bath at 37 °C.

    6. Exchange the media completely to 100% of M3 (sm+) medium (5-6 ml final volume).

    7. Transfer EBs back to the same Corning Ultra-Low Attachment 60 mm/15 mm dish using a 1 ml pipette tip (cut off the tip using a sterile scissor!).

    8. Coat a 6-well plate with Matrigel (procedure described separately) as preparation for day 7.


    Day 7 (Monday): Transfer the cells to Matrigel-coated plates
    1. Prepare M3 (sm+) medium (Table 2).

    2. Transfer EBs from the Corning Ultra-Low Attachment 60 mm/15 mm dish to a 15 ml Falcon tube. Use a 1 ml tip (cut off the tip!).

      If necessary, detach neural tube-like formations using a cell spatula as described for EBs.

    3. Let EBs settle down in the water bath at 37 °C (10-15 min).

    4. Remove old media.

    5. Add new sm+ medium.

    6. Shred the cell formations by pipetting 5-10 times using a normal 1 ml tip.

    7. Transfer the whole suspension to the Matrigel-coated 6-well plate, distribute equally to 2-X wells depending on the amount of EBs.

    8. Fill up to 2 ml sm+ each 6-well.

    9. Add ROCK inhibitor (RI) (10 µM final concentration).

      Note: To increase cell survival, we recommend adding RI to the cells for the first 1-2 splittings. Afterwards, RI is not needed!

    10. Check the next day, NPC-like cells should be already visible (see Figure 1A–right image, and Figure 1E).

    11. Try to keep the cells in the first well for 1 week.

    12. If necessary, split the cells as described in the maintenance protocol.

    Note: NPCs should appear homogenous after 4-5 passages, expressing NESTIN, PAX6, and SOX2. NPCs usually also express the immature pan-neuronal marker TUJ1. Spontaneous differentiation can occur, and some cells can express the neuronal marker MAP2. Cells do not necessarily need to be cultured in 6-well plates.


    Figure 1.  Generation and characterization of human iPSC-derived NPCs.  A. Representative images of neurospheres, neuroepithelial cells, and neural progenitor cells (NPCs). B. Image of a matrigel coated well. Scale bar: 200 µm. C. Immunocytochemistry of NPCs obtained from the control iPSC line XM001 (Wang et al. 2018) stained for the neural progenitor markers NESTIN, DACH1, PAX6, and SOX2. Spontaneous differentiation of cells expressing TUJ1 or MAP2 can be present. Scale bar: 100µm. D. Quantitative real-time reverse transcription PCR (qRT-PCR) of neural progenitor markers SOX1, NESTIN, PAX6, and SOX2, and of pluripotency-associated markers OCT4, NANOG, DNMT3B, and DPPA4. Transcription levels in NPCs were normalized using GAPDH housekeeper gene (mean=SD +/-; n=3 technical replicates) and reported in relation to the transcription level of the respective control iPSC line XM001. E. Representative images of neural progenitor cells (NPCs) from passage 3 (p3) and passage 7 (p7) demonstrating the change in morphology towards homogenous NPC cultures. Scale bar: 100 µm.


  2. Matrigel preparation

    1. Thaw frozen Matrigel (stored at -20 °C) OVERNIGHT (ON) on ice at +4 °C.

    2. Check protein concentration (stock concentration) of Matrigel as provided in Lot datasheet.

    3. Calculation: Add a specific volume (=x) of cold KnockOut-DMEM to the 10 ml thawed Matrigel in the vial to dilute it to 5 mg/ml final concentration. The stock concentration of your vial (= y) should be written on the lot datasheet. Calculate the needed volume using the formula: x = y * 2-10 ml.

    4. Perform next steps under the hood and on ice!

    5. Prepare a pre-chilled 50 ml Falcon tube with specific volume of cold KnockOut-DMEM.

    6. Prepare pre-chilled 15 ml Falcon tubes on ice labeled with 1 M.

    7. Transfer Matrigel quickly to KnockOut-DMEM and mix carefully.

    8. Aliquot 1 ml of the diluted Matrigel in each pre-chilled 15 ml Falcon tube.

    9. Store Aliquots immediately at -20 °C.

    10. Final concentration in 6/12-well-plates: 200 µg/ml.


  3. Coating plates with Matrigel

    1. Transfer 30 ml cold KnockOut-DMEM into a 50 ml Falcon tube.

    2. Dilute 1 ml aliquot 1:30 in cold KnockOut-DMEM.

    3. Resuspend the 1 ml Matrigel aliquot (15 ml Falcon tube, -20 °C) with 5 ml of the 30 ml cold KnockOut-DMEM in the 15 ml Falcon tube until pellet is resolved! (It may take a while).

    4. 1 ml Matrigel aliquot resuspended in 30 ml cold KnockOut-DMEM as described in C2 is enough for 4 plates.

      1. 6-well: 1.25 ml.

      2. 12-well: 500 µl.

      3. 24-well: 300 µl.

    5. Add Matrigel-medium to the wells (see point 4).

    6. Polymerization:

      1. Store at 4 °C in the fridge, ON (preferentially!).

      2. 2 h at 37 °C is possible.

    7. Check polymerization in phase contrast before use! The evenly distributed formation of an extracellular matrix should be visible (Figure 1B). Coated plates can be stored for 1-2 weeks (4 °C). For storage seal the plates with Parafilm.

    8. Before use, pre-warm the plate either at RT or 37 °C.


  4. Maintenance of NPC cultures

    1. Media needs to be changed every 2nd day.

    2. Media should be pre-warmed before applying to the cells. Do not keep media at 37 °C for long periods but rather pre-warm at RT.

    3. Typically, cells were split using Accutase in ratios of 1:3 up to 1:10 every 4-5 days depending on the cell density.


  5. Splitting NPCs

    1. Aspirate growth media.

    2. Add 500 μl Accutase and incubate for 2-5 min at RT or 37 °C if cells do not detach easily.

    3. Dilute Accutase activity by addition of 2 ml sm- (without any supplementation) or PBS.

    4. Gently detach the cells by pipetting.

    5. Transfer the cells to a 15 ml Falcon tube and centrifuge at 200 × g for 3 min at RT.

    6. In the meantime, prepare Matrigel-coated plates for plating the cells.

      1. Aspirate Matrigel-mix.

      2. Add 1 ml sm+ and store in the incubator until further use.

      3. Fill blank wells with 2 ml DPBS.

        Note: Matrigel coating should not dry out.

    7. Aspirate the supernatant.

    8. Resuspend the pellet in sm+ medium.

    9. Distribute cells to Matrigel-coated 6-well plates.

    10. Fill up with media to 2 ml sm+.

      Note: To increase cell survival, we recommend adding RI (10 µM) to the cells for the first 1-2 splittings. Afterwards, RI is not needed!


  6. Freezing NPCs

    1. Detach NPCs according to the splitting procedure.

    2. Resuspend NPCs in 0.5 ml of sm+. Add freezing medium (2× sm+ freeze:sm+, 1:1).

      Note: 10 ml of 2× sm+ freeze is prepared by mixing 2 ml of sm+, 6 ml of KnockOut-SR, and 2 ml of DMSO.

    3. Alternatively, use commercial freezing media according to manufacturer’s protocol.

      Note: For example, Bambanker (Nippon Genetics, catalog number: BB03).

    4. Transfer the resuspended NPCs into a cryovial and place the vials at -80 °C in a cryo-freezing container.

      Note: Use a freezing container such as Mr. FrostyTM (Nalgene) to freeze the cells, which allows slow freezing at a rate of -1 °C per minute.

    5. Store NPCs at -80 °C or in a liquid nitrogen tank for long term storage (1-3 years).


  7. Thawing NPCs

    1. Quickly but gently thaw the cryovial containing NPCs in a 37 °C pre-warmed water bath.

    2. Transfer NPCs into a 15 ml Falcon tube set with 1 ml of sm+ and up to 10 ml of pre-warmed PBS.

    3. Centrifuged the cells at 200 × g for 3 min.

    4. Remove the supernatant and resuspend NPCs in sm+ medium.

    5. Seed NPCs on previously prepared Matrigel-coated plates 1:1 (see Coating plates with Matrigel). Note: Add 10 µM RI after thawing to support cell survival.


  8. Characterization of NPCs by immunofluorescence

    1. After 4-6 passages, seed NPCs on Matrigel-coated 24-well plates containing 12 mm coverslips. When reaching approximately 80% of confluence, rinse NPCs with PBS, and fix the cells with a 4% paraformaldehyde (PFA)/PBS solution for 15 min at RT.

    2. Remove the PFA and rinse NPCs with PBS calcium/magnesium free (PBS -/-) 3 times for 5 min.

    3. Add blocking solution to the cells for 1 h at RT.

      Note: Blocking solution is prepared with 10% donkey serum and 1% Triton X-100 in PBS -/- with 0.1% Tween-20.

    4. Incubate the cells with specific primary antibodies overnight at 4 °C on an orbital shaker (60-80 rpm).

      Note: Dilute primary antibody in blocking solution. Following antibodies are commonly used in our laboratory: NESTIN (Millipore, 1:200), PAX6 (Covance, 1:200), SOX2 (Santa Cruz, 1:100), DACH1 (ProteinTech, 1:100), TUJ-1 (Sigma-Aldrich, 1:1,000), MAP2 (Synaptic system, 1:1,000).

    5. Rinse the cells with PBS -/-, 3 times for 5 min.

    6. Incubate the cells with a specific secondary antibody and Hoechst staining (1:2500) for nucleus visualization for at least 1 h at RT.

      Note: Dilute secondary antibodies 1:300 using blocking solution. Following secondary antibodies are commonly used in our laboratory: Alexa FluorTM 488 donkey anti-rabbit IgG (H+L) (Thermo Fisher Scientific), Alexa FluorTM 647 donkey anti-mouse IgG (H+L) (Thermo Fisher Scientific), donkey anti-goat Cy 3 (Sigma-Aldrich) and donkey anti-guinea pig Cy 3 (Sigma-Aldrich).

    7. Rinse the cells with PBS -/-, 3 times for 5 min.

    8. Invert coverslips and mount them to a glass slide using mounting medium (IMMU-MOUNTTM or similar). After the mounting medium is dry the samples can be analyzed by confocal imaging.


  9. Characterization of NPCs by quantitative real-time reverse transcription PCR (qRT-PCR)

    1. Prepare SYBR Green PCR Master Mix according to the manufacturer’s description.

    2. Pipette the PCR master mix and the cDNA samples into the 384-Well Optical Reaction Plates (Applied Biosystems).

      Note: RNA was extracted from cell pellets using the RNeasy Mini Kit (50) from Qiagen according to the manufacturers protocol. cDNA was generated using the Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase kit from Thermo Fisher Scientific according to the manufacturers protocol. For each target gene, cDNA samples and negative controls should be measured in biological triplicates.

    3. Set the PCR cycle conditions as follows: 1 cycle 50 °C 2 min, 1 cycle 95 °C 10 min, 40 cycles 95 °C 15 s  60 °C 30 s  72 °C 30 s, 1 cycle 72 °C 10 min.

    4. Primers used were:

      For OCT4

      F: GTGGAGGAAGCTGACAACAA and R: ATTCTCCAGGTTGCCTCTCA

      For NANOG

      F: CCTGTGATTTGTGGGCCTG and R: GACAGTCTCCGTGTGAGGCAT

      For SOX2

      F: GTATCAGGAGTTGTCAAGGCAGAG and R: TCCTAGTCTTAAAGAGGCAGCAAAC

      For DNMT3B

      F: GCTCACAGGGCCCGATACTT and R: GCAGTCCTGCAGCTCGAGTTTA

      For DPP4

      F: TGGTGTCAGGTGGTGTGTGG and R: CCAGGCTTGACCAGCATGAA

      For VIM

      F: GGAGCTGCAGGAGCTGAATG and R: GACTTGCCTTGGCCCTTGAG

      For NESTIN

      F: TTCCCTCAGCTTTCAGGAC and R: GAGCAAAGATCCAAGACGC

      For PAX6

      F: GAATTCTGCAGACCCATGC and R TCTCGTAATACCTGCCCAG

      For SOX1

      F: TTGGCATCTAGGTCTTGGCTCA and R: CGGGCGCACTAACTCAGCTT

      For SOX2

      F: GTATCAGGAGTTGTCAAGGCAGAG and R: TCCTAGTCTTAAAGAGGCAGCAAAC

      For GAPDH

      F: CTGGTAAAGTGGATATTGTTGCCAT and R: TGGAATCATATTGGAACATGTAAACC

Data analysis

Fluorescent images were visualized using the Zeiss Axio Imager Z1 confocal microscope in combination with AxioVision V4.6.3.0 software. Images were merged either directly during image acquisition using AxioVision V4.6.3.0 software, or afterwards using Adobe Photoshop CS6 (Adobe, California, USA).

  Gene expression analysis was performed by qRT-PCR using SYBR Green PCR Master Mix and the ViiATM 7 Real-Time PCR System (Applied Biosystems, California, USA). Relative transcript levels of each gene were calculated based on the 2−ΔΔCT method. Data were normalized to the housekeeping gene GAPDH and presented as mean LOG2 ratios in relation to control iPSCs.

Recipes

Media formulation (Table 1 and Table 2):

1. All media should be prepared fresh and used within one week.

2. Do not thaw N-2 Supplement and B-27 Supplement in the water bath. Instead thaw them at RT for 2-4 h or in the fridge (4 °C) over night.

3. All media should be pre-warmed before applying to the cells. Do not keep media at 37 °C for long periods but rather pre-warm them at RT.


Table 1. Basic media formulation

Medium Components Volume Stock conc. Final conc.

Basic Medium 1 (BM1)
KnockOut-DMEM 40 ml
KnockOut-SR 10 ml
Pen/Strep 500 µl 10 mg/ml; 10.000 U/ml 0.1 mg/ml;
100 U/ml
Glutamine 500 µl 200 mM 2 mM
NEAA 500 µl 100×
Pyruvate 500 µl 100 mM 1 mM
MycoZap 100 µl 500×

Basic Medium
sm-
(base for sm+)
DMEM/F12 240 ml 0.5×
Neurobasal 240 ml 0.5×
N-2 Supplement 2.5 ml 100× 0.5×
B-27 Supplement without vitamin A 5 ml 50× 0.5×
Pen/Strep 5 ml 10 mg/ml; 10.000 U/ml 0.1 mg/ml;
100 U/ml
Glutamine 5 ml 200 mM 2 mM
MycoZap 1 ml 500×

Table 2. Culture media formulation
Medium Components Volume Stock conc. Final conc.

Medium 1 (M1); day 0-day 2
BM1 10 ml
CHIR 5 µl 6 mM 3 µM
SB 10 µl 10 mM 10 µM
Dorsomorphin 2 µl 5 mM 1 µM
Purmorphamine 7.69 µl 0.65 mM 500 nM

Medium 2 (M2); day 2-day 4
sm- 10 ml
CHIR 5 µl 6 mM 3 µM
SB 10 µl 10 mM 10 µM
Dorsomorphin 2 µl 5 mM 1 µM
Purmorphamine 7.69 µl 0.65mM 500 nM

M3/sm+(maintenance of NPCs)
sm- 10 ml
CHIR 5 µl 6 mM 3 µM
Purmorphamine 7.69 µl 0.65 mM 500 nM
Vitamin C 7.5 µl 200 mM 150 µM


Acknowledgments

We acknowledge support from the Deutsche Forschungsgemeinschaft (DFG) (PR1527/5-1 to A.P.), Spark-Berlin Institute of Health (BIH Validation Funds to A.P.), National Science Center, Poland (No. 2016/22/M/NZ2/00548; No. 2017/27/B/NZ1/02401 to P.L), the United Mitochondrial Disease Foundation (UMDF to A.P.), and the German Federal Ministry of Education and Research (BMBF) (AZ.031A318 & 031L0211 to A.P.). The original research paper was: Lorenz et al.(2017).

Competing interests

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

Ethics

The study was approved by the ethic committee of the Medical Faculty of Heinrich Heine University (study number 2019/681).

References

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简介

[摘要]药物开发过程中的高流失率要求使用其他基于人的模型系统。但是,在脑部疾病的情况下,不适合对活的神经元细胞进行采样以进行化合物测试。人类诱导的多能干细胞(iPSC )的使用彻底改变了神经元疾病建模和药物发现领域。由于基于iPSC的神经元分化方案(包括三维脑类器官)的发展,现在可以在一个碟子中分子解剖人神经元发育和人脑疾病的发病机理。这些方法可以允许在与疾病相关的细胞环境中解剖患者特异性的治疗功效。但是,对于药物发现方法,需要高度可复制且具有成本效益的细胞模型。在这里,我们描述了一种一步-步骤,用于从人产生健壮和可膨胀的神经祖细胞(NPC)工艺的iPSC 。用此协议生成的NPC是同质的且高度增殖。这些功能使NPC适合开发用于药物发现的高通量化合物筛选。人iPSC衍生的NPC示出了代谢依赖于线粒体活性,因此可也用于研究神经病症,其中线粒体功能受到影响。该协议涵盖了制备,培养和表征人iPSC来源的NPC所需的所有步骤。


图形摘要:


示意性的协议的所述发电机密封的离子人类源自iPSC的的NPC

[背景技术]近年来,目标为中心的药物发现的缺点已经用于寻址的神经系统疾病的方案变得明显,特别是(保罗等人,2010) 。基于靶标的药物发现的挑战导致表型药物发现的复兴。表型筛选需要鉴定可在细胞或生物的生理环境内调节的疾病特异性特征(表型)(Khurana等,2015)。该方法的优点是鉴定出在复杂的生物环境中显示出作用的化合物。同时,表型药物的发现要求存在与疾病相关的强大表型,而该表型可以使用可靠的高通量检测方法来有效地调节。当前的药物开发流程强烈依赖缺乏脑细胞功能特性的源自癌症的永生化细胞系。此外,永生的细胞系大多具有糖酵解作用,比神经元细胞对线粒体损伤的敏感性较低(Bénit等,2016)。因此,永生细胞不是神经疾病和线粒体氧化磷酸化(OXPHOS)功能异常的疾病(例如线粒体疾病和神经退行性疾病)的有效药物发现模型(Cunnane等,2020)。

细胞重编程技术代表了神经元疾病建模和药物发现领域的重要突破。细胞重编程可以转换给定细胞的身份,从而可以从患者特定的体细胞中生成脑细胞。细胞转化可以通过诱导多能干细胞(iPSC )的衍生(Takahashi et al。,2007)或通过直接转化的过程实现,直接转化可以将皮肤细胞直接重编程为有丝分裂后诱导的神经元(iNs )(Vierbuchen et al。等人,2010)。iPSC还可以用于衍生三维(3D)脑器官,可以概括人类大脑的组织结构(Lancaster等,2013)。

为了进行有意义的表型筛选,有必要使用与疾病相关的细胞模型。在重编程来源的细胞类型中,患者特异性神经祖细胞(NPC)似乎特别适合神经疾病的表型药物发现,因为它们具有同质特征,经济高效的培养条件和轻度的增殖状态。实际上,源自iPSC的NPC已经被成功地用于神经系统疾病和神经精神疾病中(Lorenz等人,2017; Readhead等人,2018; Walter等人,2019)。

在神经发生过程中,其特征是代谢从糖酵解转变为OXPHOS (Zheng等人,2016),NPC代表第一种依赖线粒体呼吸的细胞类型(Beckervordersandforth,2017; Lorenz and Prigione,2017)。由于NPC对损害OXPHOS的疾病表现出敏感性,因此它们可以用作有效的细胞模型,以进行神经和线粒体疾病的高通量筛选。因此,来自线粒体疾病患者的iPSC衍生的NPC表现出有意义的表型,可以利用这些表型进行表型化合物筛选(Lorenz等,2017)。总而言之,源自iPSC的NPC可能有助于识别神经系统疾病和线粒体疾病中难以用传统药物发现系统建模的新疗法(Inak等人,2017)。

在这里,我们详细描述了人类iPSC衍生的NPC的生成和维护。可以使用不同的协议来获取这些细胞。一些方案产生具有有限增殖特征的NPC群体(Elkabetz等,2008),而另一些则可能依赖于人LIF的添加,这使得培养细胞非常耗费成本(Li等,2011)。然而,最有用的方法仅依赖于小分子来产生高度增殖的NPC群体(Reinhardt等人,2013)。这些NPC的关键特征是其强大且同质的扩展能力。同时,NPC应该能够有效分化为神经元和神经胶质细胞类型(Reinhardt等,2013 ;Lorenz等,2017 ) 。

我们的方法基于Reinhardt等人的协议。(2013),并发表在Lorenz等人的文章中。(2017)在神经性线粒体疾病背景下的药物发现应用。该协议允许生成NPC,这些NPC在化学定义的条件下经过数次传代仍可保持其特性,并且可以轻易地分化为所需的脑细胞类型。该方案是高度可重复的,并导致同质的细胞群。从人胚胎干细胞,健康对照iPSC和患者特异性iPSC开始,它同样有效(Lorenz等,2017)。

为了发起神经诱导,骨形态发生蛋白(BMP)抑制剂dorsomorphin (DM) ,和转化生长因子β (TGF β )-7号信令抑制剂SB43152(SB)被补充到基本NPC媒体(BM1)。加入嘌吗啡胺(PU)和CHIR99021(CHIR)以刺激声刺猬(SHH)信号传导途径,抑制糖原合酶激酶3(GSK3)并激活经典WNT信号传导(Chambers等,2009; Kim等, 2010)。在诱导培养基中放置第一天后,胚状体(EB)形成神经上皮结构(图1 A )。6天后,将EB轻轻解离并接种在Matrigel包被的板上进行纯化。后4-6通道后神经诱导神经祖形态变得均匀(图1 A) 。NPC应该表达典型的NPC标记,例如NESTIN,PAX6,SOX1,SOX2和DACH1。表达泛神经元标记TUJ1的细胞自发分化始终存在(图1 C - 1 D )。相反,与亲本iPSC品系相比,OCT4,NANOG,DNMT3B和DPPA4等多能性标记应下调。NPC在多次传代(> 30)以及冷冻和解冻后仍保持其多能身份,而细胞形态没有变化。

以下分步协议允许实验人员在4-8周内获得具有高度同质性的人iPSC衍生NPC,并对其进行培养和表征,以用于疾病建模或药物发现应用。

关键字:人的诱导性万能干细胞, 神经前体细胞, 药物发现, 干细胞分化, 神经疾病建模, 线粒体疾病



材料和试剂


以下列表提供了我们在实验室中日常使用的材料和设备的示例。但是,具有类似规格的试剂和设备也可以使用。


蜂星细胞培养的多孔板(6-24孔)(格雷纳生物一个,Ç atalog数小号:657 160 [6-孔; 665 180 [12孔]; 662 160 [24孔] )
康宁60毫米超低附着培养皿(Corning,目录号:3261)
锅铲(TPP ,目录号99010)
血清移液管(格雷纳生物ø NE,目录号小号:76018 0 [ 2毫升]; 606180 [ 5毫升]; 607180 [ 10毫升]; 760107 [ 25毫升] ; 768160 [ 50毫升] )
Bemis TM Curwood Parafilm TM M实验胶片(Fisher Scientific,产品代码HS234526C,目录号:10018130)
Greiner离心管(50 ml和15 ml)(Sigma - Aldrich,制造商:Greiner; 15 ml的目录号:T1818,50 ml的目录号:T2318)
液氮
康宁®基质胶®生长因子降低(GFR)基底膜基质,酚红-自由,自由LDEV-(康宁,C AS号:356231,格式10 ml的,储存在-20 ℃下,并按照供应商从用于制备说明)
CHIR 99021(开曼化工,CAS号:252917-06-9,Ç atalog号码:13122;产品形式:2.5毫克/毫升的在DMSO中;存储在-20 ℃下为6毫米的库存;稳定性:≥2年)
SB431542在溶液(MACS Miltenyi公司,CAS号:130-106-543,Ç atalog号:130-106-543;产品格式:10 mM的在DMSO中;存储在-20 ℃下为10 mM的库存,避光;稳定性:6个月小号)
Dorsomorphin (干细胞; CAS号:866405-64-3,Ç atalog号码:72102;产品格式10毫克固体;存储在-20 ℃下为5 mM的库存在DMSO(热施加)或乙醇;稳定性:12个月小号)
Purmorphamin (MAC小号Miltenyi公司,CAS号:483367-10-8; Ç atalog号:130-104-465的产品形式:5毫克固体;存储在-20℃下在DMSO中的10mM股票,避光)
(+) - L-抗坏血酸钠(维生素C)(默克,Sigma-Aldrich公司,Ç atalog号:A4034;产品格式固体粉末;存储作为在水中200μM库存在-20℃下)
N-2补充(100 × )(热Fisher Scientific公司,Gibco公司,Ç atalog号:17502-048; p RODUCT格式:5 ml的液体;储存在-20 ℃下,避光;稳定性:18个月小号)
B27无维生素A(50 × )(热Fisher Scientific公司,Gibco公司,Ç atalog号:12587010; p RODUCT格式10 ml的液体;储存在-20 ℃下避光)             
的Neurobasal ®培养基(热Fisher Scientific公司,Gibco公司,Ç atalog号:21103049; p RODUCT格式:500 ml的液体;储存在2-8 ℃下,避光;保质期:12个月S)
DMEM / F12 ,HEPES(热Fisher Scientific公司,Gibco公司,Ç atalog号:31330038;产品格式:500 ml的液体;储存于2-8 ℃,避光;保质期:12个月)             
淘汰赛DMEM(热Fisher Scientific公司,Gibco公司,Ç atalog号:10829018;产品格式:500 ml的液体;储存于2-8 ℃下避光
淘汰赛血清替代(热Fisher Scientific公司,Gibco公司,Ç atalog号:10828010;产品格式:500 ml的液体;储存在-20 ℃下避光
StemMACS TM iPS- Brew XF,人类(MACS Miltenyi Biotec ,目录号:130-104-368)
mTeSR TM 1(STEMCELL Technologies,500毫升试剂盒的目录号:85850)
青霉素-链霉素(P / S; 10 ,000 U / ml的青霉素,10 ,000微克/毫升链霉素)(热Fisher Scientific公司,Gibco公司,Ç atalog号15140122;产品格式:100毫升液体;储存在-20 ℃;保质期:12个月)
L-谷氨酰胺(200 mM )(Thermo Fisher Scientific,Gibco ,目录号:25030081;产品格式:100 ml液体;储存在-20 °C ,避光;保质期:24个月)
非必需氨基酸(NEAA,100 × )(Thermo Fisher Scientific,Gibco ,目录号:11140035 ,产品形式:100毫升,储存于2-8 °C;货架寿命:12个月)
丙酮酸钠(100 mM )(Thermo Fisher Scientific,Gibco ,目录号:11360070;产品格式:100 ml ;避光保存在2-8 °C;保质期:12个月)
MycoZap Plus-CL(L onza ,产品目录号VZA-2012;产品格式:20毫升液体;储存在-20 °C ,避光)
的StemPro TM的Accutase TM细胞解离试剂(热Fisher Scientific公司,Gibco公司/的StemPro ,目录号:A1110501;产品格式:100 ml的液体,储存在-20 ℃下,避光)
DPBS(不含钙和镁)(Thermo Fisher Scientific,Gibco ,目录号:14190250;产品格式:500毫升液体;储存于15-30 °C ;货架寿命:36个月)
ROCK抑制剂(RI),Y-27632 ,二盐酸盐(Enzo,CAS号:129830-38-2;目录号:ALX-270-333-M005;产品形式5 mg粉末;储存在-20 °C;可溶于DMSO浓度为25 mg / ml ;稳定性:原液在-20 °C稳定1个月)
UltraPure TM 0.5 M EDTA(Invitrogen,目录号:15575020)
Bambanker (NIPPON Genetics,目录号:BB03)
Shandon TM Immu-Mount TM (Thermo Scientific TM ,目录号:77-86-1;参考号9990402;正式产品:20 ml )
抗巢蛋白抗体,克隆10C2(Millipore公司,目录编号:MAB5326;品牌系列:Chemicon公司® )
抗Pax-6抗体(Covance,目录号:901301)
SOX2抗体(R&D Systems,目录号:AF2018)
DACH1抗体(武汉三鹰,目录号:10914-1-AP)
抗β-管蛋白III(TUJ-1)(Sigma-Aldrich,目录号:T8578)
MAP2(突触系统,目录号:188004)
Hoechst 33342(Hoechst)(Invitrogen TM ,目录号:H3570)
16%多聚甲醛水溶液(PFA)(电子显微镜科学;目录号:50-980-487)
Tween-20(Sigma-Aldrich,目录号:P9416)
Triton TM -X100(Sigma-Aldrich,目录号:T8532)
的Alexa氟TM 488驴抗兔IgG(H + L)二抗(热Fisher Scientific公司,目录号:A-21206)
的Alexa氟TM 647驴抗小鼠IgG(H + L)二抗(热Fisher Scientific公司,目录号:A-31571)
驴抗山羊Cy 3(Sigma - Aldrich,目录号:AP180C)
驴抗豚鼠Cy 3(Sigma - Aldrich,目录号:AP193C)
SYBR TM Green PCR Master Mix(Thermo Fisher Scientific,目录号:4309155)
M- ML V逆转录酶(Invitrogen,货号:28025013)
RNeasy Mini Kit(50)(Qiagen ,目录号:74104)
RNA酶-免费DNA酶组(50)(Qiagen公司,目录号:79254)
寡(dT)12-18引物(Invitrogen公司,CATAL OG号:18418012)
的dNTP混合(10毫摩尔)(Invitrogen公司,CATAL OG号:18427088)
带条形码的MicroAmp TM Optical 348孔反应板(Thermo Fisher Scientific,目录号4438814)
基本介质1(BM1)(请参阅配方,表1)
BM2(sm- )培养基(请参见配方,表1)
M1介质(请参见配方,表2)
M2介质(请参见配方,表2)
M3(sm +)培养基(请参见配方,表2)


设备


离心机(Eppendorf,型号:5810R)
水浴(赛飞世尔科技,FISHERBRAND TM ISOTEMP TM数控恒温水浴:中号奥德尔220)
用于细胞培养的CO 2培养箱(Thermo Fisher Scientific,型号:Heraeus BBD 6220)
用于iPSC培养的缺氧培养箱(Binder GmbH,型号:Binder BCB 160)
qPCR机(Thermo Fisher Scientific,型号:ViiA 7)
共焦显微镜系统(Zeiss,型号:Axio Imager Z1)
冷冻容器(Nalgene,型号:Frosty先生; Corning CoolCell TM )
-80°C冰柜(新不伦瑞克省,型号:Innova 4725)              
层流罩(BDK,Luft und Reinraumtechnik GMBH)
无菌剪刀(例如:锋利的手术剪刀,Sigma - Aldrich,目录号:Z265977)
摇床(洛克菲特,LTF劳工技术公司)


程序


一般来说:


在无菌条件下(例如,层流罩)工作。
在引擎盖下传送之前,请对试剂和消耗品进行消毒。
所有介质应准备新鲜,并在一周之内使用。
不要在水浴中解冻N-2 S补充剂和B-27补充剂。而是将它们在室温(RT,20-22 °C)下解冻2-4小时,或在冰箱(4°C)中过夜解冻。
在将所有介质应用于细胞之前,应预热所有介质。不要长时间将介质保持在37°C下,而应在室温下预热。
在37°C和5%CO 2的受控环境中培养NPC和iPSC 。
可选:我F可体,培养iPSC的低氧条件下(37℃,5%CO 2 ,5%氧气2 ),其更接近地模拟体内条件。
我们在StemMACS TM iPS- Brew XF或mTeSR TM 1上于无饲养层的条件下,在Matrigel包被的6孔板上培养iPSC 。
我们使用0.5 mM EDTA / PBS以1:4 -1:12的比率传代iPSC 。为了增加细胞存活率,我们建议每次分裂后以10 µM的浓度添加ROCK抑制剂(RI)。


NPC生成的分步协议
第0天(星期一):ħ arvesting所述的iPSC的上和转印康宁超低吸附60毫米/ 15mm宽的菜肴。


准备基本介质1(BM1 )(表1)。
准备M1培养基(表2)。
选择一到两口iPSC (约80%汇合)。
除去培养基,并用DPBS冲洗孔两次,以除去死细胞和碎片。
使用Accutase (1 mg / ml)从板上分离细胞。添加0.5 - 1毫升/孔孵育2-5分钟,在37℃,在之间目视检查。
当菌落主要从板中分离时,继续进行。
添加4 - 10毫升PBS或培养基以降低的Accutase活性。
              可选:中号echanically分离细胞用细胞刮刀,如果细胞不能完全分离的Accutase活动。
              将细胞悬浮液转移至15 ml F alcon管(将两个孔合并),在水浴中使细胞沉降至底部(10-15分钟)。
              可选:Ç在120 entrifuge ×克5分钟(潜在地更损坏但在一个更加固体丸粒更快的结果)。
通过移液(例如用5 ml血清移液管)从沉淀细胞中去除尽可能多的培养基。确保不以驱散或大吃细胞。
向细胞中加入5 ml M1,将细胞转移至未经处理的康宁超低附着60毫米/ 15毫米培养皿中(1:1 :1;或者在高汇合的情况下增加X孔)。
              在37°C,5%CO 2下孵育2天。
              与M1一起孵育将形成漂浮在培养基中的胚状体(EBs)。如果他们在第1天附接至康宁超低附件60毫米/15毫米培养皿的底部,其分离由gentl ÿ使用1ml移液管吹吸(切断了用无菌剪刀尖!)。确保不给一丝一毫的浮动日圆。或者,使用细胞刮铲。将培养基保存在孔中,并使用细胞刮刀将EB轻轻刮出整个孔。


第2天(星期三):50%的媒体交换到M2


准备BM2(sm- )培养基(表1)。
准备M2培养基(表2)。
使用无菌剪刀剪掉1毫升移液器吸头的吸头。
转移的EB至15ml ˚F爱尔康使用1ml移液管末端管(切断的尖!)。
可选:如果已连接一些EB,请使用细胞刮铲分离EB。
让EB在37°C的水浴中静置15分钟。
删除50%的M1,然后添加50%的M2 。
可选:- [R EMOVE所有M1,并添加100%M2(但可能更具破坏性的细胞)。


8.转移将EB回到同一康宁ù LTRA-低附着使用1ml移液管末端60毫米/ 15 mm皿(切断的使用无菌剪刀尖端!)。           



第3天(星期四):100%的媒体交换到M2


准备M2培养基(表2)。
使用无菌剪刀剪掉1毫升移液器吸头的吸头。
转移的EB至15ml ˚F爱尔康使用1ml移液管末端管(切断的尖!)。
可选:我˚F必要,分离的EB使用细胞刮刀如前所述。
让EB在37°C的水浴中静置15分钟。
将培养基完全交换为100%的M2培养基(最终体积5-6 ml)。
转移的EB回到同一康宁超低附件使用1ml移液管末端60毫米/ 15 mm皿(切断的使用无菌剪刀尖端!)。


第4天(周五):100%培养基交换至M3(sm +)并制备Matrigel包被的板


准备M3(sm +)培养基(表2)。
使用无菌剪刀剪掉1毫升移液器吸头的吸头。
转移的EB至15ml ˚F爱尔康使用1ml移液管末端管(切断的尖!)。
可选:我˚F必要,分离的EB使用细胞刮刀如前所述。
让EB在37°C的水浴中静置15分钟。
完全交换培养基到100%的M3(sm +)培养基(最终体积5-6 ml)。
转移的EB回到同一康宁超低附件使用1ml移液管末端60毫米/ 15 mm皿(切断的使用无菌剪刀尖端!)。
为第7天的准备工作涂上6孔板的Matrigel (步骤另行说明)。


第7天(星期一):将细胞转移至基质胶包被的板中


准备M3 (sm +)培养基(表2)。
将EBs从康宁超低附件60 mm / 15 mm皿中转移到15 ml F alcon管中。使用1个毫升尖端(切断的尖!)。
我˚F必要,分离神经管样使用细胞刮刀作为EB中描述的地层。


              让EB在37°C(10-15分钟)的水浴中沉淀下来。
              删除旧媒体。
添加新的sm +介质。
              使用普通的1 ml吸头将5-10次移液以切碎细胞。
              将整个悬浮液转移到Matrigel涂层的6孔板中,根据EB的数量平均分配到2-X孔中。
最多填充2 ml sm +每6孔一次。
              加入ROCK抑制剂(RI)(终浓度10 µM)。
注意:为增加细胞存活率,我们建议在前1-2次分裂中将RI添加到细胞中。之后,不需要RI!


检查第二天,类似NPC的细胞应该已经可见(请参见图1A)–右图,以及图1E)。
尝试将细胞保留在第一个孔中1周。
              如有必要,请按照维护协议中的说明拆分电池。
注:筹备应该出现4-均匀后5代,表达巢蛋白,PAX6,和SOX2。NPC通常还表达未成熟的泛神经标记TUJ1。可以发生自发分化,并且某些细胞可以表达神经元标记MAP2。细胞不一定需要在6孔板中培养。




图1.人类iPSC衍生的NPC的生成和特征化。A.代表性图像的神经球,神经上皮细胞和神经祖细胞(NPC)。B.基质胶涂层良好的图像。比例尺:200 µm。C.从对照iPSC品系XM001 (Wang等人,2018)获得的NPC的免疫细胞化学染色后,对神经祖细胞标记物NESTIN,DACH1,PAX6和SOX2进行了染色。可以存在表达TUJ1或MAP2的细胞的自发分化。比例尺:100µm。D. Q的定量的实时逆转录PCR(定量RT -PCR)神经祖细胞标记SOX1的,巢蛋白,PAX6,和SOX2,和多能性相关标记OCT4,NANOG,DNMT3B,和DPPA4。转录水平在的NPC用GAPDH标准化housekeep ER基因(平均值= SD +/-; n = 3个的技术重复)并且相对于报告到所述转录的各控制iPSC系XM001的水平。E.来自第3代(p3)和第7代(p7)的神经祖细胞(NPC)的代表性图像,显示了形态向同质NPC培养的变化。比例尺:100 µm。


基质胶制备
在+4°C的冰上解冻冷冻的Matrigel (储存在-20°C)过夜(开)。
如批次数据表中所述,检查基质胶的蛋白质浓度(原液浓度)。
计算:将特定体积(= x)的冷KnockOut -DMEM添加到小瓶中10 ml解冻的Matrigel中,以将其稀释至5 mg / ml最终浓度。小瓶的库存浓度(= y)应写在批次数据表上。使用以下公式计算所需的体积:x = y * 2-10 ml。
在引擎盖下和冰上执行下一步!
准备一个预冷的50 ml F alcon管,其中装有一定体积的冷KnockOut -DMEM。
在标有1 M的冰上准备预冷的15 ml F alcon管。
快速将Matrigel转移至KnockOut -DMEM并仔细混合。
在每个预冷的15 ml F alcon管中分装1 ml稀释的Matrigel 。
将等分试样立即储存在-20°C下。
6/12孔板中的终浓度:200 µg / ml。


基质胶涂层板
将30 ml冷的KnockOut -DMEM转移到50 ml F alcon管中。
在冷的KnockOut -DMEM中按1:30的比例稀释1毫升等分试样。
重悬1毫升基质胶等份(15毫升˚F爱尔康管,-20℃)用5ml 30毫升冷淘汰赛-DMEM在15毫升˚F爱尔康管直到沉淀物解决了!(它中号AY需要一段时间)。
如C 2中所述,将1 ml Matrigel等分试样重新悬浮在30 ml冷KnockOut -DMEM中足以用于4个板。
6孔:1.25毫升。
12孔:500 µl 。
24孔:300 µl 。
              将Matrigel -medium添加到孔中(请参见第4点)。
              聚合:
存放在4 °C的冰箱中(优先!)。
在37°C下2 h是可能的。
              使用前,请检查相衬中的聚合反应!细胞外基质的均匀分布形成应该是可见的(图1B)。涂层板可保存1-2周(4°C)。为了存储,用Parafilm密封板。
              使用前,请在室温或37°C下预热板。


              维护人大文化
媒体需要被改变每2次一天。
应用于细胞之前,应预热培养基。不要将介质长时间保持在37°C下,而应在室温下预热。
通常,根据细胞密度,每隔4-5天使用Accutase将细胞以1:3至1:10的比例分裂。


分裂NPC
1.吸出生长培养基。           

2.加入500微升的Accutase和孵化在室温下2-5分钟或37℃,如果细胞不容易脱离。           

3.稀的Accutase通过加入2毫升活性SM - (无任何补充)或PBS。           

4.通过移液轻轻地分离细胞。           

5.将细胞转移到15 ml F alcon管中,在室温以200 × g离心3分钟。           

6.同时,准备Matrigel涂层板以铺板细胞。           

吸基质胶-mix。
加入1 ml sm +,并保存在培养箱中直至进一步使用。
用2 ml DPBS填充空白孔。
注意:Matrigel涂层不应变干。


7.吸出上清液。           

8.将沉淀重悬于sm +培养基中。           

9.将细胞分配到Matrigel包被的6孔板中。           

10.装满2 ml sm +的介质。           

注意:为增加细胞存活率,我们建议在前1-2次分裂中向细胞中添加RI(10 µM)。之后,不需要RI!


冻结NPC
1.按照拆分步骤分离NPC。           

2.重悬的NPC于0.5 ml的的SM + 。加入冷冻培养基(2 × sm +冷冻:sm + ,1:1)。           

注10毫升的2 × SM +冻结通过混合2制备毫升的SM + 6毫升的淘汰赛-SR,和2毫升DMSO中。


3.或者,根据制造商的协议使用商业冷冻介质。           

注意:例如F ,例如Bambanker (Nippon Genetics,目录号:BB03)。


4.转让再悬浮的NPC到冷冻管并放置小瓶在-80 ℃的低温-freezing容器。           

注意:请使用诸如Frosty TM (Nalgene)先生之类的冷冻容器冷冻细胞,以使其以-1 °C /分钟的速度缓慢冷冻。


5.将NPC存放在-80 °C或液氮罐中,以长期存储(1-3年)。           



解冻NPC
1.在37 °C的预热水浴中快速但轻轻地解冻含有NPC的冷冻管。           

2.转移的NPC到15 ml的˚F爱尔康管组用1 ml的的SM +和高达10 ml的预温热的PBS中。           

3.将细胞以200 × g离心3分钟。           

4.除去上清液,将NPC重悬于sm +培养基中。           

5.将NPC接种在先前准备的Matrigel包被的1:1平板上(请参阅使用Matrigel包被的平板)。注:一解冻,以支持细胞存活后DD 10μMRI。           



              NPC的免疫荧光表征
经过4-6次传代后,将NPC接种在含有12 mm盖玻片的Matrigel包被的24孔板上。当达到约80%的汇合度时,用PBS冲洗NPC,并在室温下用4%多聚甲醛(PFA)/ PBS溶液固定细胞15分钟。
除去PFA,并用不含钙/镁的PBS(PBS-/-)冲洗NPC 3次,每次5分钟。
在室温下将封闭溶液添加到细胞中1小时。
注意:封闭溶液是用10%驴血清和1%Triton X-100的PBS-/-和0.1%Tween - 20制备的。

孵育具有特定初级抗体的细胞过夜,在4℃在定轨振荡器(60 - 80转)。
注意:在封闭溶液中稀释一抗。本实验室常用的抗体如下:NESTIN(Millipore,1:200),PAX6(Covance,1:200),SOX2(Santa Cruz,1:100),DACH1(ProteinTech ,1:100),TUJ-1( Sigma-Aldrich公司,1:1 ,000),MAP2(突触系统,1:1 ,000)。


              用PBS-/-漂洗细胞3次,每次5分钟。
              用特定的二抗和Hoechst染色(1:2500)将细胞孵育以在RT进行细胞核可视化至少1 h 。
注意:使用封闭液稀释二抗1:300。我们的实验室通常使用以下二抗:Alexa Fluor TM 488驴抗兔IgG(H + L)(Thermo Fisher Scientific),Alexa Fluor TM 647驴抗小鼠IgG(H + L)(Thermo Fisher Scientific),驴抗山羊Cy 3(Sigma-Aldrich)和驴抗豚鼠Cy 3(Sigma-Aldrich)。


              用PBS-/-漂洗细胞3次,每次5分钟。
              倒置盖玻片,并使用固定介质(IMMU-MOUNT TM或类似介质)将其固定在载玻片上。固定介质干燥后,可以通过共聚焦成像分析样品。


通过定量实时逆转录PCR(qRT -PCR )鉴定NPC
根据制造商的说明准备SYBR Green PCR Master Mix。
将PCR主混合物和cDNA样品移液到384孔光学反应板(Applied Biosystems)中。
注意:根据制造商的协议,使用Qiagen的RNeasy Mini Kit(50)从细胞沉淀中提取RNA 。根据生产商协议,使用Thermo Fisher Scientific的莫洛尼鼠白血病病毒(M- ML V)反转录酶试剂盒生成cDNA 。对于每个靶基因,应一式三份测量cDNA样品和阴性对照。


如下设置PCR循环条件:1个循环50°C 2分钟,1个循环95°C 10分钟,40个循环95°C 15 s60 °C 30 s72 °C 30 s,1个循环72°C 10分钟
使用的引物是:
F或OCT4


F:GTGGAGGAAGCTGACAACAA和R:ATTCTCCAGGTTGCCTCTCA


F或NANOG


F:CCTGTGATTTGTGGGCCTG和R:GACAGTCTCCGTGTGAGGCAT


F或SOX2


F:GTATCAGGAGTTGTCAAGGCAGAG和R:TCCTAGTCTTAAAGAGGCAGCAAAC


F或DNMT3B


F:GCTCACAGGGCCCGATACTT和R:GCAGTCCTGCAGCTCGAGTTTA


F或DPP4


F:TGGTGTCAGGTGGTGTGTGTGG和R:CCAGGCTTGACCAGCATGAA


F或VIM


F:GGAGCTGCAGGAGCTGAATG和R:GACTTGCCTTGGCCCTTGAG


F或NESTIN


F:TTCCCTCAGCTTTCAGGAC和R:GAGCAAAGATCCAAGACGC


F或PAX6


F:GAATTCTGCAGACCCATGC和R TCTCGTAATACCTGCCCAG


F或SOX1


F:TTGGCATCTAGGTCTTGGCTCA和R:CGGGCGCACTAACTCAGCTT


F或SOX2


F:GTATCAGGAGTTGTCAAGGCAGAG和R:TCCTAGTCTTAAAGAGGCAGCAAAC


F或GAPDH


F:CTGGTAAAGTGGATATTGTTGCCAT和R:TGGAATCATATTGGAACATGTAAACC


数据分析


使用Zeiss Axio Imager Z1共焦显微镜结合AxioVision V4.6.3.0软件可以可视化荧光图像。可以在使用AxioVision V4.6.3.0软件进行图像采集期间直接合并图像,或者之后使用Adobe Photoshop CS6(美国加利福尼亚州的Adobe)合并图像。


使用SYBR Green PCR Master Mix和ViiA TM 7 Real-Time PCR System(Applied Biosystems,California,USA )通过qRT -PCR进行基因表达分析。再各基因的lative转录物水平来计算基于所述2 - ΔΔCT方法。将数据标准化为管家基因GAPDH,并表示为与对照iPSC有关的平均LOG2比。


菜谱


媒体FORMUL通货膨胀(表1和表2) :


1.所有介质应准备新鲜,并在一周内使用。           

2.请勿在水浴中解冻N - 2补充剂和B - 27补充剂。而是将它们在室温下融化2-4小时,或在冰箱(4 °C)中过夜解冻。           

3.在将所有介质应用于细胞之前,应预热所有介质。不要将介质长时间保持在37 °C下,而应在室温下对其进行预热。           



表1.基本培养基配方
 
表2.培养基配方


致谢


我们承认,从支持的德意志研究联合会(DFG)(PR1527 / 5-1 AP),健康(BIH验证资金AP),国家科学中心的火花柏林研究所,波兰(第22分之2016/ M / NZ 2 / 00548 ;第二十七分之二千零十七/ B / NZ1 / 02401至PL),美国线粒体疾病基金会(UMDF到AP),以及对教育的德国联邦与研究部(BMBF)(AZ.031A318 &031L0211到AP)。最初的研究论文是:Lorenz等。(2017年)。


利益争夺


作者宣称没有相互竞争的金融和非金融相互竞争的利益。


伦理


这项研究得到了海因里希·海涅大学医学院的伦理委员会的批准(研究编号2019/681)。


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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zink, A., Lisowski, P. and Prigione, A. (2021). Generation of Human iPSC-derived Neural Progenitor Cells (NPCs) as Drug Discovery Model for Neurological and Mitochondrial Disorders. Bio-protocol 11(5): e3939. DOI: 10.21769/BioProtoc.3939.
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Pashma Nawaz
University of Central Punjab
what is M3 (sm+)? and what stands for sm+ ?
2021/5/3 9:03:45 回复
hadia mujahid
hadia mujahid

this is a media used for culturing the neural cells. it could be used for freezing, moreover you can look for the functions through the composition provided in this article.

2021/5/8 22:11:24 回复


Annika Zink
Heinrich Heine University Düsseldorf

Dear Pashma,

hadia mujahid is right. M3, also called sm+, is the final culturing media for the NPCs. Once the generation of the NPCs is completed, cells are kept in sm+ throughout the culture. You can find the media composition in Tab. 1 (basic media) and Tab. 2.

2021/5/10 1:15:28 回复