搜索

Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24+iNKT Cells
诱导多能干细胞高效生成功能性人NKT细胞 - 人Vα24+iNKT细胞的重新编程   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24+iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8+ cytotoxic T lymphocytes to eliminate MHC- and MHC+ tumor cells, respectively. Here we describe a robust protocol to reprogram human Vα24+iNKT cells into iPSC, and then to re-differentiate them into Vα24+iNKT cells (iPS-Vα24+iNKT). We further provide a protocol to measure the activity of iPS-Vα24+iNKT cells.

Keywords: Induced pluripotent stem cell(诱导多能干细胞), iPSC(iPSC细胞), Vα24+invariant natural killer T cell(Vα24+恒定自然杀伤T细胞), Vα24+iNKT(Vα24+iNKT), Anti-tumor activity(抗肿瘤活性), IFN-γ production(IFN-γ生成), Tumor immunotherapy(肿瘤免疫治疗)

Background

It was previously reported that clinical trials of Vα24+iNKT cell cancer immunotherapy targeting advanced non-small cell lung cancer (NSCLC) and head and neck cancer showed efficacy and were well-tolerated (Motohashi et al., 2009; Yamasaki et al., 2011). However, it has been known that the cell yield from ex vivo expansion of Vα24+iNKT cells from peripheral blood mononuclear cells (PBMCs) is often low (Motohashi et al., 2006). Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) using Yamanaka factors (Oct4, Sox2, Klf4 and c-Myc) has contributed greatly to the goals of regenerative medicine. The technology has recently been used to regenerate tumor-specific cytotoxic T lymphocytes and murine invariant natural killer T (iNKT) cells from iPSCs, thus opening up a new approach for cancer immunotherapy. Here, we have established a robust protocol to reprogram human Vα24+iNKT cells. We showed that iPS-derived Vα24+iNKT cells acted as cellular adjuvants and exerted anti-tumor activity, further extending their therapeutic potential. The complementation of other therapies with functionally validated Vα24+iNKT cells derived from iPSC could be valuable for cancer patients.

Materials and Reagents

  1. Pipette tips
  2. 24-well plate (Corning, Falcon®, catalog number: 353047 )
  3. 15 ml centrifuge tube (Corning, Falcon®, catalog number: 352196 )
  4. G27 needle (Terumo, catalog number: NN-2719S )
  5. 12-well plate (Corning, Falcon®, catalog number: 353043 )
  6. Cell strainer (size: 100 μm) (Greiner Bio One International, catalog number: 542000 )
  7. 10 cm dish (Corning, Falcon®, catalog number: 353003 )
  8. 6-well plate (Corning, Falcon®, catalog number: 353046 )
  9. 6 cm dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150288 )
  10. 96-well round-bottomed plate (Corning, Falcon®, catalog number: 351190 )
  11. 96-well round-bottomed plate for Procedures C and D (Corning, Falcon®, catalog number: 353077 )
  12. StemPro EZ passage tool (Thermo Fisher Scientific, GibcoTM, catalog number: 23181010 )
  13. Cell scraper (IWAKI, catalog number: 9000-220 )
  14. 0.22 μm bottle top filter (EMD Millipore, catalog number: SCGVU05RE )
  15. Human Vα24+iNKT cells
  16. K562 (ATCC, catalog number: CCL-243 )
  17. OP9 feeder cells (obtained from RIKEN BRC Cell No. RCB2926)
  18. SeV-KOS and SeV-c-MYC from CytoTune-iPS 2.0 (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: DV-0305-3A )
  19. OP9DLL1 feeder cells (obtained from RIKEN BRC Cell No. RCB2927)
  20. Peripheral blood mononuclear cell (PBMC)
  21. Cord blood mononuclear cell (CBMC)
  22. Trypan blue stain 0.4% (Thermo Fisher Scientific, InvitrogenTM, catalog number: T10282 )
  23. SeV vector with a SV40 large T antigen (T) insertion (SeV-SV40) (ID Pharma, custom order)
  24. Mitomycin-C (Sigma-Aldrich, catalog number: M4287 )
  25. iMatrix-511 (Nippi, catalog number: 892 012 )
  26. StemFit AK02N (ReproCELL, catalog number: RCAK02N )
  27. Freezing medium for human ES/iPS cells (DAP213) (ReproCELL, catalog number: RCHEFM001 )
  28. Stem-cell banker GMP grade (Nippon Zenyaku Kogyo, Zenoaq, catalog number: CB045 )
  29. 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  30. TripLE select (Thermo Fisher Scientific, GibcoTM, catalog number: 12563011 )
  31. Y-27632, inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK) (Wako Pure Chemical Industries, catalog number: 253-00513 )
  32. Hanks’ balanced salt solution without phenol red (HBSS+) (Wako Pure Chemical Industries, catalog number: 084-08965 )
  33. Collagenase (Wako Pure Chemical Industries, catalog number: 036-23141 )
  34. Dulbecco’s phosphate buffered saline (D-PBS) (Wako Pure Chemical Industries, catalog number: 045-29795 )
  35. Stem cell factor (SCF) (R&D Systems, catalog number: 255-SC-050 )
  36. Recombinant human interleukin-7 (IL-7) (PeproTech, catalog number: 200-07 )
  37. Fms-related tyrosine kinase 3 (Flt-3) ligand (R&D Systems, catalog number: 3008-FK-025 )
  38. Recombinant human IL-15 (PeproTech, catalog number: 200-15 )
  39. 7-AAD staining solution (BD, BD Biosciences, catalog number: 559925 )
  40. V450 mouse anti-human CD3 clone UCHT1 (BD, BD Biosciences, catalog number: 560365 )
  41. Anti-TCR Vβ11-APC (Beckman Coulter, catalog number: A66905 )
  42. Anti-TCR Vα24-PE (Beckman Coulter, catalog number: IM2283 )
  43. Lactate dehydrogenase (LDH) cytotoxicity detection kit (Takara Bio, catalog number: MK401 )
  44. BD OptELISA human IFN-γ enzyme-linked immunosorbent assay (ELISA) set (BD, BD Biosciences, catalog number: 555142 )
  45. BD OptELISA Human IL-4 ELISA Set (BD, BD Biosciences, catalog number: 555194 )
  46. Triton X-100 (Sigma-Aldrich, catalog number: T8787-100ML )
  47. RPMI1640 (Sigma-Aldrich, catalog number: R8758 )
  48. Fetal bovine serum (Sigma-Aldrich, catalog number: 172012-500ML )
  49. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  50. Hanks’ balanced salt solution without phenol red, without calcium, without magnesium (HBSS-) (Wako Pure Chemical Industries, catalog number: 085-09355 )
  51. 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H3375-250G )
  52. Human IL-2 (Shionogi, catalog number: 4987058697900 )
  53. Primate ES Cell Medium (ReproCELL, catalog number: RCHEMD001 )
  54. Fibroblast growth factor basic (bFGF) (Wako Pure Chemical Industries, catalog number: 062-06661 )
  55. MEMα (Thermo Fisher Scientific, GibcoTM, catalog number: 11900-073 )
  56. Sodium hydrogen carbonate (NaHCO3) (Nacalai Tesque, catalog number: 31213-15 )
  57. Recombinant mouse GM-CSF (PeproTech, catalog number: 315-03 )
  58. α-galactosylceramide (α-GalCer) (Funakoshi, catalog number: KRN7000 )
  59. Lipopolysaccharide (LPS) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 00-4976-93 )
  60. Dulbecco’s modified Eagle’s medium (D-MEM) (Wako Pure Chemical Industries, catalog number: 044-29765 )
  61. R10 medium (see Recipes)
  62. NKT Media (see Recipes)
  63. Human pluripotent stem cell medium (see Recipes)
  64. OP9 medium (see Recipes)
  65. DC/Gal (see Recipes)
  66. MEF medium (see Recipes)

Equipment

  1. CO2 incubator (35 °C, 37 °C, 38 °C) (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  2. Inverted microscope (Leica Microsystems, model: DM IL LED )
  3. Stereomicroscope (Leica, model: MZ75 )
  4. P200-pipette (Gilson, catalog number: FA10005P )
  5. Flow cytometers (BD, BD Biosciences, model: FACSCanto II )
  6. Centrifuge (KUBOTA, model: 2800 )
  7. Cell counter (Thermo Fisher Scientific, InvitrogenTM, catalog number: C10227 )
  8. Controlled rate freezer (Grant Instruments, model: EF600M )
  9. OptEIA (BD, BD Biosciences, San Jose, CA)
  10. Microplate reader (Molecular Devices, model: SpectraMax 190 )

Software

  1. FlowJo software (FlowJo, LLC)

Procedure

  1. iPSC induction from human Vα24+iNKT cells
    Day -3
    1. Human Vα24+iNKT cells are seeded at 1 x 106 cells/ml in NKT medium into a 24-well plate (1 x 106 cells/well) and stimulated with 1 x 105 DC/Gal as previously described (Shimizu et al., 2006; Yamada et al., 2016).

    Day 0

    1. Collect stimulated human Vα24+iNKT cells (Figure 1) into a 15 ml centrifuge tube by pipetting and count the viable cell number by trypan blue staining. Resuspend the cells in 1 ml R10 medium without antibiotics and seed at 1 x 106 cells/ml into a 24-well plate.


      Figure 1. Representative microscopic image for the stimulated human Va24+iNKT cells. Human Va24+iNKT cells were stimulated with DC/Gal for several days.

    2. Add SeV (MOI: KOS, c-MYC, SV40 = 30, 3, 30) into the 24-well plate with the human Vα24+iNKT cells and mix gently.
    3. Incubate at 35 °C in an incubator with 5% CO2.

    Day 1

    1. Replace the medium with 1 ml complete R10 medium.
    2. Incubate at 35 °C in an incubator with 5% CO2.

    Day 6

    1. Collect the cells and count the viable cell number by trypan blue staining.
    2. Resuspend the cells with complete R10 medium and seed at 3 x 105 cells on mitomycin C-treated MEFs or iMatrix-511 coated 6 cm dish.
    3. Incubate at 35 °C in an incubator with 5% CO2.

    Day 7

    1. Replace the medium with human pluripotent stem cell medium (on mitomycin C-treated MEF) or StemFit AK02N (on iMatrix-511 coated).
    2. Incubate at 35 °C in an incubator with 5% CO2.
    3. Replace the medium every other day until human ESC-like colonies (Figure 2) appear (Takahashi and Yamanaka, 2006; Takahashi et al., 2007).


      Figure 2. Representative ESC-like colony shape. A diameter of ESC-like colony is about 1 to 1.5 mm.

    Day 21-28

    1. Divide each human ESC-like colony (the diameter will be about 1 to 1.5 mm) into two clumps using a G27 needle under a stereomicroscope. Collect one clump (about 500 cells) into a tube by pipetting and subject to PCR for human Vα24+iNKT cell-specific TRAV (Vα24-Jα18) recombination detection (Yamada et al., 2016).
    2. Transfer the other clump onto mitomycin C-treated MEFs or iMatrix-511 coated 12 well-plate for cell proliferation after dissociation using gentle pipetting.
    3. Incubate at 37 °C in an incubator with 5% CO2.
    4. Maintain the cells using a StemPro EZ passage tool (on mitomycin C-treated MEF) or TripLE select (on iMatrix-511 coated) plates as previously described (Vizcardo et al., 2013; Nakagawa et al., 2014). Store the cells using freezing medium for human ES/iPS cells (for cells on mitomycin C-treated MEF) or Stem cell banker (for cells on iMatrix-511 coated plates).

  2. Differentiation of Vα24+iNKT cells from Vα24+iNKT-iPSC (iPS-NKT) (Figure 3)


    Figure 3. Schematic representation of iPS-NKT differentiation from NKT-iPSCs. NKT derived iPSCs (NKT-iPSC) were separated into small clumps and seeded on OP9 feeder cells at day 0. On Day 13, cells were transferred to co-culture with OP9DLL1 feeder cells. On Day 33, cells were collected for feeder-free culture another 10 days.

    Day -7
    1. Dissociate OP9 cells (around 90% confluent) with 0.25% trypsin-EDTA.
    2. Collect dissociated OP9 cells by passing through a cell strainer (size: 100 μm), and seed at 6 x 105 cells in a 10 cm dish in 10 ml OP9 medium.
    3. Incubate at 37 °C in an incubator with 5% CO2.

    Day -5

    1. Add 10 ml OP9 medium.
    2. Incubate at 37 °C in an incubator with 5% CO2.

    Day 0

    1. Replace OP9 medium with fresh OP9 medium supplemented with 10 μM Y-27632.
    2. Dissociate human iPS cells (80% confluent) into clumps using a StemPro EZ passage tool under a stereomicroscope (Figure 4A).
    3. Collect about 100 clumps (Figure 4B) using a P200-pipette under a stereomicroscope and plate them onto an OP9-seeded 10 cm dish.
    4. Incubate at 37 °C in an incubator with 5% CO2.


      Figure 4. Representative NKT-iPSC morphology for iPS-NKT differentiation. NKT-iPSC were divided into a few clumps using EZ-passage tool and pipetting. A. Colony shape of NKT-iPSC after cut using StemPro EZ-passage tool; B. Small NKT-iPSC clumps were prepared and collected by pipetting and passage into OP9 feeder cells.

    Day 1

    1. Replace the medium with 20 ml OP9 medium.
    2. Incubate at 37 °C in an incubator with 5% CO2.

    Day 9

    1. Discard half of the supernatant (media).
    2. Add 10 ml OP9 medium.
    3. Incubate at 37 °C in an incubator with 5% CO2.

    Day 12

    1. Dissociate OP9DLL1 cells (around 90% confluent) with 0.25% trypsin-EDTA.
    2. Collect dissociated OP9DLL1 cells by passing through a cell strainer (size: 100 μm), and seed at 2 x 106 cells on a 10 cm dish in 10 ml OP9 medium.
    3. Incubate at 37 °C in an incubator with 5% CO2.

    Day 13

    1. Discard medium and rinse the cells with 10 ml HBSS+.
    2. Add 6 ml collagenase (20 mg/ml).
    3. Incubate at 37 °C, 45 min in a CO2 incubator.
    4. Discard collagenase and rinse the cells with 10 ml D-PBS.
    5. Add 6 ml 0.25% trypsin-EDTA.
    6. Incubate at 37 °C, 30 min in a CO2 incubator.
    7. Add 4 ml OP9 medium and collect dissociated cells passing through a cell strainer (size: 100 μm).
    8. Resuspend the collected cells with 10 ml OP9 medium supplemented with 10 ng/ml of SCF, 5 ng/ml human IL-7 and 5 ng/ml Flt3-ligand and plate on an OP9DLL1 cell-seeded 10 cm dish (prepared at Day 12).

    Day 15

    1. Dissociate OP9DLL1 cells (around 90% confluent) with 0.25% trypsin-EDTA.
    2. Collect the dissociated OP9DLL1 cells by passing through a cell strainer (size: 100 μm), and seed at 2 x 106 cells in a 10 cm dish in 10 ml OP9 medium.
    3. Incubate at 37 °C in an incubator with 5% CO2.

    Day 16

    1. Preparation of mitomycin-C treated OP9DLL1 cells
      1. Replace medium of OP9DLL1 cells with 6 ml OP9 medium supplemented with 1 μg/ml of mitomycin-C.
      2. Incubate for 2 h, at 37 °C, 5% CO2 in a CO2 incubator.
      3. Rinse mitomycin-C treated OP9DLL1 cells with D-PBS twice, and add 6 ml OP9 medium.
      4. Incubate at 37 °C in an incubator with 5% CO2.
    2. Passage of differentiating iPS-NKT cells.
      1. Collect the cells (prepared at Day 13) by pipetting and dissociate by passing through a cell strainer (size: 100 μm).
      2. Resuspend collected cells with 10 ml OP9 medium supplemented with10 ng/ml SCF, 5 ng/ml human IL-7 and 5 ng/ml Flt3-ligand and plate onto a mitomycin-C treated OP9DLL1 cell-seeded 10 cm dish.
      3. Incubate at 37 °C in an incubator with 5% CO2.

    Day 24

    1. Dissociate OP9DLL1 cells (around 90% confluent) with 0.25% trypsin-EDTA.
    2. Collect the dissociated cells by passing through a cell strainer (size: 100 μm), and seed at 2 x 106 cells on 10 cm dish in 10 ml OP9 medium.
    3. Incubate at 37 °C in an incubator with 5% CO2.

    Day 25

    1. Preparation of mitomycin-C treated OP9DLL1 cells
      1. Replace medium of OP9DLL1 cells with 6 ml OP9 medium supplemented with 1 μg/ml of mitomycin-C.
      2. Incubate for 2 h at 37 °C, 5% CO2 in a CO2 incubator.
      3. Rinse mitomycin-C treated OP9DLL1 cells with D-PBS twice, and add 6 ml OP9 medium.
      4. Incubate at 37 °C in an incubator with 5% CO2.
    2. Passage of differentiating iPS-NKT cells
      1. Collect the cells (prepared at Day 16) by pipetting and dissociate by passing through a cell strainer (size: 100 μm).
      2. Resuspend collected cells with 10 ml OP9 medium supplemented with 10 ng/ml SCF, 5 ng/ml human IL-7 and 5 ng/ml Flt3-ligand and plate onto a mitomycin-C treated OP9DLL1 cell-seeded 10 cm dish.
      3. Incubate at 37 °C in an incubator with 5% CO2.

    Day 30

    1. Add 5 ml OP9 medium supplemented with10 ng/ml SCF, 5 ng/ml human IL-7 and 5 ng/ml Flt3-ligand (total 15 ml).
    2. Incubate at 37 °C in an incubator with 5% CO2.

    Day 33

    1. Collect the cells by passing through a cell strainer (size: 100 μm).
    2. Count the viable cell number by trypan blue staining and use 1 x 105 cells to analyze iPS-NKT marker (CD3, TRAV24 and TRBV11) expression by flow cytometry.
    3. Seed the iPS-NKT cells at 5 x 105 cells/well into a 6 well-plate in 2 ml OP9 medium supplemented with 5 ng/ml human IL-7 and 10 ng/ml human IL-15.
    4. Incubate at 37 °C in an incubator with 5% CO2.

    Day 35, 37, 39 and 41

    1. Add 1 ml OP9 medium supplemented with 5 ng/ml human IL-7 and 10 ng/ml human IL-15.
    2. Incubate at 37 °C in an incubator with 5% CO2.

    Day 43

    1. Collect iPS-NKT cells (Figure 5) by pipetting and passing through a cell strainer (size: 100 μm).


      Figure 5. Representative proliferating iPS-NKT cells. Activated T cell-like morphology is observed.

    2. Count the viable iPS-NKT cell number by trypan blue staining and use 1 x 105 cells analyze iPS-NKT marker (CD3, TRAV24 and TRBV11) expression by flow cytometry (Figure 6).
    3. iPS-NKT cells can be used at this point for functional assays such as anti-tumor cell line assay or cytokine production assay.


      Figure 6. Representative iPS-NKT cells analysis using flow cytometer. A. Lymphocyte fraction was gated using FSC and SSC as indicated by ellipse. B. Live cells were selected by 7-AAD negative fraction. C. CD3 positive fraction was selected and D. TRAV24 and TRBV11 positive cells were indicated as square.

  1. Cytokine production assay
    1. iPS-NKT cells are seeded on a 96-well round-bottomed plate (1 x 105 cells/well/200 μl).
    2. Cells are co-cultured with or without murine DC/Gal (1 x 105 cells/well) for 24 h at 37 °C at 37 °C, 5% CO2 in a CO2 incubator. The amount of IFN-γ and IL-4 in the culture supernatants is measured with OptEIA.

  2. Anti-tumor cell line assay
    1. Effector cells are cultured with 1 x 104 of target cells at an E/T ratio of 5 or 10 in a 96-well round-bottomed plate.
    2. Six hours after incubation at 37 °C, 5% CO2 in a CO2 incubator, 100 μl of supernatant is collected and LDH activity is measured with an LDH cytotoxicity detection kit in a 96-well flat-bottomed plate.
      1. As a positive control, target cells lysed with 2% of Triton X-100 are used.
      2. As background controls, effector cells or target cells alone are used.
    3. In anti-tumor analysis using the LDH kit, O.D. at 490 nm and 600 nm is measured with a SpectraMax 190. O.D. at 490 nm is subtracted from the O.D. at 600 nm as noise. Killing activity is calculated with following formula: Killing activity = [{(O.D. of ‘effector + target’ - O.D. of medium) - (O.D. of target alone - O.D. of medium)} - (O.D. of effector alone - O.D. of medium)]/(O.D. of positive control - O.D. of target alone) x 100 (%).

Data analysis

We performed reprogramming of Vα24+iNKT cells into iPSCs from 4 donors and used karyotype stability for iPSC line selection. For assessing the phenotypes, we use FlowJo software (FlowJo, LLC) to process flow cytometry data of iPS-NKT cells obtained using a FACSCanto II. In the cytokine producing assay, we performed 4 to 7 independent experiments with 2 to 3-well replicates and pooled results were statistically analyzed with the Student’s t-test. In the anti-tumor cell line assay, we performed 8 independent experiments with triplicate replicates and statistically analyzed the data with the Student’s t-test. We describe details of replicates in the cytokine production assay in the Figure 2A legend in our original paper (Yamada et al., 2016) and those of the anti-tumor cell line assay in Figure 2B (Yamada et al., 2016).

Notes

  1. We verified the reproducibility of our iPS-NKT cell flow cytometry data, cytokine producing assay and anti-tumor effects in at least 4 independent experiments in all studies. In all, we successfully reprogrammed four different Vα24+iNKT cells into iPSCs and regenerated three of them into Vα24+iNKT cells. These iPS-NKT cells always showed high IFN-γ production activity (more than 30 ng/ml) and low IL-4 production (lower than 0.3 ng/ml) upon stimulation with DC/Gal. After stimulation with DC/Gal or cytokines (IL-7+IL-15), iPS-NKT cells always show high cytotoxic activity against tumor cell lines (more than 50% against the K562 leukemia cell line). These results are very reproducible. In our hands, we observed high killing activity of iPS-NKT cells, i.e., 50-70%, against K562 leukemia cells.
  2. As extra notes and technical tips, we addressed the following important items. Since the cell proliferation may depend on the lot of FCS, we have tested different lots for culturing OP9 feeder cells (Figure 7). In the anti-tumor cell line assay, we paid attention to removing dead cells before culturing iPS-NKT cells and target tumor cells since the dead cells may lead to a high background.


    Figure 7. Representative OP9 feeder cells morphology. After 48 h cultivation (seeded at 6 x 105 cells in a 10 cm dish), more than 3 x 106 OP9 feeder cells (about 90% confluent) in the 10 cm dish were observed.

  3. At Day 6 of iPSC induction, there are 1 x 106 to 1.5 x 106 cells in a 24-well plate. We usually observe 200-300 ES-like colonies by Day 21-28.
  4. At Day 33, about 1 x 106 iPS-NKT cells are obtained from about 5 x 104 NKT-iPSC and finally these cells can proliferate around 10-fold 8 days after being stimulated with IL-7 and IL-15. For clinical use, we can increase the OP9DLL1-seeded 10 cm dish to ten dishes, yielding ~5 x 107 iPS-NKT cells.
  5. Mitomycin-C treated MEFs are prepared as previously described (Conner, 2001) with a small modification.
    1. Replace medium of the MEFs with 6 ml MEF medium supplemented with 1 μg/ml of mitomycin-C.
    2. Incubate for 2 h at 37 °C, 5% CO2 in a CO2 incubator.
    3. Rinse mitomycin-C treated MEF with D-PBS twice, and add 6 ml MEF medium.
    4. Incubate at 37 °C in an incubator with 5% CO2.
    5. Mitomycin C-treated MEFs purchased from ReproCELL can also be used.
  6. We usually start with 2-4 x 107 mononuclear cells from peripheral blood or cord blood that can be stored in liquid nitrogen until use. PBMCs or CBMCs are cultured in RPMI supplemented with 10% FBS and 100 U/ml of hIL-2 and stimulated with α-GalCer (100 ng/ml) for 10-14 days. Cells are stained with FITC-conjugated anti-human Va24 antibody followed by anti-FITC MACS beads (Miltenyi Biotech, 10 μl beads to 107 cells). According to manufacturer’s protocol, Vα24+iNKT cells are positively purified by using an LS column (Miltenyi Biotech, purity > 95%). Purified Vα24+iNKT cells are cultured in complete NKT medium. We usually obtain around 2 million Vα24+iNKT cells from one donor.

Recipes

  1. R10 medium
    RPMI1640 containing the following supplements:
    10% fetal bovine serum (FBS)
    100 U/ml penicillin
    100 μg/ml streptomycin
    10 mM HEPES
  2. NKT medium
    R10 medium containing human IL-7 (5 ng/ml), human IL-15 (10 ng/ml) and human IL-2 (100 U/ml)
  3. Human pluripotent stem cell medium
    Primate ES Cell Medium containing bFGF (10 ng/ml)
  4. OP9 medium
    MEMα powder and 2.2 g of NaHCO3 are dissolved into 1 L of distilled water and sterilized using a 0.22 μm bottle top filter
    Finally, the following supplements are added:
    100 U/ml penicillin
    100 μg/ml streptomycin
    20% fetal bovine serum (FBS)
  5. DC/Gal
    Mouse bone marrow cells which are depleted of CD4, CD8, Class II and B220 positive cells are cultured in a 24-well plate in the presence of recombinant GM-CSF (20 ng/ml)
    Bone marrow-derived DCs are pulsed with 100 ng/ml α-GalCer for 48 h on Day 6 and stimulated by adding LPS (100 ng/ml) for the last 24 h
  6. MEF medium
    D-MEM containing the following supplements:
    15% fetal bovine serum (FBS)
    100 U/ml penicillin
    100 μg/ml streptomycin

Acknowledgments

We are grateful to prof. P.D. Burrows for the critical reading of the manuscript. We would like to thank Genta Kitahara, Momoko Okoshi, Midori Kobayashi, Maki Sakurai for their technical assistance. This work was supported by the Research Center Network for Realization of Regenerative Medicine from Japan Agency for Medical Research and Development (AMED) and CREST, Japan Science and Technology Agency. This protocol was modified from previous works that we had done with murine iPS-NKT cells and human iPS-T cells (Watarai et al., 2010; Vizcardo et al., 2013).

References

  1. Conner, D. A. (2001). Mouse embryo fibroblast (MEF) feeder cell preparation. Curr Protoc Mol Biol 23(2): Unit 23.2.
  2. Motohashi, S., Ishikawa, A., Ishikawa, E., Otsuji, M., Iizasa, T., Hanaoka, H., Shimizu, N., Horiguchi, S., Okamoto, Y., Fujii, S., Taniguchi, M., Fujisawa, T. and Nakayama, T. (2006). A phase I study of in vitro expanded natural killer T cells in patients with advanced and recurrent non-small cell lung cancer. Clin Cancer Res 12(20 Pt 1): 6079-6086.
  3. Motohashi, S., Nagato, K., Kunii, N., Yamamoto, H., Yamasaki, K., Okita, K., Hanaoka, H., Shimizu, N., Suzuki, M., Yoshino, I., Taniguchi, M., Fujisawa, T. and Nakayama, T. (2009). A phase I-II study of alpha-galactosylceramide-pulsed IL-2/GM-CSF-cultured peripheral blood mononuclear cells in patients with advanced and recurrent non-small cell lung cancer. J Immunol 182(4): 2492-2501.
  4. Nakagawa, M., Taniguchi, Y., Senda, S., Takizawa, N., Ichisaka, T., Asano, K., Morizane, A., Doi, D., Takahashi, J., Nishizawa, M., Yoshida, Y., Toyoda, T., Osafune, K., Sekiguchi, K. and Yamanaka, S. (2014). A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep 4: 3594.
  5. Shimizu, K., Hidaka, M., Kadowaki, N., Makita, N., Konishi, N., Fujimoto, K., Uchiyama, T., Kawano, F., Taniguchi, M. and Fujii, S. (2006). Evaluation of the function of human invariant NKT cells from cancer patients using alpha-galactosylceramide-loaded murine dendritic cells. J Immunol 177(5): 3484-3492.
  6. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K. and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5): 861-872.
  7. Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4): 663-676.
  8. Vizcardo, R., Masuda, K., Yamada, D., Ikawa, T., Shimizu, K., Fujii, S., Koseki, H. and Kawamoto, H. (2013). Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD8+ T cells. Cell Stem Cell 12(1): 31-36.
  9. Watarai, H., Fujii, S., Yamada, D., Rybouchkin, A., Sakata, S., Nagata, Y., Iida-Kobayashi, M., Sekine-Kondo, E., Shimizu, K., Shozaki, Y., Sharif, J., Matsuda, M., Mochiduki, S., Hasegawa, T., Kitahara, G., Endo, T. A., Toyoda, T., Ohara, O., Harigaya, K., Koseki, H. and Taniguchi, M. (2010). Murine induced pluripotent stem cells can be derived from and differentiate into natural killer T cells. J Clin Invest 120(7): 2610-2618.
  10. Yamasaki, K., Horiguchi, S., Kurosaki, M., Kunii, N., Nagato, K., Hanaoka, H., Shimizu, N., Ueno, N., Yamamoto, S., Taniguchi, M., Motohashi, S., Nakayama, T. and Okamoto, Y. (2011). Induction of NKT cell-specific immune responses in cancer tissues after NKT cell-targeted adoptive immunotherapy. Clin Immunol 138(3): 255-265.
  11. Yamada, D., Iyoda, T., Vizcardo, R., Shimizu, K., Sato, Y,,Endo, T. A., Kitahara, G., Okoshi, M., Kobayashi, M., Sakurai, M., Ohara, O., Taniguchi, M., Koseki, H. and Fujii, S. I. (2016). Efficient regeneration of human Vα24+ invariant natural killer T cells and their anti-tumor activity in vivo. Stem Cells 34(12): 2852-2860.

简介

抗原特异性T细胞来源的诱导多能干细胞(iPSCs)已显示重新分化为功能性T细胞,从而提供可用于癌症免疫治疗的T细胞的潜在来源。不变性自然杀伤T(Vα24 + iNKT)细胞的人Vα24 + 细胞是T细胞的子集,其特征在于与Vβ11配对的不变Vα24-Jα18的表达,其识别糖脂,如α-半乳糖神经酰胺(α-GalCer),由MHC I类分子CD1d呈递。据报道能够产生IFN-γ的Vα24 + i / KT细胞增加抗肿瘤反应,其影响NK细胞和CD8 +细胞毒性T淋巴细胞以消除MHC - 和MHC + 肿瘤细胞。在这里,我们描述了将人Vα24 + iNKT细胞重编程到iPSC中的鲁棒方案,然后将其重新分化为Vα24 + iNKT细胞(iPS-Vα24功能的iNKT)。我们进一步提供了测定iPS-Vα24 + iNKT细胞活性的方案。

背景 以前有报道说,针对晚期非小细胞肺癌(NSCLC)和头颈部癌症的Vα24 + iNKT细胞癌免疫治疗的临床试验显示疗效,耐受性良好(Motohashi et al。等人,2009; Yamasaki等人,2011)。然而,已知来自外周血单核细胞(PBMC)的Vα24 iNKT细胞的离体扩增的细胞产量通常较低(Motohashi等人等人,2006)。使用山中因素(Oct4,Sox2,Klf4和c-Myc)将体细胞重编程为诱导多能干细胞(iPSCs),对再生医学的目标有很大贡献。该技术最近被用于从iPSC再生肿瘤特异性细胞毒性T淋巴细胞和鼠不变的自然杀伤T(iNKT)细胞,从而开辟了一种新的癌症免疫治疗方法。在这里,我们已经建立了一个强大的协议来重新编程人类Vα24 + iNKT细胞。我们显示iPS衍生的Vα24 + iNKT细胞作为细胞佐剂起作用并发挥抗肿瘤活性,进一步延长其治疗潜力。来自iPSC的功能验证的Vα24 iNKT细胞的其他疗法的补充可能对于癌症患者是有价值的。

关键字:诱导多能干细胞, iPSC细胞, Vα24+恒定自然杀伤T细胞, Vα24+iNKT, 抗肿瘤活性, IFN-γ生成, 肿瘤免疫治疗

材料和试剂

  1. 移液器提示
  2. 24孔板(Corning,Falcon ®,目录号:353047)
  3. 15ml离心管(Corning,Falcon ®,目录号:352196)
  4. G27针(Terumo,目录号:NN-2719S)
  5. 12孔板(Corning,Falcon ®,目录号:353043)
  6. 细胞过滤器(尺寸:100μm)(Greiner Bio One International,目录号:542000)
  7. 10厘米盘(Corning,Falcon ®,目录号:353003)
  8. 6孔板(Corning,Falcon ®,目录号:353046)
  9. 6厘米盘(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:150288)
  10. 96孔圆底板(Corning,Falcon ®,目录号:351190)
  11. 用于程序C和D(Corning,Falcon ®,目录号:353077)的96孔圆底板
  12. StemPro EZ通道工具(Thermo Fisher Scientific,Gibco TM ,目录号:23181010)
  13. 电池刮刀(IWAKI,目录号:9000-220)
  14. 0.22μm瓶顶过滤器(EMD Millipore,目录号:SCGVU05RE)
  15. 人类Vα24 + iNKT细胞
  16. K562(ATCC,目录号:CCL-243)
  17. OP9饲养细胞(从RIKEN BRC细胞编号RCB2926获得)
  18. 来自CytoTune-iPS 2.0的SeV-KOS和SeV-c-MYC(MEDICAL& BIOLOGICAL LABORATORIES,目录号:DV-0305-3A)
  19. OP9DLL1饲养细胞(从RIKEN BRC细胞编号RCB2927获得)
  20. 外周血单核细胞(PBMC)
  21. 脐带血单核细胞(CBMC)
  22. 台盼蓝染色0.4%(Thermo Fisher Scientific,Invitrogen TM,目录号:T10282)
  23. 具有SV40大T抗原(T)插入的SeV载体(SeV-SV40)(ID Pharma,定制顺序)
  24. 丝裂霉素C(Sigma-Aldrich,目录号:M4287)
  25. iMatrix-511(Nippi,目录号:892 012)
  26. StemFit AK02N(ReproCELL,目录号:RCAK02N)
  27. 用于人类ES/iPS细胞的冷冻培养基(DAP213)(ReproCELL,目录号:RCHEFM001)
  28. 干细胞银行家GMP级(Nippon Zenyaku Kogyo,Zenoaq,目录号:CB045)
  29. 0.25%胰蛋白酶 - 乙二胺四乙酸(EDTA)(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  30. TripLE选择(Thermo Fisher Scientific,Gibco TM ,目录号:12563011)
  31. Y-27632,Rho相关的含盘绕圈蛋白激酶(ROCK)的抑制剂(和光纯药,目录号:253-00513)
  32. Hanks不含酚红的平衡盐溶液(HBSS + )(Wako Pure Chemical Industries,目录号:084-08965)
  33. 胶原酶(Wako Pure Chemical Industries,目录号:036-23141)
  34. Dulbecco的磷酸盐缓冲盐水(D-PBS)(和光纯药工业公司,目录号:045-29795)
  35. 干细胞因子(SCF)(R& D Systems,目录号:255-SC-050)
  36. 重组人白细胞介素-7(IL-7)(PeproTech,目录号:200-07)
  37. Fms相关的酪氨酸激酶3(Flt-3)配体(R& D Systems,目录号:3008-FK-025)
  38. 重组人IL-15(PeproTech,目录号:200-15)
  39. 7-AAD染色溶液(BD,BD Biosciences,目录号:559925)
  40. V450小鼠抗人CD3克隆UCHT1(BD,BD Biosciences,目录号:560365)
  41. 抗TCRVβ11-APC(Beckman Coulter,目录号:A66905)
  42. 抗TCRVα24-PE(Beckman Coulter,目录号:IM2283)
  43. 乳酸脱氢酶(LDH)细胞毒性检测试剂盒(Takara Bio,目录号:MK401)
  44. BD OptELISA人IFN-γ酶联免疫吸附测定(ELISA)组(BD,BD Biosciences,目录号:555142)
  45. BD OptELISA人IL-4 ELISA组(BD,BD Biosciences,目录号:555194)
  46. Triton X-100(Sigma-Aldrich,目录号:T8787-100ML)
  47. RPMI1640(Sigma-Aldrich,目录号:R8758)
  48. 胎牛血清(Sigma-Aldrich,目录号:172012-500ML)
  49. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  50. 汉克斯平衡盐溶液无酚红,无钙,无镁(HBSS - )(和光纯药公司,目录号:085-09355)
  51. 2- [4-(2-羟基乙基)-1-哌嗪基]乙磺酸(HEPES)(Sigma-Aldrich,目录号:H3375-250G)
  52. 人IL-2(Shionogi,目录号:4987058697900)
  53. 灵长类ES细胞培养基(ReproCELL,目录号:RCHEMD001)
  54. 成纤维细胞生长因子碱性(bFGF)(Wako Pure Chemical Industries,目录号:062-06661)
  55. MEMα(Thermo Fisher Scientific,Gibco TM ,目录号:11900-073)
  56. 碳酸氢钠(NaHCO 3)(Nacalai Tesque,目录号:31213-15)
  57. 重组小鼠GM-CSF(PeproTech,目录号:315-03)
  58. α-半乳糖神经酰胺(α-GalCer)(Funakoshi,目录号:KRN7000)
  59. 脂多糖(LPS)(Thermo Fisher Scientific,Invitrogen TM,目录号:00-4976-93)
  60. Dulbecco改性Eagle's培养基(D-MEM)(Wako Pure Chemical Industries,目录号:044-29765)
  61. R10培养基(见食谱)
  62. NKT媒体(见食谱)
  63. 人类多能干细胞培养基(见食谱)
  64. OP9培养基(见食谱)
  65. DC/Gal(参见食谱)
  66. MEF培养基(见食谱)

设备

  1. CO 3培养箱(35℃,37℃,38℃)(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heracell TM 150i)
  2. 倒置显微镜(Leica Microsystems,型号:DM IL LED)
  3. 立体显微镜(Leica,型号:MZ75)
  4. P200移液器(Gilson,目录号:FA10005P)
  5. 流式细胞仪(BD,BD Biosciences,型号:FACSCanto II)
  6. 离心机(KUBOTA,型号:2800)
  7. 细胞计数器(Thermo Fisher Scientific,Invitrogen TM,目录号:C10227)
  8. 控制率冷冻机(Grant Instruments,型号:EF600M)
  9. OptEIA(BD,BD Biosciences,San Jose,CA)
  10. 酶标仪(Molecular Devices,型号:SpectraMax 190)

软件

  1. FlowJo软件(FlowJo,LLC)

程序

  1. 来自人Vα24 + iNKT细胞的iPSC诱导 第-3天
    1. 将人类Vα24细胞在NKT培养基中以1×10 6个细胞/ml接种到24孔板(1×10 6个/细胞/孔),并如先前所述(Shimizu等人,2006; Yamada等人)用1×10 5 DC/Gal刺激。 ,2016)。

    第0天

    1. 通过移液将受刺激的人Vα24 + iNKT细胞(图1)收集到15ml离心管中,并通过台盼蓝染色计数活细胞数。将细胞重悬于1ml不含抗生素的R10培养基中,以1×10 6细胞/ml种子进入24孔板。


      图1.刺激的人类Va24 + iNKT细胞的代表性显微镜图像使用DC/Gal刺激人类Va24 iNKT细胞数天。

    2. 使用人Vα24 + iNKT细胞将SeV(MOI:KOS,c-MYC,SV40 = 30,3,30)加入到24孔板中并轻轻混合。
    3. 在5%CO 2培养箱中在35℃下孵育。

    第1天

    1. 用1 ml完整的R10培养基更换培养基。
    2. 在5%CO 2培养箱中在35℃下孵育。

    第6天

    1. 收集细胞并通过台盼蓝染色计数活细胞数。
    2. 在丝裂霉素C处理的MEF或iMatrix-511涂布的6cm培养皿上,用3×10 5个细胞将完整的R10培养基和种子重悬细胞。
    3. 在5%CO 2培养箱中在35℃下孵育。

    第七天

    1. 用人多能干细胞培养基(丝裂霉素C处理的MEF)或StemFit AK02N(涂覆在iMatrix-511上)更换培养基。
    2. 在5%CO 2培养箱中在35℃下孵育。
    3. 每隔一天更换培养基,直到出现人类ESC样菌落(图2)(Takahashi和Yamanaka,2006; Takahashi等人,2007)。


      图2.代表性的ESC样菌落形状。 ESC样菌落的直径约为1〜1.5mm

    第21-28天

    1. 在立体显微镜下使用G27针将每个人类ESC样菌落(直径约为1至1.5毫米)分成两个团块。通过移液将一个聚集体(约500个细胞)收集到管中,并进行人Vα24 iNKT细胞特异性TRAV(Vα24-Jα18)重组检测(Yamada等人, em>,2016)。
    2. 将其他团块转移到丝裂霉素C处理的MEF或iMatrix-511涂覆的12孔板上,以便使用温和的移液法解离后进行细胞增殖。
    3. 在37℃,5%CO 2培养箱中孵育。
    4. 使用StemPro EZ通道工具(丝裂霉素C处理的MEF)或TripLE选择(在iMatrix-511涂覆的)平板上维持细胞,如先前所述(Vizcardo等人,2013; Nakagawa et al。,2014)。使用人ES/iPS细胞(用于丝裂霉素C处理的MEF上的细胞)或干细胞储存器(用于iMatrix-511涂布板上的细胞)的冷冻培养基存储细胞。

  2. Vα24 iNKT-iPSC(iPS-NKT)的Vα24 + iNKT细胞的分化(图3)


    图3.来自NKT-iPSC的iPS-NKT分化的示意图。将NKT衍生的iPSC(NKT-iPSC)分离成小团块,并在第0天接种在OP9饲养细胞上。在第13天,细胞转移到与OP9DLL1饲养细胞共培养。在第33天,收集细胞用于无饲养层培养另外10天。

    第七天
    1. 用0.25%胰蛋白酶-EDTA分离OP9细胞(约90%汇合)
    2. 通过细胞过滤器(大小:100μm)收集离解的OP9细胞,并在10ml的OP9培养基中于10cm培养皿中以6×10 5个细胞种子。
    3. 在37℃,5%CO 2培养箱中孵育。

    第五天

    1. 加入10 ml OP9培养基
    2. 在37℃,5%CO 2培养箱中孵育。

    第0天

    1. 用补充有10μMY-27632的新鲜OP9培养基代替OP9培养基。
    2. 使用立体显微镜下的StemPro EZ通道工具将人iPS细胞(80%融合)分离成团块。
    3. 在立体显微镜下使用P200移液管收集约100个胶囊(图4B),并将其放置在OP9种子的10厘米盘上。
    4. 在37℃,5%CO 2培养箱中孵育。


      图4. iPS-NKT分化的代表性的NKT-iPSC morophology。使用EZ通道工具和移液将NKT-iPSC分成几个团块。 A.使用StemPro EZ通道工具切割后的NKT-iPSC的集落形状; B.通过移液和通入OP9饲养细胞制备和收集小NKT-iPSC团块。

    第1天

    1. 用20 ml OP9培养基更换培养基。
    2. 在37℃,5%CO 2培养箱中孵育。

    第9天

    1. 丢弃一半的上清(培养基)。
    2. 加入10 ml OP9培养基
    3. 在37℃,5%CO 2培养箱中孵育。

    第12天

    1. 用0.25%胰蛋白酶-EDTA分离OP9DLL1细胞(约90%汇合)
    2. 通过细胞过滤器(大小:100μm)收集离解的OP9DLL1细胞,并在10 ml OP9培养基中的10cm皿上以2×10 6个细胞种子。
    3. 在37℃,5%CO 2培养箱中孵育。

    第13天

    1. 弃去培养基,用10ml HBSS 冲洗细胞
    2. 加入6毫升胶原酶(20毫克/毫升)
    3. 在CO 2培养箱中37℃孵育45分钟。
    4. 弃去胶原酶,并用10ml D-PBS冲洗细胞
    5. 加入6 ml 0.25%胰蛋白酶-EDTA
    6. 在CO 2培养箱中37℃,30分钟孵育。
    7. 加入4ml OP9培养基,并收集通过细胞过滤器(大小:100μm)的离解细胞
    8. 在补充有10ng/ml SCF,5ng/ml人IL-7和5ng/ml Flt3-配体和板的10ml OP9培养基上重新悬浮收集的细胞,在OP9DLL1细胞接种的10cm皿(在第12天制备) )。

    第15天

    1. 用0.25%胰蛋白酶-EDTA分离OP9DLL1细胞(约90%汇合)
    2. 通过穿过细胞过滤器(大小:100μm)收集解离的OP9DLL1细胞,并在10 ml的OP9培养基中的10cm培养皿中以2×10 6个细胞种子。
    3. 在37℃,5%CO 2培养箱中孵育。

    第16天

    1. 丝裂霉素C处理的OP9DLL1细胞的制备
      1. 用补充有1μg/ml丝裂霉素C的6ml OP9培养基更换OP9DLL1细胞培养基。
      2. 在CO 2培养箱中孵育2小时,37℃,5%CO 2。
      3. 用D-PBS冲洗丝裂霉素C处理的OP9DLL1细胞两次,加入6ml OP9培养基。
      4. 在37℃,5%CO 2培养箱中孵育。
    2. 差异化iPS-NKT细胞的通过。
      1. 通过移液并通过细胞过滤器(尺寸:100μm)分离收集细胞(在第13天制备)。
      2. 将收集的细胞用10μl补充有10ng/ml SCF,5ng/ml人IL-7和5ng/ml Flt3-配体和平板的OP9培养基重新悬浮在丝裂霉素-C处理的OP9DLL1细胞种植的10cm培养皿上。 >
      3. 在37℃,5%CO 2培养箱中孵育。

    第24天

    1. 用0.25%胰蛋白酶-EDTA分离OP9DLL1细胞(约90%汇合)
    2. 通过通过细胞过滤器(尺寸:100μm)收集解离的细胞,并在10ml的OP9培养基中的10cm皿上以2×10 6个细胞种子。
    3. 在37℃,5%CO 2培养箱中孵育。

    第25天

    1. 丝裂霉素C处理的OP9DLL1细胞的制备
      1. 用补充有1μg/ml丝裂霉素C的6ml OP9培养基更换OP9DLL1细胞培养基。
      2. 在CO 2培养箱中37℃,5%CO 2孵育2小时。
      3. 用D-PBS冲洗丝裂霉素C处理的OP9DLL1细胞两次,加入6ml OP9培养基。
      4. 在37℃,5%CO 2培养箱中孵育。
    2. 差异化iPS-NKT细胞的通过
      1. 通过移液并通过细胞过滤器(尺寸:100μm)分离收集细胞(在第16天制备)。
      2. 将收集的细胞重新悬浮在补充有10ng/ml SCF,5ng/ml人IL-7和5ng/ml Flt3-配体的平板的10ml OP9培养基和平板上的丝裂霉素C处理的OP9DLL1细胞种植的10cm培养皿上。 />
      3. 在37℃,5%CO 2培养箱中孵育。

    第30天

    1. 加入补充有10ng/ml SCF,5ng/ml人IL-7和5ng/ml Flt3-配体(总共15ml)的5ml OP9培养基。
    2. 在37℃,5%CO 2培养箱中孵育。

    第33天

    1. 通过细胞过滤器(尺寸:100μm)收集细胞。
    2. 通过台盼蓝染色计数活细胞数,并使用1×10 5个细胞通过流式细胞术分析iPS-NKT标记(CD3,TRAV24和TRBV11)表达。
    3. 将5×10 5个细胞/孔的iPS-NKT细胞接种到补充有5ng/ml人IL-7和10ng/ml人IL-7的2ml OP9培养基中的6孔板中15.
    4. 在37℃,5%CO 2培养箱中孵育。

    第35,37,39和41天

    1. 加入1ml补充有5ng/ml人IL-7和10ng/ml人IL-15的OP9培养基
    2. 在37℃,5%CO 2培养箱中孵育。

    第43天

    1. 通过移液并通过细胞过滤器(尺寸:100μm)收集iPS-NKT细胞(图5)。


      图5.代表性增殖的iPS-NKT细胞观察到活化的T细胞样形态。

    2. 通过台盼蓝染色计数可行的iPS-NKT细胞数,并使用1×10 5个细胞通过流式细胞术分析iPS-NKT标记(CD3,TRAV24和TRBV11)表达(图6)。 >
    3. 此时可以使用iPS-NKT细胞进行功能测定,如抗肿瘤细胞系检测或细胞因子生成测定。


      图6.使用流式细胞仪分析代表性的iPS-NKT细胞。使用FSC和SSC如椭圆所示选择淋巴细胞分数。 B.活细胞由7-AAD阴性部分选择。 C.选择CD3阳性部分,将TRAV24和TRBV11阳性细胞表示为正方形
  1. 细胞因子生产测定
    1. 将iPS-NKT细胞接种在96孔圆底板(1×10 5个细胞/孔/200μl)上。
    2. 细胞与或不与小鼠DC/Gal(1×10 5个细胞/孔)共同培养24小时,37℃,37℃,5%CO 2 >在CO 2 孵化器中。用OptEIA测量培养上清液中IFN-γ和IL-4的量。

  2. 抗肿瘤细胞系检测
    1. 在96孔圆底板中,E/T比为5或10的效应细胞与1×10 4个靶细胞一起培养。
    2. 在CO 2培养箱中37℃,5%CO 2孵育6小时后,收集100μl上清液,用LDH细胞毒性检测试剂盒测定LDH活性在96孔平底板中。
      1. 作为阳性对照,使用用2%Triton X-100裂解的靶细胞。
      2. 作为背景控制,使用效应细胞或靶细胞。
    3. 在使用LDH试剂盒的抗肿瘤分析中在490nm和600nm处用SpectraMax 190测量。在490nm处从O.D.中减去。在600 nm作为噪声。杀灭活性用下列公式计算:杀死活性= [{("效应子+靶"的OD - 培养基的OD) - (单独的OD的OD - 培养基的OD)} - (单独的效应物的OD - 培养基的OD)] /(阳性对照OD值 - 单独OD值)×100(%)

数据分析

我们将Vα24 + iNKT细胞重新编程到来自4个供体的iPSC中,并使用iPSC系选择的核型稳定性。为了评估表型,我们使用FlowJo软件(FlowJo,LLC)来处理使用FACSCanto II获得的iPS-NKT细胞的流式细胞术数据。在细胞因子产生测定中,我们进行4到7次独立实验,其中2至3个孔重复,并且用Student's检验统计分析汇总结果。在抗肿瘤细胞系检测中,我们进行了一式三份重复的8次独立实验,并用Student's检验统计分析了数据。我们在我们的原始论文(Yamada等人,2016)中描述了图2A中的细胞因子产生测定中的重复细节以及图2B中抗肿瘤细胞系测定(Yamada < em> et al。,2016)。

笔记

  1. 在所有研究中,我们验证了至少4个独立实验中我们的iPS-NKT细胞流式细胞术数据,细胞因子产生测定和抗肿瘤效应的重现性。总之,我们成功地将四种不同的Vα24 + iNKT细胞重新编程到iPSC中,并将其中的三个再生成Vα24 + iNKT细胞。在用DC/Gal刺激后,这些iPS-NKT细胞总是表现出高的IFN-γ产生活性(大于30ng/ml)和低IL-4产生(低于0.3ng/ml)。在用DC/Gal或细胞因子(IL-7 + IL-15)刺激后,iPS-NKT细胞总是显示出对肿瘤细胞系的高细胞毒性(对K562白血病细胞系超过50%)。这些结果非常重现。在我们手中,我们观察到针对K562白血病细胞的iPS-NKT细胞的高杀伤活性,即50-70%。
  2. 作为额外的笔记和技术提示,我们讨论了以下重要项目。由于细胞增殖可能取决于FCS的大量,我们已经测试了不同批次培养OP9饲养细胞(图7)。在抗肿瘤细胞系测定中,我们注意在培养iPS-NKT细胞和靶向肿瘤细胞之前去除死细胞,因为死细胞可能导致高背景。


    图7.代表性的OP9饲养细胞形态培养48小时(在10cm培养皿中以6×10 5个细胞接种),超过3×10 4个观察10cm皿中的OP9饲养细胞(约90%汇合)
  3. 在iPSC诱导的第6天,在24孔板中有1×10 6个至1.5×10 6个细胞。我们通常在21-28日观察200-300个ES样殖民地。
  4. 在第33天,从约5×10 4个NKT-iPSC获得约1×10 6个iPS-NKT细胞,最后这些细胞可以在10天8天内增殖在用IL-7和IL-15刺激后。对于临床使用,我们可以将OP9DLL1种子10厘米的菜增加到十个菜,产生约5×10 7个iPS-NKT细胞。
  5. 丝裂霉素C处理的MEF如前所述(Conner,2001)以小的修饰制备。
    1. 用补充有1μg/ml丝裂霉素C的6ml MEF培养基代替MEF的培养基
    2. 在CO 2培养箱中37℃,5%CO 2孵育2小时。
    3. 用D-PBS冲洗丝裂霉素C处理的MEF两次,加入6ml MEF培养基
    4. 在37℃,5%CO 2培养箱中孵育。
    5. 也可以使用从ReproCELL购买的丝裂霉素C治疗的MEF。
  6. 我们通常从可以储存在液氮中直到使用的外周血或脐血的2-4×10 7个单核细胞开始。将PBMC或CBMC在补充有10%FBS和100U/ml hIL-2的RPMI中培养,并用α-GalCer(100ng/ml)刺激10-14天。细胞用FITC缀合的抗人Va24抗体,然后用抗FITC MACS珠(Miltenyi Biotech,10μl珠至10μg/ml)细胞染色。根据制造商的方案,通过使用LS柱(Miltenyi Biotech,纯度> 95%)对Vα24 + iNKT细胞进行阳性纯化。纯化的Vα24细胞在完全的NKT培养基中培养。我们通常从一个供体获得约200万个Vα24 + iNKT细胞。

食谱

  1. R10中等
    RPMI1640包含以下补充:
    10%胎牛血清(FBS)
    100 U/ml青霉素
    100μg/ml链霉素
    10 mM HEPES
  2. NKT培养基
    含有人IL-7(5ng/ml),人IL-15(10ng/ml)和人IL-2(100U/ml)的R10培养基
  3. 人多能干细胞培养基
    含有bFGF(10ng/ml)的灵长类ES细胞培养基
  4. OP9中等
    将MEMα粉末和2.2g NaHCO 3溶解在1L蒸馏水中,并使用0.22μm瓶顶过滤器
    最后,添加以下补充:
    100 U/ml青霉素
    100μg/ml链霉素
    20%胎牛血清(FBS)
  5. DC/Gal
    在重组GM-CSF(20ng/ml)存在下,在24孔板中培养耗尽CD4,CD8,II类和B220阳性细胞的小鼠骨髓细胞。 骨髓来源的DC在第6天用100ng/ml的α-GalCer脉冲48小时,并通过在最后24小时加入LPS(100ng/ml)刺激
  6. MEF中等 D-MEM包含以下补充:
    15%胎牛血清(FBS)
    100 U/ml青霉素
    100μg/ml链霉素

致谢

我们非常感谢教授。 P.D.对于手稿的批判性阅读而言。我们要感谢Genta Kitahara,Momoko Okoshi,小林小林,Maki Sakurai的技术援助。这项工作得到了日本医学研究与发展机构(AMED)和日本科技局CREST研究中心实现再生医学网络的支持。该方案是从以前的研究中修改过的,我们用鼠iPS-NKT细胞和人iPS-T细胞(Watarai等人,2010; Vizcardo等人) 2013)。

参考

  1. Conner,DA(2001)。  小鼠胚胎成纤维细胞(MEF )饲养细胞制备。 Curr Protoc Mol Biol 23(2):Unit 23.2。
  2. Motohashi,S.,Ishikawa,A.,Ishikawa,E.,Otsuji,M.,Iizasa,T.,Hanaoka,H.,Shimizu,N.,Horiguchi,S.,Okamoto,Y.,Fujii, Taniguchi,M.,Fujisawa,T。和Nakayama,T。(2006)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/17028247"靶向="_ blank">在体外的I期研究扩大了晚期和复发性非小细胞肺癌患者的自然杀伤T细胞。 em> 12(20 Pt 1):6079-6086。
  3. Motohashi,S.,Nagato,K.,Kunii,N.,Yamamoto,H.,Yamasaki,K.,Okita,K.,Hanaoka,H.,Shimizu,N.,Suzuki,M.,Yoshino, Taniguchi,M.,Fujisawa,T.和Nakayama,T。(2009)。晚期和复发性非小细胞肺癌患者的α-半乳糖神经酰胺脉冲IL-2/GM-CSF培养的外周血单核细胞的I期II期研究。 > J Immunol 182(4):2492-2501。
  4. Nakagawa,M.,Taniguchi,Y.,Senda,S.,Takizawa,N.,Ichisaka,T.,Asano,K.,Morizane,A.,Doi,D.,Takahashi,J.,Nishizawa, Yoshida,Y.,Toyoda,T.,Osafune,K.,Sekiguchi,K.and Yamanaka,S。(2014)。< a class ="ke-insertfile"href ="https://www.ncbi。 nlm.nih.gov/pubmed/24399248"target ="_ blank">一种用于衍生人诱导多能干细胞的新型无效饲料培养系统。 4:3594 。
  5. Shimizu,K.,Hidaka,M.,Kadowaki,N.,Makita,N.,Konishi,N.,Fujimoto,K.,Uchiyama,T.,Kawano,F.,Taniguchi,M。和Fujii,S。( 2006)。 使用α的癌症患者评估人类不变NKT细胞的功能 - 半乳糖神经酰胺负载的鼠树突状细胞。 J Immunol 177(5):3484-3492。
  6. Takahashi,K.,Tanabe,K.,Ohnuki,M.,Narita,M.,Ichisaka,T.,Tomoda,K.and Yamanaka,S。(2007)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/18035408"target ="_ blank">通过确定的因素从成年人成纤维细胞诱导多能干细胞。细胞 131(5):861-872。
  7. Takahashi,K.和Yamanaka,S。(2006)。通过限定因子从小鼠胚胎和成年成纤维细胞培养物诱导多能干细胞。细胞 126(4):663-676。
  8. Vizcardo,R.,Masuda,K.,Yamada,D.,Ikawa,T.,Shimizu,K.,Fujii,S.,Koseki,H.and Kawamoto,H。(2013)。< a class = ke-insertfile"href ="http://www.cell.com/cell-stem-cell/abstract/S1934-5909(12)00711-4"target ="_ blank">人肿瘤抗原特异性T细胞的再生来自成熟CD8 + T细胞的iPSC。细胞干细胞 12(1):31-36。
  9. Watarai,H.,Fujii,S.,Yamada,D.,Rybouchkin,A.,Sakata,S.,Nagata,Y.,Iida-Kobayashi,M.,Sekine-Kondo,E.,Shimizu,K.,Shozaki ,Y.,Sharif,J.,Matsuda,M.,Mochiduki,S.,Hasegawa,T.,Kitahara,G.,Endo,TA,Toyoda,T.,Ohara,O.,Harigaya,K.,Koseki, H.和Taniguchi,M。(2010)。鼠诱导的多能干细胞可衍生并分化为天然杀伤T细胞。 J Clin Invest 120(7):2610-2618。
  10. Yamasaki,K.,Horiguchi,S.,Kurosaki,M.,Kunii,N.,Nagato,K.,Hanaoka,H.,Shimizu,N.,Ueno,N.,Yamamoto,S.,Taniguchi, Motohashi,S.,Nakayama,T.和Okamoto,Y。(2011)。 NKT细胞靶向过继免疫治疗后,在癌组织中诱导NKT细胞特异性免疫应答。 Clin Immunol 138(3):255-265。 >
  11. Yamada,D.,Iyoda,T.,Vizcardo,R.,Shimizu,K.,Sato,Y ,, Endo,TA,Kitahara,G.,Okoshi,M.,Kobayashi,M.,Sakurai,M.,Ohara ,O.,Taniguchi,M.,Koseki,H.and Fujii,SI(2016)。  人类Vα24 + 不变天然杀伤T细胞的有效再生及其在体内的抗肿瘤活性。干细胞 34(12):2852-2860。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yamada, D., Iyoda, T., Shimizu, K., Sato, Y., Koseki, H. and Fujii, S. (2017). Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24+iNKT Cells. Bio-protocol 7(10): e2277. DOI: 10.21769/BioProtoc.2277.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。