Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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

DY Daisuke Yamada
TI Tomonori Iyoda
KS Kanako Shimizu
YS Yusuke Sato
HK Haruhiko Koseki
SF Shin-ichiro Fujii

发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2277 浏览次数: 13802

评审: Lian Qun QiuAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Stem Cells, featuring study using the protocol.

Dec 2016

Advertisement

High-throughput T-cell antigen discovery technology platform and its applications
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

利用气液界面培养从人多能干细胞（hPSCs）构建患者特异性功能性胸腺类器官

利用气液界面培养从人多能干细胞（hPSCs）构建患者特异性功能性胸腺类器官

Stephan A. Ramos and Holger A. Russ

2026年05月05日 1183 阅读

人小气道上皮细胞气液界面培养及基于 TEER 的屏障完整性评估操作方案

人小气道上皮细胞气液界面培养及基于 TEER 的屏障完整性评估操作方案

Dominika Jakubczyk [...] Sabina Górska

2026年06月05日 320 阅读

人脐带血造血干细胞的分离及向巨核细胞定向分化

人脐带血造血干细胞的分离及向巨核细胞定向分化

Wenjun Xia [...] Yahan Fan

2026年09月20日 118 阅读

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) search iPSC (iPSC细胞) search Vα24+invariant natural killer T cell (Vα24+恒定自然杀伤T细胞) search Vα24+iNKT (Vα24+iNKT) search Anti-tumor activity (抗肿瘤活性) search IFN-γ production (IFN-γ生成) search Tumor immunotherapy (肿瘤免疫治疗) search

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

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

ris ris Download Citation in RIS Format

分类

干细胞 > 多能干细胞 > 细胞诱导

免疫学 > 免疫细胞分化 > T 细胞

细胞生物学 > 细胞分离和培养 > 细胞分化

您对这篇实验方案有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件