JoVE Logo
Faculty Resource Center

Sign In





Representative Results





Developmental Biology

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Published: November 11th, 2016



1Department of Surgery, University of Michigan, 2Department of Biochemistry II, Kanazawa Medical University, 3Center for Immunotherapy, Roswell Park Cancer Institute, 4DNAVEC Corporation, 5Department of Ophthalmology, Keio University School of Medicine, 6Department of Surgical Oncology, Roswell Park Cancer Institute

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

Cellular reprogramming technology that allows generation of induced pluripotent stem cells (iPSCs) via overexpression of a defined set of transcription factors holds great promise in the field of cell-based therapies1,2. These iPSCs exhibit transcriptional and epigenetic features and have the capacity for self-renewal and pluripotency, similarly to embryonic stem cells (ESCs)3-5. Remarkable progress made in reprogramming technology over the past decade has allowed us to generate human iPSCs even from terminally differentiated cells, such as T cells6-8. T cell-derived iPSCs (TiPSCs) retain the same rearranged configuration of T cell rec....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NOTE: Patients should give informed consent to participate in the Institutional Review Board and Human Pluripotent Stem Cell Committee approved study.

1. Isolation and Culture of TILs

  1. Obtain tumor material that is not required for histopathologic diagnosis from the pathology service/tissue procurement core. Place 20-100 g of tumor specimens in a 50-ml tube with 30 ml tumor collecting media (Table 1).
  2. Dissect solid, firm, normal tissue of the tumor specime.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Figure 1 shows the overview of the procedure that involves the initial expansion of melanoma TILs with rhIL-2, which is followed by activation with anti-CD3/CD28 and gene transfer of OCT3/4, KLF4, SOX2, and c-MYC to TILs for the generation of iPSCs. Usually, TILs on culture with rhIL-2 start to form spheres 21-28 days after initiation of culture. At this point, TILs are ready to be activated with anti-CD3/CD28. Figure 2A shows TILs, on culture with rhIL-2.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Here, we demonstrated a protocol for reprogramming melanoma TILs to iPSCs by SeV-mediated transduction of the four transcription factors OCT3/4, SOX2, KLF4, and c-MYC. This approach, using a SeV system to reprogram T cells, offers the advantage of a non-integrating method7.

A previous study showed that a SeV reprogramming system was highly efficient and reliable to reprogram not only fibroblasts but also peripheral blood T cells7,17. In addition, we have recently shown th.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We thank Ms. Deborah Postiff and Ms. Jackline Barikdar in the Tissue Procurement Core and Dr. Cindy DeLong in the Pluripotent Stem Cell Core Laboratory at the University of Michigan for her technical assistance. This study was supported by University of Michigan startup funding and grants from the Central Surgical Association, American College of Surgeons, Melanoma Research Alliance, and NIH/NCI (1K08CA197966-01) to F. Ito.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
gentle MACS C Tubes Miltenyi Biotec 130-093-237
gentle MACS Dissociator Miltenyi Biotec 130-093-235
Tumor Dissociation Kit, human Miltenyi Biotec 130-095-929
RPMI 1640 Life technologies 11875-093
Falcon 70 um Cell Strainer BD 352350
BD Falcon 50ml Conical Cntrifuge tubes BD 352070
IMDM Life technologies 12440053
human AB serum Life technologies 34005100
L-glutamine (200mM) Life technologies 25030-081
2-mercaptoethanol (1000x, 55mM) Life technologies 21985-023
Penicillin-Streptomycin  Life technologies 15140-122
gentamicin Life technologies 15750-060
Ficoll-Paque PLUS GE 17-1440-02
D-PBS (-) Life technologies 14040-133
recombinant human (rh) IL-2 Aldesleukin, Prometheus Laboratories Inc.
Purified NA/LE Mouse Anti-Human CD3 BD 555329
Purified NA/LE Mouse Anti-Human CD28 BD 555725
X-VIVO 15 Lonza 04-418Q
FBS Gibco 26140-079
HEPES Life technologies 15630-080
N-Acetylcysteine Cumberland Pharmaceuticals Inc. NDC 66220-207-30
Falcon Tissue Culture Plates (6-well) Corning 353046
Falcon Tissue Culture Plates (24-well) Corning 353047
Sendai virus vector DNAVEC
SNL feeder cells Cell Biolabs, Inc CBA-316
mitomycin C SIGMA M4287 soluble in water (0.5 mg/ml)
gelatin SIGMA G1890
Primate ES Cell Medium Reprocell RCHEMD001 warm in 37 ℃ water bath before use
basic fibroblast growth factor (bFGF) Life technologies PHG0264
ReproStem Reprocell RCHEMD005 warm in 37 ℃ water bath before use

  1. Takahashi, K., et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131, 861-872 (2007).
  2. Takahashi, K., Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126, 663-676 (2006).
  3. Wernig, M., et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 448, 318-324 (2007).
  4. Maherali, N., et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell. 1, 55-70 (2007).
  5. Okita, K., Ichisaka, T., Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature. 448, 313-317 (2007).
  6. Loh, Y. H., et al. Reprogramming of T cells from human peripheral blood. Cell Stem Cell. 7, 15-19 (2010).
  7. Seki, T., et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell. 7, 11-14 (2010).
  8. Staerk, J., et al. Reprogramming of human peripheral blood cells to induced pluripotent stem cells. Cell Stem Cell. 7, 20-24 (2010).
  9. Nishimura, T., et al. Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation. Cell Stem Cell. 12, 114-126 (2013).
  10. Vizcardo, R., et al. Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8(+) T Cells. Cell Stem Cell. 12, 31-36 (2013).
  11. Wakao, H., et al. Expansion of functional human mucosal-associated invariant T cells via reprogramming to pluripotency and redifferentiation. Cell Stem Cell. 12, 546-558 (2013).
  12. Dudley, M. E., Wunderlich, J. R., Shelton, T. E., Even, J., Rosenberg, S. A. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. J Immunother. 26, 332-342 (2003).
  13. Gros, A., et al. PD-1 identifies the patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors. J Clin Invest. 124, 2246-2259 (2014).
  14. Rosenberg, S. A., et al. Durable Complete Responses in Heavily Pretreated Patients with Metastatic Melanoma Using T-Cell Transfer Immunotherapy. Clinical Cancer Research. 17, 4550-4557 (2011).
  15. Restifo, N. P., Dudley, M. E., Rosenberg, S. A. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol. 12, 269-281 (2012).
  16. Saito, H., et al. Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells. Stem Cells International. 2016, 11 (2016).
  17. Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-362 (2009).
  18. Ban, H., et al. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc Natl Acad Sci U S A. 108, 14234-14239 (2011).
  19. Fujie, Y., et al. New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood. PLoS One. 9, e113052 (2014).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved