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

Summary

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

Abstract

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.

Introduction

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 therapies^1,2. 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 cells^6-8. T cell-derived iPSCs (TiPSCs) retain the same rearranged configuration of T cell rec....

Protocol

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 specimen from fragile and/or bloody necrotic areas using scissors. After removing the necrotic tissue, use the scissors to mince the specimen as small as pos....

Results

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.......

Discussion

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 cells^7,17. In addition, we have recently shown th.......

Materials

List of materials used in this article

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
basic fibroblast growth factor (bFGF)	Life technologies	PHG0264