Published: November 11th, 2016
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....
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
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.......
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.......
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.....
|gentle MACS C Tubes
|gentle MACS Dissociator
|Tumor Dissociation Kit, human
|Falcon 70 um Cell Strainer
|BD Falcon 50ml Conical Cntrifuge tubes
|human AB serum
|2-mercaptoethanol (1000x, 55mM)
|recombinant human (rh) IL-2
|Aldesleukin, Prometheus Laboratories Inc.
|Purified NA/LE Mouse Anti-Human CD3
|Purified NA/LE Mouse Anti-Human CD28
|Cumberland Pharmaceuticals Inc.
|Falcon Tissue Culture Plates (6-well)
|Falcon Tissue Culture Plates (24-well)
|Sendai virus vector
|SNL feeder cells
|Cell Biolabs, Inc
|soluble in water (0.5 mg/ml)
|Primate ES Cell Medium
|warm in 37 ℃ water bath before use
|basic fibroblast growth factor (bFGF)
|warm in 37 ℃ water bath before use
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