Name: generation of induced pluripotent stem cells from human melanoma tumor-infiltrating lymphocytes
Uploaded: 2016-11-11T13:00:00
Description: Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes at JoVE.com

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.

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
<ol>
	<li>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).</li>
	<li>Dissect solid, firm, normal tissue of the tumor specime...

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+T+cells+from+human+peripheral+blood.">Reprogramming of T cells from human peripheral blood.</a> Cell Stem Cell. 7, 15-19 (2010).</li><li>Seki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+from+human+terminally+differentiated+circulating+T+cells.">Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.</a> Cell Stem Cell. 7, 11-14 (2010).</li><li>Staerk, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell Stem Cell. 7, 20-24 (2010).</li><li>Nishimura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+rejuvenated+antigen-specific+T+cells+by+reprogramming+to+pluripotency+and+redifferentiation.">Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation.</a> Cell Stem Cell. 12, 114-126 (2013).</li><li>Vizcardo, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+Human+Tumor+Antigen-Specific+T+Cells+from+iPSCs+Derived+from+Mature+CD8(+)+T+Cells.">Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8(+) T Cells.</a> Cell Stem Cell. 12, 31-36 (2013).</li><li>Wakao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+functional+human+mucosal-associated+invariant+T+cells+via+reprogramming+to+pluripotency+and+redifferentiation.">Expansion of functional human mucosal-associated invariant T cells via reprogramming to pluripotency and redifferentiation.</a> Cell Stem Cell. 12, 546-558 (2013).</li><li>Dudley, M. E., Wunderlich, J. R., Shelton, T. E., Even, J., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+tumor-infiltrating+lymphocyte+cultures+for+use+in+adoptive+transfer+therapy+for+melanoma+patients.">Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients.</a> J Immunother. 26, 332-342 (2003).</li><li>Gros, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1+identifies+the+patient-specific+CD8(+)+tumor-reactive+repertoire+infiltrating+human+tumors.">PD-1 identifies the patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors.</a> J Clin Invest. 124, 2246-2259 (2014).</li><li>Rosenberg, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durable+Complete+Responses+in+Heavily+Pretreated+Patients+with+Metastatic+Melanoma+Using+T-Cell+Transfer+Immunotherapy.">Durable Complete Responses in Heavily Pretreated Patients with Metastatic Melanoma Using T-Cell Transfer Immunotherapy.</a> Clinical Cancer Research. 17, 4550-4557 (2011).</li><li>Restifo, N. P., Dudley, M. E., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+for+cancer:+harnessing+the+T+cell+response.">Adoptive immunotherapy for cancer: harnessing the T cell response.</a> Nat Rev Immunol. 12, 269-281 (2012).</li><li>Saito, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+Melanoma+Tumor-Infiltrating+Lymphocytes+to+Induced+Pluripotent+Stem+Cells.">Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells.</a> Stem Cells International. 2016, 11 (2016).</li><li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-362 (2009).</li><li>Ban, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+transgene-free+human+induced+pluripotent+stem+cells+(iPSCs)+by+temperature-sensitive+Sendai+virus+vectors.">Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors.</a> Proc Natl Acad Sci U S A. 108, 14234-14239 (2011).</li><li>Fujie, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Type+of+Sendai+Virus+Vector+Provides+Transgene-Free+iPS+Cells+Derived+from+Chimpanzee+Blood.">New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood.</a> PLoS One. 9, e113052 (2014).</li></ol>

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...

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 receptor (TCR) chain genes as the original T cells, which allows regeneration of antigen-specific T cells from TiPSCs9-11.
Nearly 80% of melanoma-infiltrating lymphocytes (TILs) obtained from a patient&#39;s tumor specifically recognize tumor-associated antigens and maintain cytotoxicity against the original cancer cells12. Notably, the expression of programmed cell death protein-1 (PD-1) on TILs was found to identify the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8+ lymphocytes13. Adoptive transfer of ex-vivo expanded autologous TILs in combination with preparative lymphodepleting regimens and systemic administration of Interleukin-2 (IL-2) can cause substantial regression of metastatic melanoma in subsets of patients14. Despite encouraging results in preclinical models and in patients, poor survival of infused T cells and the existence of immune suppressive pathways appear to compromise the full potential of adoptive T cell therapy. Current clinical protocols require extensive ex vivo manipulation of autologous T cells in order to obtain large numbers. This results in the generation of terminally differentiated T cells that have poor survival, reduced proliferative capacity, and high levels of PD-115.
This limitation of adoptive T cell therapy can be theoretically overcome by using iPSCs that can provide an unlimited source of autologous T cells for immunotherapy. We have recently reported the reprogramming of melanoma TILs expressing high levels of PD-1 by Sendai virus (SeV)-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC16. While retrovirus vectors require integration into host chromosomes to express reprogramming genes, SeV vectors are non-integrating and are eventually eliminated from the cytoplasm. Reprogramming efficiency is much higher with a SeV system compared with lentivirus or retrovirus vectors6-8. Furthermore, SeV can specifically reprogram T cells in peripheral blood mononuclear cells (PBMCs), while some iPSC clones generated by lentivirus or retrovirus vectors can be from nonlymphoid lineages6-8. Here, we detail the procedures implemented for the isolation and activation of human melanoma TILs and for the generation of TIL-derived iPSCs using a SeV reprogramming system.

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			<td height="20" style="height:20px;width:271px;">gentle MACS C Tubes</td>
			<td style="width:287px;">Miltenyi Biotec</td>
			<td style="width:132px;">130-093-237</td>
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			<td height="20" style="height:20px;width:271px;">gentle MACS Dissociator</td>
			<td style="width:287px;">Miltenyi Biotec</td>
			<td style="width:132px;">130-093-235</td>
			<td></td>
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			<td height="20" style="height:20px;width:271px;">Tumor Dissociation Kit, human</td>
			<td style="width:287px;">Miltenyi Biotec</td>
			<td style="width:132px;">130-095-929</td>
			<td></td>
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			<td height="20" style="height:20px;width:271px;">RPMI 1640</td>
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			<td height="20" style="height:20px;width:271px;">Falcon 70 um Cell Strainer</td>
			<td style="width:287px;">BD</td>
			<td style="width:132px;">352350</td>
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			<td height="20" style="height:20px;width:271px;">BD Falcon 50ml Conical Cntrifuge tubes</td>
			<td style="width:287px;">BD</td>
			<td style="width:132px;">352070</td>
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			<td height="20" style="height:20px;width:271px;">human AB serum</td>
			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">34005100</td>
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			<td height="20" style="height:20px;width:271px;">L-glutamine (200mM)</td>
			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">25030-081</td>
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			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">15140-122</td>
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			<td height="20" style="height:20px;width:271px;">gentamicin</td>
			<td style="width:287px;">Life technologies</td>
			<td>15750-060</td>
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			<td height="20" style="height:20px;width:271px;">Ficoll-Paque PLUS</td>
			<td style="width:287px;">GE</td>
			<td style="width:132px;">17-1440-02</td>
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			<td height="20" style="height:20px;width:271px;">D-PBS (-)</td>
			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">14040-133</td>
			<td></td>
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			<td height="20" style="height:20px;">recombinant human (rh) IL-2</td>
			<td style="width:287px;">Aldesleukin, Prometheus Laboratories Inc.</td>
			<td style="width:132px;"></td>
			<td></td>
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			<td height="20" style="height:20px;width:271px;">Purified NA/LE Mouse Anti-Human CD3</td>
			<td style="width:287px;">BD</td>
			<td style="width:132px;">555329</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Purified NA/LE Mouse Anti-Human CD28</td>
			<td style="width:287px;">BD</td>
			<td style="width:132px;">555725</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">X-VIVO 15</td>
			<td style="width:287px;">Lonza</td>
			<td style="width:132px;">04-418Q</td>
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			<td height="20" style="height:20px;width:271px;">FBS</td>
			<td style="width:287px;">Gibco</td>
			<td style="width:132px;">26140-079</td>
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			<td height="20" style="height:20px;width:271px;">HEPES</td>
			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">15630-080</td>
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			<td height="20" style="height:20px;width:271px;">N-Acetylcysteine</td>
			<td style="width:287px;">Cumberland Pharmaceuticals Inc.</td>
			<td style="width:132px;">NDC 66220-207-30</td>
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			<td height="20" style="height:20px;width:271px;">Falcon Tissue Culture Plates (6-well)</td>
			<td style="width:287px;">Corning</td>
			<td>353046</td>
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			<td height="20" style="height:20px;width:271px;">Falcon Tissue Culture Plates (24-well)</td>
			<td style="width:287px;">Corning</td>
			<td style="width:132px;">353047</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Sendai virus vector</td>
			<td style="width:287px;">DNAVEC</td>
			<td style="width:132px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:271px;">SNL feeder cells</td>
			<td style="width:287px;">Cell Biolabs, Inc</td>
			<td style="width:132px;">CBA-316</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mitomycin C</td>
			<td style="width:287px;">SIGMA</td>
			<td style="width:132px;">M4287</td>
			<td>soluble in water (0.5 mg/ml)</td>
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		<tr height="20">
			<td height="20" style="height:20px;">gelatin</td>
			<td style="width:287px;">SIGMA</td>
			<td style="width:132px;">G1890</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primate ES Cell Medium</td>
			<td style="width:287px;">Reprocell</td>
			<td style="width:132px;">RCHEMD001</td>
			<td>warm in 37 &#8451; water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">basic fibroblast growth factor (bFGF)</td>
			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">PHG0264</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ReproStem</td>
			<td style="width:287px;">Reprocell</td>
			<td style="width:132px;">RCHEMD005</td>
			<td>warm in 37 &#8451; water bath before use</td>
		</tr>
	</tbody>
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generation of induced pluripotent stem cells from human melanoma tumor-infiltrating lymphocytes

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.

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...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes at JoVE.com

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

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 ...

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