Name: Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes (Video) | JoVE Journal
Uploaded: 2016-11-11T13:00:00.000+00:00
Duration: 10 min 3 s
Description: Learn to generate induced pluripotent stem cells from melanoma tumor-infiltrating lymphocytes using Sendai virus reprogramming. This method enables unlimited production of tumor-specific T cells for cancer immunotherapy research.

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5 minutes

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 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 possible.</li>
	<li>Dissociate the minced specimen into a single-cell suspension using a dissociator and tumor dissociation kit (human) according to the manufacturer&#39;s instructions. Filter the suspension with a 70 &#181;m cell strainer that is set on a 50 ml tube and wash the strainer 2 times with 2 ml of RPMI 1640.</li>
	<li>Centrifuge at 200 x g at room temperature for 5 min. Discard the supernatant and resuspend the cells in 10 ml of T cell media (Table 1).</li>
	<li>Prepare a two-step gradient solution such as Ficoll (gradient solution) in a 50-ml tube, with a lower step of 10 ml of 100% gradient solution and a middle step of 30 ml of 75% gradient solution diluted with Dulbecco&#39;s phosphate-buffered saline, no calcium, no magnesium (D-PBS(-)).</li>
	<li>Layer the cell suspension (from Step 1.4) on the gradient solution (from Step 1.5). Centrifuge at 400 x g at 20 &#176;C for 45 min.</li>
	<li>Collect the layer containing the enriched TILs at the interface between the 100% gradient solution and the 75% gradient solution in a 50 ml tube. Dilute the layer of the solution with the enriched TILs by adding 20-30 ml of D-PBS(-).</li>
	<li>Centrifuge at 200 x g at room temperature for 5 min. Discard the supernatant and resuspend the TIL-enriched fraction in 10 ml of T cell media.</li>
	<li>Culture the fraction (from Step 1.8) with 2 ml of T cell media and 6,000 IU/ml recombinant human (rh) IL-2 in a 6-well plate at 37 &#176;C, 5% CO2.</li>
	<li>Change half the media on day 5 after culture initiation and every 2-3 days thereafter.</li>
	<li>Split the cells at a ratio of 1:2 without trypsinization after softly suspending, doubling the number of wells when reaching 80-90% confluency. Add a half volume (1 ml) of fresh T cell media and rhIL-2 at 6,000 IU/ml.</li>
</ol>
2. Preparation of Mitomycin-C-treated SNL Feeder Cell Plate
<ol>
	<li>Culture SNL feeder cells in 10 ml of SNL feeder cell media (Table 1) on a 0.1% gelatin-coated 10 cm dish until 80-90% confluence (3-4 x 106 cells) is reached.</li>
	<li>Add 310 &#181;l of 0.4 mg/ml mitomycin C solution directly into the SNL feeder cell culture dish and incubate at 37 &#176;C, 5% CO2 for 2 hr and 15 min. Aspirate the media and wash the cells twice with 5 ml D-PBS(-).</li>
	<li>Add 0.5 ml of 0.25% trypsin/1 mM EDTA and incubate at room temperature for 1 min to dissociate the cells. Add 4.5 ml of SNL feeder cell media to neutralize and re-suspend the cells into a single suspension.</li>
	<li>Transfer the cells into a 15 ml tube and centrifuge at 200 x g for 5 min. Aspirate the media and count the cells with a hemocytometer after re-suspending in 10 ml of SNL feeder cell media.</li>
	<li>Plate 1.5 x 106 cells per well of the SNL feeder cells on a 10 cm dish and incubate at 37 &#176;C, 5% CO2. Use the SNL feeder cells within 3 days.</li>
</ol>
3. Generation of iPSCs Using the Sendai Virus (SeV) Vector
<ol>
	<li>Harvest the TILs (from Step 1.11) and activate 5 x 105 cells of TILs with plate-bound mouse anti-human CD3 (1 &#181;g/ml) and soluble mouse anti-human CD28 (5 &#181;g/ml), with rhIL-2 (6,000 IU/ml) in 2 ml of T cell media in a 6-well plate at 37 &#176;C, 5% CO2 for 5 days.</li>
	<li>Harvest the TILs (from Step 3.1) in a 15 ml tube using a pipette and count the cell number with a hemocytometer.</li>
	<li>Reactivate 1 x 105 cells of TILs with plate-bound mouse anti-human CD3 (1 &#181;g/ml) and soluble mouse anti-human CD28 (5 &#181;g/ml), with rhIL-2 (60 IU/ml) in 500 &#181;l of reprogramming media (Table 1) per well in a 24-well plate at 37 &#176;C, 5% CO2 for 24 hr. Prepare 3 wells for SeV infection: OCT3/4, SOX2, KLF4, and c-MYC (1 well); SeV infection (GFP: green fluorescent protein) (1 well); and cell count (1 well).</li>
	<li>After 24 hr (from Step 3.3), count the cell number with a hemocytometer and determine the volume of each virus for various (3-20) multiplicities of infection (MOI). Remove one set of Sendai virus vector tubes from the -80 &#176;C storage. Thaw the SeV vectors quickly in a water bath (37 &#176;C) and place them on ice. 
	Volume of virus (&#181;l) = MOI (CIU/cell) x number of cells/titer of virus (CIU/ml) &#215; 10-3 (&#181;l/ml)</li>
	<li>Add the calculated volumes of each of the four Sendai virus vector tubes that individually carried OCT3/4, SOX2, KLF4, and c-MYC by adding a mixture of SeV vectors at 10-20 MOI into the media, including activated TILs.</li>
	<li>To determine the transduction efficiency, add the calculated volumes of SeV-GFP into a well of activated TILs.</li>
	<li>Place the dish (from Step 3.5 and Step 3.6) in the incubator at 37 &#176;C, 5% CO2 for 24 hr. After 24 hr, estimate the infection efficiency using TILs infected with SeV-GFP under fluorescence microscopy.</li>
	<li>Collect the cells (from Step 3.5) in a 15 ml tube. Centrifuge at 200 x g at room temperature for 5 min.</li>
	<li>Discard the supernatant and resuspend the pellet in 0.5 ml of hESC media with Primate ES Cell Medium and basic fibroblast growth factor (bFGF, 4 ng/ml).</li>
	<li>Transfer the suspension into a 10 cm dish that contains mitomycin-C-treated SNL feeder cells in 10 ml of hESC media. Change the hESC media every other day until the colonies are grown.</li>
	<li>Around day 18 to 21, remove the SNL feeder cells around the colonies under a microscope using a 10-&#181;l tip in the clean bench.</li>
	<li>Scrape the colonies using a 10-&#181;l tip and transfer them using a 200-&#181;l tip into 100 &#181;l of iPSCs media per well of a 96-well tissue culture plate. Pipet up and down at 2-3 times to break the colonies into small clumps with an average size of 50-100 &#181;m.</li>
	<li>Transfer the small clumps into 6-well tissue culture plates with mitomycin-C-treated SNL feeder cells (from Step 2) in 2 ml per well of iPSCs media with basic fibroblast growth factor (bFGF, 4 ng/ml). Change iPSCs media every day.</li>
	<li>Transfer the iPSCs to 6-well tissue culture plates with fresh mitomycin-C-treated SNL feeder cells with 1 mg/ml collagenase IV every 5-6 days. Prepare 2 wells per plates of each clone for immunostaining.</li>
	<li>Determine the full reprogramming of each clone by immunohistochemical staining of pluripotency markers (SSEA3, SSEA4, TRA-1-60, TRA-1-81, and OCT3/4)1. 
	NOTE: For further evaluation of pluripotency potential, confirm that fully-reprogrammed iPSC lines can differentiate into three germ layers by an embryoid body formation and differentiation assay or a teratoma formation assay16.</li>
	<li>Confirm that fully-reprogrammed iPSC lines demonstrate a normal karyotype by cytogenetic analysis.</li>
	<li>For teratoma formation, inject an undifferentiated TIL-derived iPSC (1 x 106) suspension in 100 &#181;l of DMEM containing 10% FCS into the subcutaneous tissue of 6-8 week old female NOD/SCID mice. Stain teratoma sections with hematoxylin and eosin.</li>
</ol>
4. Immunofluorescence Staining of iPSCs for SSEA3, SSEA4, TRA1-60 and TRA1-81
<ol>
	<li>Wash iPSCs with 1 ml of PBS and fix the iPSCs with 1 ml of 4% paraformaldehyde solution for 10 min. Remove the 4% paraformaldehyde solution and wash the iPSCs with 1 ml of PBS 3 times. 
	NOTE: Be careful when using paraformaldehyde because it is toxic.</li>
	<li>Pretreat the iPSCs with blocking buffer (Table 1) for 30 min. Meanwhile, dilute the primary antibodies (rat anti-human SSEA3, mouse anti-human SSEA4, mouse anti-human TRA1-60, and mouse anti-human TRA1-81) 1:50 in 1.5% goat serum solution diluted with PBS and TritonX-100 (total volume: 1 ml).</li>
	<li>Remove the blocking buffer (Table 1). Add the diluted primary antibody solution and incubate for 1 hr at room temperature. Meanwhile, dilute the secondary antibody (anti-mouse IgG and IgM or anti-rat IgM) 1:50 in 1.5% goat serum solution.</li>
	<li>Remove the diluted primary antibody solution and wash the iPSCs with 1 ml PBS 3 times, each for 5 min. Add the diluted secondary antibody solution and incubate for 45 min at room temperature in a dark room.</li>
	<li>Remove the diluted secondary antibody solution and wash the iPSCs with 1 ml of PBS 3 times, each for 5 min. Observe the iPSCs with immunofluorescence microscopy.</li>
</ol>
5. Immunofluorescence Staining of iPSCs for Oct3/4
<ol>
	<li>Wash iPSCs with 1 ml of PBS and fix the iPSCs with 1 ml of 4% paraformaldehyde solution for 10 min. Remove the 4% paraformaldehyde solution and wash the iPSCs with PBS 3 times.</li>
	<li>Add permeabilization buffer (Table 1) and incubate at room temperature for 10 min. Remove the permeabilization buffer and add blocking buffer (Table 1).</li>
	<li>Incubate at room temperature for 30 min. Meanwhile, dilute the primary antibody (mouse anti-human Oct3/4) 1:50 in blocking buffer.</li>
	<li>Add the diluted primary antibody solution and incubate at 4 &#176;C overnight.</li>
	<li>Remove the diluted primary antibody solution and wash the iPSCs with 1ml of PBS 3 times, each for 5 min. Meanwhile, dilute the secondary antibody (goat anti-mouse IgG/IgM) 1:100 in blocking buffer.</li>
	<li>Add the diluted secondary antibody solution and incubate at room temperature in a dark room for 45 min.</li>
	<li>Remove the diluted secondary solution and wash the iPSCs with 1 ml of PBS 3 times, each for 5 min. Observe the iPSCs under immunofluorescence microscopy.</li>
</ol>

<ol><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li>
<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li>
<li>Wernig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li>
<li>Maherali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directly+reprogrammed+fibroblasts+show+global+epigenetic+remodeling+and+widespread+tissue+contribution%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.</a> Cell Stem Cell. 1, 55-70 (2007).</li>
<li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+germline-competent+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li>
<li>Loh, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reprogramming+of+T+cells+from+human+peripheral+blood%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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/pubmed?term=((Generation+of+induced+pluripotent+stem+cells+from+human+terminally+differentiated+circulating+T+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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/pubmed?term=((Reprogramming+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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/pubmed?term=((Generation+of+rejuvenated+antigen-specific+T+cells+by+reprogramming+to+pluripotency+and+redifferentiation%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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/pubmed?term=((Regeneration+of+Human+Tumor+Antigen-Specific+T+Cells+from+iPSCs+Derived+from+Mature+CD8%28%2B%29+T+Cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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/pubmed?term=((Expansion+of+functional+human+mucosal-associated+invariant+T+cells+via+reprogramming+to+pluripotency+and+redifferentiation%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">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/pubmed?term=((Generation+of+tumor-infiltrating+lymphocyte+cultures+for+use+in+adoptive+transfer+therapy+for+melanoma+patients%5BTitle%5D)+AND+%22J+Immunother%22%5BJournal%5D)">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/pubmed?term=((PD-1+identifies+the+patient-specific+CD8%28%2B%29+tumor-reactive+repertoire+infiltrating+human+tumors%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">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/pubmed?term=((Durable+Complete+Responses+in+Heavily+Pretreated+Patients+with+Metastatic+Melanoma+Using+T-Cell+Transfer+Immunotherapy%5BTitle%5D)+AND+%22Clinical+Cancer+Research%22%5BJournal%5D)">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/pubmed?term=((Adoptive+immunotherapy+for+cancer%3A+harnessing+the+T+cell+response%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">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/pubmed?term=((Reprogramming+of+Melanoma+Tumor-Infiltrating+Lymphocytes+to+Induced+Pluripotent+Stem+Cells%5BTitle%5D)+AND+%22Stem+Cells+International%22%5BJournal%5D)">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/pubmed?term=((Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus%2C+an+RNA+virus+that+does+not+integrate+into+the+host+genome%5BTitle%5D)+AND+%22Proc+Jpn+Acad+Ser+B+Phys+Biol+Sci%22%5BJournal%5D)">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/pubmed?term=((Efficient+generation+of+transgene-free+human+induced+pluripotent+stem+cells+%28iPSCs%29+by+temperature-sensitive+Sendai+virus+vectors%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((New+Type+of+Sendai+Virus+Vector+Provides+Transgene-Free+iPS+Cells+Derived+from+Chimpanzee+Blood%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood.</a> PLoS One. 9, e113052(2014).</li>
</ol>

The authors declare that they have no competing financial interests.

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.

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

generation of induced pluripotent stem cells from human melanoma tumor-infiltrating lymphocytes

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...

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 ...

The overall goal of this experiment is to generate induced pluripotent stem cells or iPSCs from melanoma infiltrating lymphocytes. This method can help answer key questions in the field of tumor immunology especially for cancer immunotherapy. The main advantage of this technique is that the ability to generate human iPS cells from tumor-infiltrating lymphocytes could provide unlimited numbers of less differentiated tumor specific T cells for immune therapy.This technique may have applications for the treatment of metastatic melanoma as currently the poor survival of T cells in a clinic has been the major limitation of adaptive T cell therapy. This method may also be useful in the investigation of the T cell receptor repertoire in the tumor microenvironment. Demonstrating the procedure will be Dr.Kumiko Iwabuchi, a post doctoral fellow from my laboratory.After obtaining the tumor material select five to 20 grams of tumor specimens and use scissors to dissect the normal, solid, and firm tissue from the fragile and or bloody necrotic areas of the tumor samples. Mince the dissected specimens as finely as possible then use a dissociator and a human tumor dissociation kit to disperse the minced tumor pieces into a single cell suspension according to the manufacturer's instructions. At the end of the dissociation filter the cells through 70 micron strainer into a 50 milliliter tube and wash the strainer two times with two milliliters of RPMI 1640.Then pellet the cells by centrifugation and re-suspend the cells in 10 milliliters of fresh T-cell medium. To separate the cells by density gradient first layer 30 milliliters of 75%gradient solution followed by 10 milliliters of 100%gradient solution in DPBS without calcium or magnesium into a new 50 milliliter tube. Then carefully overlay the cells onto the gradient solution taking care not to disrupt the layers and centrifuge the sample.At the end of separation, transfer the enriched tumor-infiltrating lymphocytes or TILs at the interface between the gradient solutions into a new 50 milliliter conical tube and wash the cells with 20 to 30 milliliters of fresh DPBS. Re-suspend the pellet in 10 milliliters of fresh T-cell medium, then add two milliliters of TILs per well to five wells of a six well plate and supplement the TIL cultures with Recombinant Human IL-2 for five days at 37 degrees Celsius and 5%carbon dioxide. On day five, change half the medium in each well and every two to three days thereafter.When the cultures reach 80 to 90%confluency use a pipette to gently re-suspend the cells in each well and split the cultures at a one-to-two ratio in one milliliter of fresh T-cell medium supplemented with Recombinant Human IL-2. To prepare mitomycin-C-treated SNL feeder plates, first culture SNL feeder cells in 10 milliliters of SNL feeder cell medium on a 0.1%gelatin coated 10 centimeter dish. When the cells are 80 to 90%confluent, treat the feeders with mitomycin C for two hours and 15 minutes at 37 degrees Celsius in 5%carbon dioxide.At the end of incubation, wash the cells two times with five milliliters of DPBS. Then detach the feeders with room-temperature Trypsin-EDTA. After one minute neutralize the dissociation reaction with 4.5 milliliters of fresh SNL feeder medium and transfer the single cell suspension into a 15 milliliter conical tube for centrifugation.Re-suspend the pellet in 10 milliliters of fresh SNL feeder cell medium for counting. Then plate 1.5 times 10 to the six SNL feeder cells per well in 10 centimeter dishes and incubate the cultures at 37 degrees Celcius in 5%carbon dioxide for up to three days. To generate the iPSCs with Sendai Virus transfer five times 10 to the fifth TILs per well to a new six well plate coated with mouse anti-human CD3 in two milliliters of fresh T cell medium supplemented with Recombinant Human IL-2 and soluble mouse anti-human CD28.Activate the TILs in the cell culture incubator for five days. Then use a pipette to pool the cultures in a 15 milliliter conical tube for counting. Next transfer one times 10 to the fifth TILs per well into a 24 well plate coated with anti-human CD3 in 500 microliters of reprogramming medium supplemented with soluble mouse anti-human CD28 in Recombinant Human IL-2.After 24 hours in the cell culture incubator enumerate the cells in each well and prepare several volumes of virus at the appropriate multiplicities of infection. Then add a mixture of Sendai Virus vectors at 10 to 20 MOI into the appropriate volume of medium and add the virus containing medium to each well of TILs. For determination of the transduction efficiency add the calculated volume of Sendai Virus GFP into a well of activated TILs and return the cultures to the incubator for 24 hours.The next day, view the Sendai Virus GFP infected cells by fluorescence microscopy to estimate the infection efficiency of the transduction. Pool the virally infected cells from each well into a 15 milliliter tube for centrifugation and re-suspend the pellet in 0.5 milliliters of human embryonic stem cell medium supplemented with primate embryonic stem cell medium and basic fibroblast growth factor. Next add 9.5 milliliters of fresh human embryonic stem cell medium to the cells and transfer the suspension onto a mitomycin C treated SNL feeder cell plate.In this image TILs on day 21 of culture with Recombinant Human IL-2 that are ready to be activated with anti CD3 and CD28 are shown. The TILs can be transfected with Sendai Virus at a multiplicity of infection of 20. Here a typical pluripotent clone on SNL feeder cells 18 to 21 days after Sendai Virus infection is shown.As observed in this representative karyogram the TIL derived iPSCs exhibit a normal karyotype. Immunofluorescence staining to confirm pluripotency marker expression on TIL-derived iPSCs demonstrates that these cells exhibit the expected levels of expression of typical pluripotency markers. In addition, iPSCs derived from melanoma TILs are able to form teratomas that contain a variety of cells from the three germ layers.Further, TIL derived iPSCs retained their T cell receptor rearrangements as assessed by capillary electrophoresis. Our first development, this technique pave the way for researchers in the field of oncology to explore more effective cancer immunotherapies for metastatic melanoma patients. While attempting this procedure, it is important to check T cell proliferation and viability before Sendai Virus infection and to evaluate infection efficiency by the contour GFP expression.After watching this video you should have a good understanding of how to generate human TIL cells from melanoma TILs.

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Research

JoVE Journal

Developmental Biology

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Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

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Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

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Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

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Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

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A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

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Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

Learn to generate induced pluripotent stem cells from melanoma tumor-infiltrating lymphocytes using Sendai virus reprogramming. This method enables unlimited production of tumor-specific T cells for cancer immunotherapy research.

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

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