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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">RPMI 1640</td>
			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">11875-093</td>
			<td></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>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
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			<td height="20" style="height:20px;width:271px;">IMDM</td>
			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">12440053</td>
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		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">2-mercaptoethanol (1000x, 55mM)</td>
			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">21985-023</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">gentamicin</td>
			<td style="width:287px;">Life technologies</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">FBS</td>
			<td style="width:287px;">Gibco</td>
			<td style="width:132px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">HEPES</td>
			<td style="width:287px;">Life technologies</td>
			<td style="width:132px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Falcon Tissue Culture Plates (6-well)</td>
			<td style="width:287px;">Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<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>
			<td></td>
		</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>
		</tr>
		<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>
</table>

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 ...

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

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...