We thank Yoshiko Miyake, Sayaka Kanaami, Chihana Fujita, Miho Yamaguchi, Natsuko Henmi, and Rei Ohno from the Keio University School of Medicine for technical assistance. This work was partly funded by an R&#38;D Systems support program to accelerate the practical use of health research outcomes, and the Highway Program for the Realization of Regenerative Medicine.

1. Prepare Activated Human T Cells
<ol>
	<li>Collect 10 ml heparinized whole blood from a donor by venipuncture.
	<ol>
		<li>Obtain informed consent of donors in advance of venipuncture. Venipuncture must be done by qualified and trained personnel.</li>
		<li>Handle obtained blood samples with sterile equipment and following appropriate biosafety guidelines.</li>
	</ol>
	</li>
	<li>Dilute 10 ml heparinized whole blood with 10 ml D-PBS(&#8211;).</li>
	<li>Put 15 ml Ficoll solution (Ficoll-Paque PREMIUM) into a new 50-ml conical tube.</li>
	<li>Layer the 20-ml blood solution prepared in Step 1.2 onto the Ficoll solution in Step 1.3. Using an electric pipette, let blood solution prepared in Step 1.2 run slowly along the inside wall of tube. Be careful not to mix the blood solution and Ficoll solution.</li>
	<li>Centrifuge at 400 &#215; g for 30 min at RT. Set the centrifugal deceleration speed to &#8220;slow&#8221;. 
	Note: After centrifugation, a white thin layer emerges between an upper milky plasma layer and a lower clear Ficoll layer. This white thin layer contains mononuclear cells.</li>
	<li>Transfer the mononuclear cell layer to a new 50-ml conical tube with a 1-ml pipette. Be careful not to include Ficoll solution.</li>
	<li>Add D-PBS(&#8211;) to the mononuclear cells to a total volume of 10 ml, and centrifuge at 200 &#215; g for 5 min at RT.</li>
	<li>Discard the supernatant. Add 10 ml of D-PBS(&#8211;) to the mononuclear cells, and centrifuge at 200 &#215; g for 5 min at RT.</li>
	<li>Discard the supernatant, add 1 ml of KBM502 medium to the collected mononuclear cells, and count the cells using a hemocytometer. Over 5 &#215; 106 mononuclear cells can be obtained from 10 ml heparinized whole blood with appropriate separation using Ficoll.</li>
	<li>Prepare an anti-human CD3 antibody solution at a concentration of 10 &#181;g/ml in D-PBS(&#8211;). Add the anti-human CD3 antibody solution to a 6-well plate, soak the surface of each well, and incubate the dish at 37 &#176;C in a 5% CO2 incubator for at least 30 min.</li>
	<li>Remove the anti-human CD3 antibody solution from each well and wash once with D-PBS(&#8211;) just prior to seeding the cells.</li>
	<li>Seed mononuclear cells that were prepared in Step 1.9 at a density of 2.5 &#215; 105 cells/cm2 on the anti-human CD3 antibody-coated 6-well plates in KBM502 medium. Incubate at 37 &#176;C in a 5%-CO2 incubator for 3&#8211;7 days without medium change until the T cells reach confluency. T cells in this period are both suspended and adhering cells.</li>
</ol>
2. Infect Human T Cells with SeV Vectors
<ol>
	<li>After 3&#8211;7 days culture, collect the activated T cells with existing medium by pipetting. Transfer them to a 15-ml conical tube and centrifuge at 200 &#215; g for 5 min at RT.</li>
	<li>Remove the supernatant and add 1 ml fresh KBM502 medium. Count the cells and plate 1.5 &#215; 106 cells (in 2 ml fresh KBM502 medium) into each well of an anti-CD3 antibody-coated 6-well plate.</li>
	<li>Thaw the SeV solutions of OCT3/4-SeV/TS&#916;F, SOX2-SeV/TS&#916;F, KLF4-SeV/TS&#916;F, and c-MYC (HNL)-SeV/TS15&#916;F on ice.</li>
	<li>Add the SeV solutions containing OCT3/4-SeV/TS&#916;F, SOX2-SeV/TS&#916;F, KLF4-SeV/TS&#916;F, and c-MYC (HNL)-SeV/TS15&#916;F individually to the wells, each at a multiplicity of infection (MOI) of 10. Incubate at 37 &#176;C in a 5%-CO2 incubator for a further 24 hr.</li>
</ol>
3. Remove SeV Vectors from Human T Cells
<ol>
	<li>24 hr after infection, collect the infected cells with a pipette. If there are adhering cells, they can easily be detached pipetting them repeatedly up and down. Transfer them to a 15-ml conical tube, and centrifuge at 200 &#215; g for 5 min at RT.</li>
	<li>Remove the supernatant containing the SeV vectors, and add 2 ml fresh KBM502 medium. Replate cells in each of the wells. Incubate at 37 &#176;C in a 5%-CO2 incubator for an additional 24 hr.</li>
</ol>
4. Reseed Cells on a Matrigel Layer
<ol>
	<li>Prepare basement membrane matrix solution by thawing matrix gel (please see the Materials List for the specific brand used in this study) at 4 &#176;C and dissolving 0.2 ml in 10 ml of DMEM/F12 medium. 
	Note: Matrix gel (please see the Materials List for the specific brand used in this study) needs to thaw O/N at 4 &#176;C to be completely thawed. Keep it on ice until just before use.</li>
	<li>Plate 5 ml basement membrane matrix solution per dish into 100-mm dishes and leave at RT for at least 30 min before seeding cells. At least two 100-mm dishes are needed for one experiment.</li>
	<li>Collect SeV-infected cells by pipetting, transfer them to a 15-ml conical tube, and centrifuge at 200 &#215; g for 5 min at RT.</li>
	<li>Remove the supernatant and add 1 ml fresh KBM502 medium. Count the number of cells using a hemocytometer, remove the basement membrane matrix solution from 100-mm dishes prepared in Step 4.2 and plate 1 &#215; 105 and 1 &#215; 106 cells (in 10 ml fresh mTeSR medium) on a 100-mm basement membrane matrix-coated dishes. Finally, use the plate in which colonies are easily picked.</li>
	<li>Incubate the dish at 37 &#176;C in a 5%-CO2 incubator O/N.</li>
	<li>Change the medium to 10 ml flesh mTeSR medium every other day. 
	Note: Approximately 20&#8211;30 days after infection, colonies that closely resemble embryonic stem cells (ESCs) will appear.</li>
</ol>
5. Expand T Cell-derived iPSCs
<ol>
	<li>Prepare basement membrane matrix solution by thawing matrix gel (please see the Materials List for the specific brand used in this study) at 4 &#176;C and dissolving 0.2 ml in 10 ml of DMEM/F12 medium.</li>
	<li>Plate 1 ml of the basement membrane matrix solution per well in a 6-well plate and leave at RT for at least 30 min before picking colonies.</li>
	<li>Fill a 96-well plate with 20 &#181;l of mTeSR medium per well.</li>
	<li>Select colonies that resemble ESCs under the stereomicroscope using a 20 &#181;l pipette. 
	Note: The colonies of ESCs and iPSCs are round and flat as shown in Figure 1B.</li>
	<li>Transfer selected colonies into the 96-well plate filled with mTeSR medium in Step 5.3.</li>
	<li>Add 200 &#181;l of mTeSR medium to each well and carefully pipette up and down to break the colony into small clumps under the stereomicroscope using a 200 &#181;l pipette. Remove the basement membrane matrix solution from the well prepared in 5.2 and put each colony into one well of the basement membrane matrix-coated 6-well plate with 2 ml of mTeSR medium per well.</li>
	<li>Change the medium to 2 ml flesh mTeSR medium every other day.</li>
</ol>
6. Maintain T Cell-derived iPSCs
The T cell-derived iPSCs can be maintained and stored using the same techniques as for human iPSCs and human ESCs. Once iPSC colonies grow up, expand them into larger dishes by repeating the same procedure.
<ol>
	<li>Change the mTeSR medium every 1&#8211;2 days.</li>
	<li>When the colonies become confluent every 5&#8211;7 days, passage the cells using dissociation solution for human iPSCs and human ESCs according to the manufacturer&#8217;s instructions.</li>
</ol>

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, 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=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318 (5858), 1917-1920 (2007).</li><li>Hayes, M., Zavazava, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+to+generate+induced+pluripotent+stem+cells.">Strategies to generate induced pluripotent stem cells.</a> Methods Mol Biol. 1029, 77-92 (2013).</li><li>Gonzalez, F., Boue, S., Izpisua Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+making+induced+pluripotent+stem+cells:+reprogramming+a+la+carte.">Methods for making induced pluripotent stem cells: reprogramming a la carte.</a> Nat Rev Genet. 12, 231-242 (2011).</li><li>Loh, Y. 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>Sampsell-Barron, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xeno-free+adaptation+and+culture+of+human+pluripotent+stem+cells.">Xeno-free adaptation and culture of human pluripotent stem cells.</a> Methods Mol Biol. 1001, 81-97 (2013).</li><li>Thomson, J. 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Minegishi, 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=Enhanced+optineurin+E50K-TBK1+interaction+evokes+protein+insolubility+and+initiates+familial+primary+open-angle+glaucoma.">Enhanced optineurin E50K-TBK1 interaction evokes protein insolubility and initiates familial primary open-angle glaucoma.</a> Hum Mol Genet. 22, 3559-353367 (2013).</li><li>Tanaka, 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=Endothelin-1+induces+myofibrillar+disarray+and+contractile+vector+variability+in+hypertrophic+cardiomyopathy-induced+pluripotent+stem+cell-derived+cardiomyocytes.">Endothelin-1 induces myofibrillar disarray and contractile vector variability in hypertrophic cardiomyopathy-induced pluripotent stem cell-derived cardiomyocytes.</a> J Am Heart Assoc. 3, e001263 (2014).</li><li>Ludwig, T. E., 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=Derivation+of+human+embryonic+stem+cells+in+defined+conditions.">Derivation of human embryonic stem cells in defined conditions.</a> Nat Biotechnol. 24, 185-187 (2006).</li><li>Kishino, 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=Derivation+of+transgene-free+human+induced+pluripotent+stem+cells+from+human+peripheral+T+cells+in+defined+culture+conditions.">Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.</a> PLoS One. 9, e97397 (2014).</li><li>Masaki, I., 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=Recombinant+Sendai+virus-mediated+gene+transfer+to+vasculature:+a+new+class+of+efficient+gene+transfer+vector+to+the+vascular+system.">Recombinant Sendai virus-mediated gene transfer to vasculature: a new class of efficient gene transfer vector to the vascular system.</a> FASEB J. 15, 1294-1296 (2001).</li><li>Li, H. O., 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=A+cytoplasmic+RNA+vector+derived+from+nontransmissible+Sendai+virus+with+efficient+gene+transfer+and+expression.">A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression.</a> J Virol. 74, 6564-6569 (2000).</li><li>Ikeda, 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=Recombinant+Sendai+virus-mediated+gene+transfer+into+adult+rat+retinal+tissue:+efficient+gene+transfer+by+brief+exposure.">Recombinant Sendai virus-mediated gene transfer into adult rat retinal tissue: efficient gene transfer by brief exposure.</a> Exp Eye Res. 75, 39-48 (2002).</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>Macarthur, C. C., 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+human-induced+pluripotent+stem+cells+by+a+nonintegrating+RNA+Sendai+virus+vector+in+feeder-free+or+xeno-free+conditions.">Generation of human-induced pluripotent stem cells by a nonintegrating RNA Sendai virus vector in feeder-free or xeno-free conditions.</a> Stem cells Int. , 564612 (2012).</li><li>Iida, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sendai+virus+vector:+vector+development+and+its+application+to+health+care+and+biotechnology.">Sendai virus vector: vector development and its application to health care and biotechnology.</a> Uirusu. 57, 29-36 (2007).</li><li>Yoshida, K., 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=In+vivo+repopulation+of+cytoplasmically+gene+transferred+hematopoietic+cells+by+temperature-sensitive+mutant+of+recombinant+Sendai+viral+vector.">In vivo repopulation of cytoplasmically gene transferred hematopoietic cells by temperature-sensitive mutant of recombinant Sendai viral vector.</a> Biochem Biophys Res Comm. 361, 811-816 (2007).</li><li>Bitzer, M., Armeanu, S., Lauer, U. M., Neubert, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sendai+virus+vectors+as+an+emerging+negative-strand+RNA+viral+vector+system.">Sendai virus vectors as an emerging negative-strand RNA viral vector system.</a> J Gene Med. 5, 543-553 (2003).</li><li>Seki, T., Yuasa, S., Fukuda, K. <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+a+small+amount+of+human+peripheral+blood+using+a+combination+of+activated+T+cells+and+Sendai+virus.">Generation of induced pluripotent stem cells from a small amount of human peripheral blood using a combination of activated T cells and Sendai virus.</a> Nat Protoc. 7, 718-728 (2012).</li><li>Okano, S., 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=Recombinant+Sendai+virus+vectors+for+activated+T+lymphocytes.">Recombinant Sendai virus vectors for activated T lymphocytes.</a> Gene Ther. 10, 1381-1391 (2003).</li><li>Okita, K., 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=An+efficient+nonviral+method+to+generate+integration-free+human-induced+pluripotent+stem+cells+from+cord+blood+and+peripheral+blood+cells.">An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.</a> Stem Cells. 31 (3), 458-466 (2013).</li><li>Zhang, X. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+reprogramming+of+human+peripheral+blood+cells.">Cellular reprogramming of human peripheral blood cells.</a> Genom Proteom Bioinform. 11 (5), 264-274 (2013).</li><li>Su, R. J., Neises, A., Zhang, X. B. <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+iPS+Cells+from+Human+Peripheral+Blood+Mononuclear+Cells+Using+Episomal+Vectors.">Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors.</a> Methods Mol Biol. , (2014).</li></ol>

The authors have no competing or conflicting interests to disclose.

We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation. 
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger. 
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.

iPSCs have attracted considerable attention as a groundbreaking source of cells for regenerative medicine1-3. To date, diverse methods for generating iPSCs have been reported4,5. Among these, iPSCs generated from human T cells have been of particular interest because of the less-invasive method of cell sampling6-8. Additionally, iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) gene and are thus a source of antigen-specific T cells9,10. Therefore, generating T cell derived iPSCs safely is useful for progressing regenerative medicine.
This method is based on the concept of reducing the risk of unpredictable pathogenicity. For safe clinical application of iPSCs it is important to reduce the risk of exposure to pathogens11. Previously in many culture systems of pluripotent stem cells, fetal bovine serum and feeder cells have been used as essential reagents12. However, removal of both these reagents from the culture system is preferable for iPSC generation to reduce the risk of unpredictable pathogenicity.
Additionally, this method has the advantage of avoiding invasive cell sampling from patients and laborious preparation of feeder cells. Because T cell derived iPSCs have already been used successfully in disease research13,14, this method is also applicable and useful for generating disease-specific iPSCs from patients.
Among T cell reprogramming methods, using the Sendai virus (SeV) vector as a gene vehicle is a method that can generate iPSCs with high efficiency7,16. Additionally, because SeV is a single-stranded RNA virus and does not need a DNA phase for replication, its use in iPSC generation avoids breaking the host genome17-19. Therefore, we have established protocols for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors.

<table border="0">
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified NA/LE mouse anti-human CD3</td>
			<td>BD Pharmingen</td>
			<td>555336</td>
			<td></td>
		</tr>
		<tr>
			<td>KBM502 medium</td>
			<td>KOHJIN BIO</td>
			<td>16025020</td>
			<td>Warm in 37 &#8451; water bath before use</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V solution</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td>Thaw completely at 4&#8451; overnight and dilute it 50 times with Dulbecco&#39;s Modified Eagle&#39;s Medium before coating culture dishes</td>
		</tr>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>STEM CELL</td>
			<td>5850</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr>
			<td>Dissociation Solution</td>
			<td>ReproCELL</td>
			<td>RCHETP002</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS(&#8211;)</td>
			<td>Wako</td>
			<td>045-29795</td>
			<td></td>
		</tr>
		<tr>
			<td>SeV Vector kit CytoTune-iPS ver.1.0</td>
			<td>DNAVEC</td>
			<td>DV-0303c</td>
			<td>Thaw on ice before use</td>
		</tr>
		<tr>
			<td>100-mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
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			<td>6-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353046</td>
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			<td>15ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>188271</td>
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			<td>50ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>227261</td>
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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.

Using this protocol, users are able to generate iPSCs from human peripheral T cells stably. iPSC generation from T cells with SeV and matrigel showed approximately 0.002% &#8211; 0.005% of efficiency of cell reprogramming in between several donor cases and SeV were not detected after several passages16. Figure 1A shows a schematic of the protocol for generating T cell-derived iPSCs in feeder-free conditions using matrigel and mTeSR medium. Around 20&#8211;30 days after infection with SeV, iPSC colonies are recognized by their ESC colony-like morphology (Figure 1B). Immunofluorescence staining revealed expression of typical pluripotent cell markers (NANOG, OCT3/4, SSEA4, TRA-1-60, and TRA-1-81) in T cell-derived iPSCs generated under feeder-free conditions (Figure 1C).
<img alt="Figure 1" src="/files/ftp_upload/53225/53225fig1highres.jpg" width="700" /> 
Figure 1. (A): A schematic of the protocol for reprogramming T cells under feeder-free conditions in this study. (B): A typical ESC-like iPSC colony on day 27 after blood sampling under feeder-free conditions. (C): ALP staining and immunofluorescence staining for pluripotency and surface markers (NANOG, OCT3/4, SSEA4, TRA-1-60, and TRA-1-81) in T cell-derived iPSCs generated under feeder-free conditions. <a href="https://www.jove.com/files/ftp_upload/53225/53225fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

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

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 ...

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Developmental Biology

11.9K Views.  Keio University School of Medicine. 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.

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

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

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 ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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.

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 ...

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 ...