We would like to thank members of the Lee lab for valuable discussions on the manuscript. Work in the Lee lab was supported by grants from Robertson Investigator Award of New York Stem Cell Foundation and from Maryland Stem Cell Research Fund (TEDCO).

1. Preparation of Cell and Media (Day 1)
<ol>
	<li>Culture and expand human fibroblast with DMEM media containing 10% FBS.</li>
	<li>Plate human fibroblasts (Figure&#160;1) onto a 24-well plate at the appropriate density per well on the day before transduction. 
		NOTE: The following serial dilutions are recommended (200K, 100K, 50K, 25K, 12.5K and 6.25K) because different cell types have different attachment ability.</li>
	<li>Incubate the cells for one more day in a 37 &#176;C, 5% CO2 incubator, ensuring the cells have fully adhered and extended.</li>
</ol>
2. Perform Transduction (Day 2)
<ol>
	<li>On the day of transduction, check the cell density and chose the most efficient density wells (Figure 2). It is better to pick three different densities: high (80~90%), middle (50~70%) and low (20~40%).</li>
	<li>At least 1 hr&#160;before transduction, aspirate the fibroblast media from the cells and change new 300 &#956;l of fibroblast medium.</li>
	<li>Remove one set of 4 different Sendai virus tubes from the -80 &#176;C storage. Thaw each tube at the same time in a 37 &#176;C water bath for few seconds, and then take the tubes out from the water bath. After that, thaw them to room temperature; centrifuge tubes at 6,000 x g for 10 sec and place them on ice till use. Do not re-freeze and thaw the virus since the titers will not maintain.</li>
	<li>Add the indicated volumes of each of the four Sendai virus tubes (Oct-4, Klf-4, c- Myc and Sox-2) to micro-centrifuge tube. Make sure that the solution is mixed well by pipetting gently. For example, if 50K cells/one well of 24-well plate lookwell for transduction: (Titer based on the Certificate of Analysis from Life Technologies, can be different from batch to batch) 
			Tube hOct-4 = 6.0 x 107 CIU,3 MOI = 5 &#956;l 
			Tube hSox-2 = 6.5 x 107 CIU, 3 MOI = 4.6 &#956;l 
			Tube hKlf-4 = 6.3 x 107 CIU, 3 MOI = 4.8 &#956;l 
			Tube hc-Myc = 7.8 x 107 CIU, 3 MOI = 3.8 &#956;l 
		Total = 18.2 &#956;l of four virus factors mixture/one well (50K cells) of 24-well plate</li>
	<li>Depending on cell densities; add 2X, 1X, and 0.5X volume of the virus mixture into the well. Gently shake the plate front to back, left and right (2X: 36.4 &#956;l of virus mixture, 1X: 18.2 &#956;l and 0.5X: 9.1 &#956;l).</li>
	<li>Place the cells in a 37 &#176;C, 5% CO2 incubator and incubate overnight.</li>
</ol>
3. Replacement of Culture Medium (Day 3 &#38; Day 5)
<ol>
	<li>24 hr&#160;after transduction, replace the medium with 500 &#956;l fresh fibroblast medium.</li>
	<li>On Day 5, change Medium with fresh fibroblast media.</li>
</ol>
4. Prepare MEF Dishes for the Co-culture (Day 8)
<ol>
	<li>Prepare MEF cells in 60 mm culture dishes with 10% FBS contained DMEM medium one day before passaging the transduced fibroblast onto MEF feeder-cells. 
		Note:&#160;We are using 5 x 105 of MEF in each&#160;60 mm dish.</li>
</ol>
5. Start Co-culture Transduced Cells with MEF Feeder Cells (Day 9)
<ol>
	<li>Before plating transduced cells, aspirate 10% FBS contained DMEM media from MEF feeder dishes and add fresh 10% FBS contained DMEM medium.</li>
	<li>Remove the medium from the transduced fibroblasts that are in the 24-well plate and wash cells once with D-PBS. Add 200 &#956;l of 0.25% Trypsin/EDTA into the fibroblast cells inthe 24-well plate. After less than 5 min&#160;incubation with Trypsin/EDTA, when the cells start to detach from the plate, collect the cells with growth medium in 15 ml conical tube. Then centrifuge them in 6,000 x g for 4 min. 
		NOTE: Pooling 2X, 1X, and 0.5X virus transduced fibroblast is recommended.</li>
	<li>Remove the supernatant medium and wash to re-suspend the cell pellet with 4-5 ml of fresh fibroblast medium for neutralization from trypsin. Then centrifuge them at 135 x g for 4 min.</li>
	<li>Plate cells as serial dilution density and start to co-culture with fibroblast media onto MEF: such as 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, and 1/128 per 60 mm dish. Depending on cell death rate during the first week of transduction, one may not need to dilute to 1/2 or 1/128 density. Put aside remaining cells to extract total RNA as a positive control for Transgene RT-PCR.</li>
	<li>Place 60 mm dishes in 37 &#176;C, 5% CO2 incubator overnight.</li>
</ol>
6. Feed Human Embryonic Stem Cell Medium and Monitor the Cells (Day 10)
<ol>
	<li>The next day, change fibroblast media to human ES media with 10 &#956;M Y-27632. After that, change medium daily with human ES medium: 800 ml&#160;DMEM/F12 + 200 ml&#160;Knockout Serum Replacement + 10 ml&#160;of L-Glutamine + 10 ml&#160;of 10 mM MEM minimum non-essential amino acids + 1 ml&#160;of 2-mercaptoethanol + 10 ng/ml of basic Fibroblast Growth Factor.</li>
	<li>Do a daily media change with fresh human ES media. After a week, check the dishes once per 2~3 days under a microscope for the formation of ES colony -like cell clumps. Depending on the cell type, the culture will take more or less time before colonies are found.</li>
</ol>
7. Picking the Induced Pluripotent Stem Cell Colonies and Expand the Cells (Day 20~)
<ol>
	<li>Three weeks after transduction, colonies should appear grown properly for picking.</li>
	<li>The day before picking the colonies, prepare MEF feeder in 24-well plate. NOTE:&#160;We are using 12.5 x 105 of MEF in one 24-well plate.</li>
	<li>The next day, change medium of MEF dishes from fibroblast media to human ES media with 10 &#956;M Y-27632. Manually pick one colony at each time under the microscope in the picking-hood and make smaller clumps by pipetting and transfer them onto newly prepared MEF dishes.Try to pick several clones.</li>
	<li>Passage and expand the each well from a 24-well plate to a 6 well plate initially. Then from a 6-well plate to a 60 mm dishes before further propagation.</li>
</ol>
8. Characterization of Human iPSCs (after 10 Passages)
<ol>
	<li>Immunofluorescence
		<ol>
			<li>Plate human iPSCs in 24-well plate and culture during 4-5 days with daily media change.</li>
			<li>For starting Immunofluorescence assay, wash the plate with D-PBS once. Then fix the cells immediately in 0.4% paraformaldehyde for 30 min. After that wash three times in D-PBS for 5 min.</li>
			<li>Treat fixed cells with 0.1% Triton X-100 solution in PBS for 15 min at room temperature.</li>
			<li>Aspirate the permeablization solution (0.1% triton X-100 solution), then add 0.5% BSA blocking solution for 1 hr&#160;at room temperature.</li>
			<li>Perform a detection of Nanog, Oct-4, SSEA (stage-specific embryonic antigen) and TRA-1-81 (tumor rejection antigen) using an anti-human antibody in 0.5% BSA solution (check the material table for antibody dilution rate). Incubate Primary Antibody for 2 hr&#160;at room temperature. Then wash three times in D-PBS for 5 min each.</li>
			<li>Check using fluorescence microscopy after 1 hr&#160;of incubation with Alexa Fluor 488 Goat anti-mouse IgG antibodies as 2nd&#160;antibody, which are diluted 1:2,000 in 0.5% BSA solution at room temperature. Stain all the nuclei of each MEF feeder cells and hiPSCs with DAPI.</li>
		</ol>
	</li>
	<li>RT-PCR of Transgene Confirmation
		<ol>
			<li>Extract total mRNA from cell pellet by using RNA extraction reagents.</li>
			<li>Do a reverse transcription on aliquots (1 &#956;g) of total RNA and the resultant cDNA for PCR amplification by Reverse Transcriptase.</li>
			<li>Prepare the PCR mixtures to contain 2 &#956;l of cDNA template, 10 &#956;l of 2X PCR master mix, 2 &#956;l of 2 pmol of primers (forward: 1 &#956;l and reverse: 1 &#956;l) and 6 &#956;l of DW. To detect the gene expression, do the RT-PCR with the primers listed in Table 1.</li>
			<li>Perform the RT-PCRs with GAPDH gene as a control for efficiency of the amplification reactions. Visualize and analyze PCR product by 1% agarose-gel electrophoresis.</li>
		</ol>
	</li>
</ol>

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	<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, 1917-1920 (2007).</li><li>Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., 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=Generation+of+mouse+induced+pluripotent+stem+cells+without+viral+vectors.">Generation of mouse induced pluripotent stem cells without viral vectors.</a> Science. 322, 949-953 (2008).</li><li>Zhou, W., Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+gene+delivery+can+reprogram+human+fibroblasts+to+induced+pluripotent+stem+cells.">Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells.</a> Stem Cells. 27, 2667-2674 (2009).</li><li>Woltjen, 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=piggyBac+transposition+reprograms+fibroblasts+to+induced+pluripotent+stem+cells.">piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.</a> Nature. 458, 766-770 (2009).</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=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nature Methods. 8, 409-412 (2011).</li><li>Yusa, K., Rad, R., Takeda, J., Bradley, 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+transgene-free+induced+pluripotent+mouse+stem+cells+by+the+piggyBac+transposon.">Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon.</a> Nature Methods. 6, 363-369 (2009).</li><li>Kim, D., 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+direct+delivery+of+reprogramming+proteins.">Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.</a> Cell Stem Cell. 4, 472-476 (2009).</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=Human+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324, 797-801 (2009).</li><li>Okita, K., Ichisaka, T., 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=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li><li>Yoshioka, N., 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+Human+iPSCs+by+a+Synthetic+Self-Replicative+RNA.">Efficient Generation of Human iPSCs by a Synthetic Self-Replicative RNA.</a> Cell Stem Cell. 13, 246-254 (2013).</li><li>Dowey, S. N., Huang, X., Chou, B. K., Ye, Z., Cheng, L. <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+integration-free+human+induced+pluripotent+stem+cells+from+postnatal+blood+mononuclear+cells+by+plasmid+vector+expression.">Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.</a> Nature Protocols. 7, 2013-2021 (2012).</li><li>Fluri, D. 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The authors have nothing to disclose.

Reprogramming human somatic cells to hiPSCs holds unprecedented promises in basic biology, personal medicine, and transplantation12. Previously, human iPSC generations required DNA virus that has integration risk into the host genome, which can create undesirable genetic mutations that limit further clinical applications such as drug development and transplantation therapies13. With this reason, many studies have been reported to generate vector- and transgene-free system human iPSCs by several alte...

Human embryonic stem cells (hESCs) have a capacity to self-renew in vitro and have pluripotency, which could be potentially useful for disease modeling, for drug screening, and to develop cell-based therapies to treat disease and tissue injuries. However, hESCs have a limitation for cell replacement therapy because of immunological, oncological and ethical barriers, and&#160;to study disease related genes, disease-specific hESCs could be isolated through pre-implantation genetic diagnosis (PGD) approaches, but it is still technically challenging and the embryo donations are pretty rare. These issues are related to the progress in stem cell biology, which has led to the development of induced pluripotent stem cells (hiPSCs).
Human iPSCs are genetically reprogrammed from human adult somatic cells and harbor pluripotent stem cell-like features similar to hESCs, which makes them a useful source for regenerative medicine such as drug discovery, disease modeling and cell therapy in patient-specific manner1,2 .
Till now, there are several methods to generate human iPSCs, including virus-mediated (retrovirus and adenovirus)3, non-virus mediated (BAC system and vectors transfection)4 gene transductions, and protein delivery system5-7.
Although a delivery of virus-mediated genes can ensure a certain level of efficiency, viral vectors could leave genetic footprint, because they integrate into host chromosomes to express reprogramming genes in an uncontrolled manner. Even when viral integration of transcription factors may activate or inactivate host genes8, it can cause an unexpected genetic aberration and the risk of tumorigenesis5,9. On the other hand, the direct introduction of proteins or RNA into somatic cells were reported, but have some disadvantages such as labor-intensive, repeated transfection, and low level of reprogramming7,10. Even episomal and non-integrating adenovirus, adeno-associated virus, and plasmid vectors are still relatively less efficient11. For these reasons, it is plausible to choose non-integration reprogramming methods with high efficacy of iPSC generation and fewer genetic abnormalities. In this study, we use a Sendai-virus based reprogramming. This method is known to be non-integrated into the host genome and consistently produces human iPSCs without transgene integrations.

<table><tbody><tr><td>CytoTune-iPS Reprogramming Kit</td><td>Invitrogen</td><td>A1378002</td><td></td></tr><tr><td>CF-6,MEFs, neomycin-resistant, mitomycin C treated&amp;nbsp;</td><td>Global stem</td><td>GSC-6105M</td><td>5 x 10&lt;sup&gt;5&lt;/sup&gt;/6 cm or 12.5 x 10&lt;sup&gt;5&lt;/sup&gt;/24-well plate</td></tr><tr><td>Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X</td><td>Invitrogen</td><td>25200114</td><td></td></tr><tr><td>DMEM/F-12 medium</td><td>Invitrogen</td><td>11330-032</td><td></td></tr><tr><td>24-well Cell Culture Plate, flat-bottom with lid</td><td>BD</td><td>353935</td><td></td></tr><tr><td>Y-27632</td><td>TOCRIS</td><td>1254</td><td>10 &amp;#956;M (Stock: 10 mM)</td></tr><tr><td>basic fibroblast growth factor</td><td>LIFE TECHNOLOGIES</td><td>PHG0263</td><td>10 ng (Stock : 100 ug)</td></tr><tr><td>Knock-out serum replacement</td><td>Gibco</td><td>10828028</td><td></td></tr><tr><td>Dulbecco&amp;#039;s Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)</td><td>Invitrogen</td><td>11965118</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Scientific Fermentas</td><td>SH30071.03</td><td></td></tr><tr><td>L-Glutamine-200 mM (100X), liquid</td><td>GIBCO</td><td>25030-081</td><td>1/100</td></tr><tr><td>MEM Non-Essential Amino Acids Solution, 100X</td><td>LIFE TECHNOLOGIES</td><td>11140050</td><td>1/100</td></tr><tr><td>2-Mercaptoethanol (1,000X), liquid</td><td>GIBCO</td><td>21985023</td><td>1/1,000</td></tr><tr><td>Hausser Phase Contrast Hemacytometers</td><td>Hausser Scientific</td><td>02-671-54</td><td></td></tr><tr><td>EmbryoMax 0.1% Gelatin Solution</td><td>Millipore</td><td>ES-006-B</td><td></td></tr><tr><td>SSEA-4</td><td>DSHB</td><td>MC-813-70</td><td>1/200</td></tr><tr><td>anti-Tra-1-81</td><td>Cell Signaling</td><td>4745S</td><td>1/200</td></tr><tr><td>mouse monoclonal Oct4 antibody</td><td>Santa Cruz</td><td>SC-5279</td><td>1/1,000</td></tr><tr><td>Nanog</td><td>R&amp;amp;D</td><td>AF1997</td><td>1/1,000</td></tr><tr><td>Alexa Flouor 488 goat anti-mouse</td><td>Invitrogen</td><td>948492</td><td>1/2,000</td></tr><tr><td>DPBS (Dulbecco&amp;#039;s Phosphate-Buffered Saline), 1X without calcium &amp;amp; magnesium</td><td>Cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>QuantiTect Reverse Transcription Kit</td><td>QIAGEN</td><td>205313</td><td></td></tr><tr><td>PCR Master Mix [2X]</td><td>Thermo Scientific Fermentas</td><td>K0171</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596018</td><td></td></tr><tr><td>picking hood</td><td>NuAire</td><td>NU-301</td><td></td></tr><tr><td>dissecting scope&amp;nbsp;</td><td>Nikon</td><td>SMZ745</td><td></td></tr></tbody></table>

efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Usually infected fibroblasts do not show any morphological changes in several days after Sendai virus transduction, but five days later, they start to have different shapes (Figure 1). As described in a right panel of Figure 1, cells do not have typical fibroblast morphology any more. They have a round shape and bigger nucleus than cytoplasm. Even when transduction is performed in 80% cellular confluency, it looks like they are less confluent in the well after they start to reprogram. If...

Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus at JoVE.com

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

efficient-generation-human-induced-pluripotent-stem-cells-from-human

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22.5K Views.  Johns Hopkins University School of Medicine. Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Video: Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

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, ...

Developmental Biology

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 ...

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 ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Bioengineering

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...