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>

<ol>
	<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. 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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 border="0" cellpadding="0" cellspacing="0" width="1109">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">CytoTune-iPS Reprogramming Kit</td>
			<td style="width:195px;">invitrogen</td>
			<td style="width:119px;">A1378002</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CF-6,MEFs, neomycin-resistant, mitomycin C treated&#160;</td>
			<td>Global stem</td>
			<td>GSC-6105M&#160;</td>
			<td>5X105/6cm or 12.5X105/24 well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X</td>
			<td style="width:195px;">invitrogen</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12 medium</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Cell Culture Plate, flat-bottom with lid</td>
			<td style="width:195px;">BD</td>
			<td>353935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Y-27632</td>
			<td style="width:195px;">TOCRIS</td>
			<td style="width:119px;">1254</td>
			<td>10 uM (Stock: 10 mM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">basic fibroblast growth factor</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>PHG0263</td>
			<td>10 ng (Stock : 100 ug)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Knock-out serum replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11965118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fetal bovine serum</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine-200 mM (100X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>25030-081</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution, 100X</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>11140050</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol (1,000X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>21985023</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hausser Phase Contrast Hemacytometers</td>
			<td>Hausser Scientific</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EmbryoMax 0.1% Gelatin Solution</td>
			<td style="width:195px;">Millipore</td>
			<td style="width:119px;">ES-006-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">SSEA-4</td>
			<td style="width:195px;">DSHB</td>
			<td>MC-813-70</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Tra-1-81</td>
			<td style="width:195px;">cell signaling</td>
			<td>4745S</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal Oct4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-5279</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Nanog</td>
			<td>R&#38;D</td>
			<td>AF1997</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Alexa Flouor 488 goat anti-mouse</td>
			<td>invitrogen</td>
			<td>948492</td>
			<td>1/2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS (Dulbecco&#39;s Phosphate-Buffered Saline), 1X without calcium &#38; magnesium</td>
			<td style="width:195px;">cellgro</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">QuantiTect Reverse Transcription Kit</td>
			<td style="width:195px;">QIAGEN</td>
			<td>205313</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">PCR Master Mix [2X]</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">K0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td style="width:195px;">invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">picking hood</td>
			<td>NuAire</td>
			<td style="width:119px;">&#160;NU-301&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">dissecting scope&#160;</td>
			<td style="width:195px;">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 ...

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Research

JoVE Journal

Biology

22.2K 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.