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. 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. 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=Derivation,+expansion+and+differentiation+of+induced+pluripotent+stem+cells+in+continuous+suspension+cultures.">Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures.</a> Nature Methods. 9, 509-516 (2012).</li><li>Nakagawa, M., 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+without+Myc+from+mouse+and+human+fibroblasts.">Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts.</a> Nature Biotechnology. 26, 101-106 (2008).</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> P Jpn Acad B-Phys. 85, 348-362 .</li><li>Silva, 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 alternative methods, but at the same time, the efficiency of isolating &#39;foot-print free&#39; human iPSCs should be considered. For example, the RNA virus should have a minimal level of genetic aberration compare to other methods14, although there is no publication for whole genome sequencing to demonstrate the &#39;genomic safety&#39; of sendai virus-mediated iPSC generation yet. Here, we present a method for generating hiPSCs with the Sendai-virus which has a high reprogramming efficiency in cost-effective way. The resulting human iPSCs are free of transgene with maintenance of cellular and molecular similarities to hESCs in proliferative and developmental potential.
We can reprogram different origins of fibroblast (&#62;4) at the same time with only one set of the Sendai-virus. And in our method, we apply various cell confluences and virus dosage differences. This &#39;mix and match&#39; combination technique can maximize the reprogramming efficacy. Fibroblasts (from a single patient) in each well have a different virus infection level, but by pooling together, the possibility of making hiPSCs colonies could increase based on our experience. Other issues of the clonal variation among hiPSC lines are proliferation rate, cell attachment/survival, status of X chromosome inactivation15, differentiation potentials16, etc. Indeed, we observed that each clone has a different neural propensity, which could be predicted by measuring the expression level of a mir-371 cluster. In addition we have succeeded in generating human iPSCs from human myoblasts with this method, which suggests that the Sendai-virus can be used for many different cell types in reprogramming process. Furthermore, using our method, we have generated hiPSCs in more than 10 different disease related fibroblasts. On average, from 5 to 10 hiPSC clones could be obtained from one fibroblast.
Although the derivation of transgene-free human iPS cells with the Sendai-virus is the most efficient reprogramming method which could be the most practical paths, there are a few limitations to be considered in this protocol; 
- Depending on each sendai-virus batch, the virus titer needs to be calculated for consistent MOI (as described in 2.4). 
- We observed the variation of infectivity among different somatic cells, which also needs additional titration for robust viral infection. 
- The Sendai virus mediated reprogramming can be considered as one of the best choices for research purposes. But for the clinical trial, it might not be the first choice, since there is a licensing issue with the company that originally developed the Sendai virus system. 
- It takes around two months until the reprogrammed somatic cells are free from transgenes, which is why we need to check the transgene expression after at least 10 passages. 
- It seems like that the passage number of the primary cultured fibroblast may affect the efficiency of reprogramming with the Sendai-virus, although we don&#39;t have a direct comparison. The proliferation rate also needs to be determined. Further studies are needed to determine the effect of fibroblasts passage numbers and their proliferation on the reprogramming efficiency. 
- In this study, we use a mouse feeder layer for hiPSC generation and maintenance, but a feeder free system may be an alternative way in a future study.
Our current protocol has&#160;been an important step toward studying patient-specific hiPSCs for disease modeling, regenerative medicine, and other applications.

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>
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			<td>15596018</td>
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			<td height="21" style="height:21px;">picking hood</td>
			<td>NuAire</td>
			<td style="width:119px;">&#160;NU-301&#160;</td>
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		<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>
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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 these kinds of morphological changes start to be seen in the most of the well, transduced fibroblasts are ready to be plated on MEF-feeder culture dishes.
In our protocol, we minimized cell culture scale and also gave various reprogramming possibilities, such as employing different fibroblast cell density and virus titer, and subsequent several re-plating ratio MEF, which will increase hiPSCs generation efficiency (Figure 2). Before transduction, the wells with different confluency (low, mid, and high) can be selected for further sendai-virus transduction; represented as red, green, and yellow color in the plate.
At the beginning of reprogramming on MEF feeders, it is hard to distinguish transduced cell types, but after over one week from co-culture, partially reprogrammed fibroblasts appear and look like loosened ES-like shape (Figure 3). Because of this reason when human iPSC colonies are ready for the passaging into new MEF feeder dish, manual picking is more efficient way for transfer instead of dissociation with enzyme.
In Figure 3 and Figure 4, under the microscope only human iPSCs have clear edges, which make a clear distinction between human iPSCs (considered as 'fully reprogrammed cells') and 'incompletely reprogrammed cells' by morphology. Fully reprogrammed cells look like compact and tightly packed. Especially the colonies have clear boundary, whereas partially reprogrammed cells look like gathering together to make colony but very lose and fragile to break down. Even if we do a pipetting gently it is easily detached. Even after excluding partially reprogrammed human iPSCs colonies(during expanding them and passaging), fully reprogrammed human iPSCs need to be maintained by manual picking.
After picking colonies and expanding hiPSC clones in several weeks, expression of stemness markers needs to be determined. After expansion of hiPSC clones, we verify them with different sets of tests, including antibody staining (SSEA4, Oct-4, TRA-81 and Nanog) and RT-PCR of pluripotent marker (data is not shown) as a quality control.
Compared to H9, two different hiPSCs are positive to SSEA4, Oct-4, TRA-81, and Nanog. This result demonstrates that isolated human iPSCs acquired pluripotent quality (Figure 5).
Finally, Figure 6 shows that we cannot detect any transgene expression in hiPSC clones by RT-PCR. At least over 10 passages hiPSCs, specific primers (Table 1) for exogenous Oct-4, Sox-2, Klf-4 and c-Myc can be used with positive control samples with strong transgene expression level.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig1highres.jpg" src="/files/ftp_upload/51406/51406fig1.jpg" /> 
Figure 1. Morphological change after Sendai virus transduction. Before the transduction, fibroblasts show typical spindle-like shape (left). Approximately eight days after transduction, morphology of transduced cell change like much more round shape (right). <a href="https://www.jove.com/files/ftp_upload/51406/51406fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig2highres.jpg" src="/files/ftp_upload/51406/51406fig2.jpg" /> 
Figure 2. Diagram of induction plan to ensure maximum efficiency. Over four different cell types can be plated in a 24-well plate (from 1 to 4). Because their attachment ability and cell survival could be different depend on the cell types, it is suggested to plate different cell density (from 200K to 6.25K cells per well). In addition, virus concentration can be adjusted (0.5X, 1X and 2X).
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig3highres.jpg" src="/files/ftp_upload/51406/51406fig3.jpg" /> 
Figure 3. Formation of human induced pluripotent stem cell colony from transduced fibroblast. In about two weeks after re-plating them into MEFs, round colonies should appear and look like human embryonic stem cell colonies. Normally the fully reprogrammed human iPSC colonies have very clear boundaries. (Arrow: fully reprogrammed human iPSC colonies, arrowhead: partially reprogrammed human iPSCs). <a href="https://www.jove.com/files/ftp_upload/51406/51406fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig4highres.jpg" src="/files/ftp_upload/51406/51406fig4.jpg" /> 
Figure 4. Picking up human induced pluripotent stem cell colonies. Human iPSC colonies are often surrounded by partially reprogrammed cells (left). Under the microscope, picking the ES-like colony only, break down into small clumps and then transfer (right). After 2~3 passages, there are undifferentiated hiPSC colonies (bottom &#38; left). (Arrow: fully reprogrammed human iPSC colonies, arrowhead: partially reprogrammed human iPSCs) <a href="https://www.jove.com/files/ftp_upload/51406/51406fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig5highres.jpg" src="/files/ftp_upload/51406/51406fig5.jpg" /> 
Figure 5. Immunofluorescence staining with stem cell pluripotent markers. Immunofluorescence assay shows human iPSCs have stemness features.SSEA-4, TRA-81, Nanog, Oct-4as pluripotent markers and DAPI is a control for nucleus staining. (A) represents H9 staining as a control sample. (B) and (C) show human iPSC clones. <a href="https://www.jove.com/files/ftp_upload/51406/51406fig5highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig6highres.jpg" src="/files/ftp_upload/51406/51406fig6.jpg" /> 
Figure 6. Confirmation of transgene expression level. At least after 10-passage culture of human iPSCs, silencing of exogenous Sox2, Klf-4, c-Myc, and Oct-4 gene expression levels is confirmed by reverse transcriptase polymerase chain reactions. hGAPDH is an internal control. For a positive control, RNA is extracted from leftover transduced fibroblasts (Day 7 after Sendai-virus infection). (Lane 1: Marker, lanes 2 - 6: GAPDH, Klf4, c-Myc, Oct-4, and Sox-2 in positive control, lanes 7 - 11: GAPDH, Klf4, c-Myc, Oct-4, and Sox-2 in hiPSCs). 
<table border="0" cellpadding="0" cellspacing="0" width="305">
	<colgroup>
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Primer Name</td>
			<td>Sequence</td>
		</tr>
		<tr>
			<td>hOCT4 (F)</td>
			<td>CCC GAA AGA GAA AGC GAA CCA G </td>
		</tr>
		<tr>
			<td>hOCT4 (R)</td>
			<td>AAT GTA TCG AAG GTG CTC AA </td>
		</tr>
		<tr>
			<td>hMYC (F) </td>
			<td>TAA CTG ACT AGC AGG CTT GTC G </td>
		</tr>
		<tr>
			<td>hMYC (R) </td>
			<td>TCC ACA TAC AGT CCT GGA TGA TGA TG </td>
		</tr>
		<tr>
			<td>hSOX2 (F) </td>
			<td>ACA AGA GAA AAA ACA TGT ATG G </td>
		</tr>
		<tr>
			<td>hSOX2 (R) </td>
			<td>ATG CGC TGG TTC ACG CCC GCG CCC AGG </td>
		</tr>
		<tr>
			<td>hKLF4 (F) </td>
			<td>ACA AGA GAA AAA ACA TGT ATG G </td>
		</tr>
		<tr>
			<td>hKLF4 (R) </td>
			<td>CGC GCT GGC AGG GCC GCT GCT CGA C </td>
		</tr>
		<tr>
			<td>hGAPDH (F)</td>
			<td>AGG TCG GAG TCA ACG GAT TTG</td>
		</tr>
		<tr>
			<td>hGAPDH (R)</td>
			<td>GTG ATG GCA TGG ACT GTG GT</td>
		</tr>
	</tbody>
</table>
Table 1. RT-PCR primers for transgene confirmation. These are the primer sequences that are used for transgene confirmation. Tm is 50.6 &#176;C in all the primer sets.

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

Universidad San Sebastian

Research

JoVE Journal

Biology

22.3K 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 Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...