Name: efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus
Uploaded: 2014-04-23T22:00:00
Description: Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus at JoVE.com

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, ...

<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. S., Rowntree, R. K., Mekhoubad, S., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-chromosome+inactivation+and+epigenetic+fluidity+in+human+embryonic+stem+cells.">X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells.</a> P Natl Acad Sci USA. 105, 4820-4825 (2008).</li><li>Kim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-371-3+expression+predicts+neural+differentiation+propensity+in+human+pluripotent+stem+cells.">miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells.</a> Cell Stem Cell. 8, 695-706 (2011).</li></ol>

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 (Video) | JoVE 

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.

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 ...

Research

JoVE Journal

Biology

Generation of Human CD40-activated B cells

Generation of Human CD40-activated B cells (Video) | JoVE 

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

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set (Video) | JoVE 

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

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells (Video) | JoVE 

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

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction (Video) | JoVE 

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

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts (Video) | JoVE 

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

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (IPSCs) Using Retroviral Vector with GFP (Video) | JoVE 

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

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells (Video) | JoVE 

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

Generating Mice from Induced Pluripotent Stem Cells | Protocol 

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

Generating Human Induced Pluripotent Stem Cells from Peripheral Blood Uing the STEMCCA Lentiviral Vector | Protocol 

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

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format (Video) | JoVE 

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