Name: induced pluripotent stem cell generation from blood cells using sendai virus and centrifugation
Uploaded: 2016-12-21T12:00:00
Description: Watch this Scientific Journal Video about Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation Video at JoVE.com

This work was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (2013R1A1A1076125).

Ethics Statement: This study protocol was approved by the institutional review board of The Catholic University of Korea (KC12TISI0861).
1. Isolation of Monocytic Cells from Blood
<ol>
	<li>Isolation of monocytic cells (Day -5)
	<ol>
		<li>Obtain at least 10 ml of fresh blood from a blood draw in a cell preparation tube (CPT).</li>
		<li>Transfer the blood to a new 50-ml conical tube and dilute it with sterile phosphate-buffered saline (PBS) at a 1:4 ratio. 
		&#8203;NOTE: A higher ratio of dilution can be ...

<ol>
	<li>Serra, M., Brito, C., Correia, C., Alves, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+engineering+of+human+pluripotent+stem+cells+for+clinical+application.">Process engineering of human pluripotent stem cells for clinical application.</a> Trends Biotechnol. 30 (6), 350-359 (2012).</li><li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+pluripotent+cell+line+from+early+mouse+embryos+cultured+in+medium+conditioned+by+teratocarcinoma+stem+cells.">Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.</a> Proc Natl Acad Sci U S A. 78 (12), 7634-7638 (1981).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Chun, Y. S., Byun, K., Lee, B. <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+cells+and+personalized+medicine:+current+progress+and+future+perspectives.">Induced pluripotent stem cells and personalized medicine: current progress and future perspectives.</a> Anat Cell Biol. 44 (4), 245-255 (2011).</li><li>Seki, T., Fukuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+induced+pluripotent+stem+cells+for+clinical+application.">Methods of induced pluripotent stem cells for clinical application.</a> World J Stem Cells. 7 (1), 116-125 (2015).</li><li>Churko, J. M., Burridge, P. W., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+iPSCs+from+human+peripheral+blood+mononuclear+cells+using+non-integrative+Sendai+virus+in+chemically+defined+conditions.">Generation of human iPSCs from human peripheral blood mononuclear cells using non-integrative Sendai virus in chemically defined conditions.</a> Methods Mol Biol. 1036, 81-88 (2013).</li><li>Loh, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+T+cells+from+human+peripheral+blood.">Reprogramming of T cells from human peripheral blood.</a> Cell Stem Cell. 7 (1), 15-19 (2010).</li><li>Ohmine, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+from+GMP-grade+hematopoietic+progenitor+cells+and+mononuclear+myeloid+cells.">Induced pluripotent stem cells from GMP-grade hematopoietic progenitor cells and mononuclear myeloid cells.</a> Stem Cell Res Ther. 2 (6), (2011).</li><li>Mae, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+and+robust+induction+of+nephrogenic+intermediate+mesoderm+from+human+pluripotent+stem+cells.">Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells.</a> Nat Commun. 4, 1367 (2013).</li></ol>

Since embryonic stem cells (ESCs) showed several shortcomings, the need of an alternative tool was required. Therefore, the development of induced pluripotent stem cells (iPSCs) by Yamanaka came under the international spotlight. It has been almost a decade since Yamanaka discovered that pluripotency can be induced by adding only four genes into adult somatic cells. Since iPSCs are &#34;induced&#34; from mature somatic cells, they can evade ethical issues that had once been the concern relating to ESCs. Unlike ESCs, iPSC...

Stem cells have been one of the most attractive materials in clinical therapy for the last several decades1. The attractive properties of stem cells are pluripotency and the ability to self-renew. In 1981, the first embryonic stem cells (ESCs) were isolated from the mouse embryo2. However, when the technique was applied to human embryos, it faced several ethical issues.
In 2006, when Dr. Yamanaka and his team reprogrammed the first pluripotent cell from mouse somatic cells, the stem cell field regained its possibility and interest was rekindled3. By delivering several defined factors, pluripotent stem cells were successfully &#34;induced&#34; from adult somatic cells, and were thus named &#34;induced pluripotent stem cells (iPSCs).&#34; In 2007, this technique was applied to human cells4, yielding cells with the exact characteristics of ESCs but none of the ethical debate. Theoretically, iPSCs can be generated from any cell type obtained from any individual or patient. Patient-specific iPSCs are rising as a potential tool that can simulate the disease phenotypes and epigenetic conditions of each individual patient. Using gene editing or other methods that can reverse the pathogenic condition, patient-specific iPSCs can also be used in personalized medicine5. Moreover, iPSCs are less associated with immune rejection because they have the same immune identity as the donor, making auto-transplantation more feasible6. Therefore, iPSCs have become the most promising platform in disease modeling, drug screening, and regenerative therapies. Given these benefits, improved protocols that can give purer and higher yields in the least amount of time from the smallest cell source are constantly under development. One major consideration of finding the most efficient protocol for future application is the primary cell type. Most of the early iPSC generation protocols are optimized for adherent cells since the original iPSC lines were induced from skin fibroblasts4. However, the isolation and preparation of these cells are labor intensive. Also, the isolation of skin fibroblasts includes invasive surgical procedures that can become a major shortcoming for broader application.
Therefore, for the further use of iPSCs, a cell source with convenient acquisition is required. Blood is regarded as an ideal cell source since it is obtained through a rather minimally invasive procedure7-9. In this study, we developed a simple modification to the protocol generating hiPSCs from peripheral blood mononuclear cells (PBMCs). Without the difficult expansion process of a specific cell type, such as CD34+ cells, whole blood cells or PBMCs were serially plated onto matrix-coated plates by centrifugation after transduction with Sendai virus containing Yamanaka factors. This method reduced the time required for the attachment of transduced floating cells and decreased the loss of reprogrammed cells that were not able to attach on their own.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plasticware</td>
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			<td>12-well Plate</td>
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			<td>24-well Plate</td>
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			<td>PBMC Isolation Materials</td>
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			<td>DPBS</td>
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			<td>StemSpan</td>
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			<td>Blood cell media supplement (100x)</td>
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			<td>iPSC Generation and Culture Materials</td>
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			<td>CytoTune-iPSC Sendai Reprogramming Kit</td>
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			<td>TeSR-E8 Media</td>
			<td>STEMCELL Technologies</td>
			<td>5940</td>
			<td>iPSC media</td>
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			<td>Vitronectin</td>
			<td>Life Technologies</td>
			<td>A14700</td>
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			<td>ReleSR</td>
			<td>STEMCELL Technologies</td>
			<td>12604-039</td>
			<td>Colony detaching solution</td>
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			<td>Quality Control Materials</td>
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			<td>18 mm Cover Glass</td>
			<td>Superior</td>
			<td>HSU-0111580</td>
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			<td>4% Paraformaldyhyde</td>
			<td>Tech &#38; Innovation</td>
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			<td>BIOSESANG</td>
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			<td>Bovine Serum Albumin&#160;</td>
			<td>Vector Lab</td>
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			<td>Millipore</td>
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			<td>Anti-Oct4 Antibody</td>
			<td>Santa Cruz</td>
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			<td>Anti-TRA-1-60 Antibody</td>
			<td>Millipore</td>
			<td>MAB4360</td>
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			<td>Anti-Sox2 Antibody</td>
			<td>Biolegend</td>
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			<td>Anti-TRA-1-81 Antibody</td>
			<td>Millipore</td>
			<td>MAB4381</td>
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			<td>Anti-Klf4 Antibody</td>
			<td>Abcam</td>
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			<td>Alexa Fluor 488 goat anti-mouse IgG (H+L) antibody</td>
			<td>Molecular Probe</td>
			<td>A11029</td>
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			<td>Alexa Fluor 594 goat anti-rabbit IgG (H+L) antibody</td>
			<td>Molecular Probe</td>
			<td>A11037</td>
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			<td>DAPI</td>
			<td>Molecular Probe</td>
			<td>D1306</td>
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			<td>Prolong gold antifade reagent</td>
			<td>Invitrogen</td>
			<td>P36934</td>
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			<td>Slide Glass, Coated&#160;</td>
			<td>Hyun Il Lab-Mate</td>
			<td>HMA-S9914</td>
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			<td>Invitrogen</td>
			<td>15596-018</td>
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			<td>Chloroform</td>
			<td>Sigma Aldrich</td>
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			<td>Isoprypylalcohol</td>
			<td>Millipore</td>
			<td>109634</td>
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			<td>Ethanol</td>
			<td>Duksan</td>
			<td>64-17-5</td>
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			<td>RevertAid First Strand cDNA Synthesis kit</td>
			<td>Thermo Scientfic</td>
			<td>K1622</td>
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			<td>i-Taq DNA Polymerase</td>
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			<td>Sigma-Aldrich</td>
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			<td>Millipore</td>
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			<td>Sodium Chloride</td>
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			<td>Rinse Buffer</td>
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			<td>Duksan</td>
			<td>1129</td>
			<td>Rinse Buffer</td>
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	</tbody>
</table>

induced pluripotent stem cell generation from blood cells using sendai virus and centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

This protocol presents a simple method to reprogram PBMCs isolated from blood. Using the combination of serial plating and centrifugation, iPSCs were successfully generated. With this method, iPSCs could be generated with a small amount of whole blood cells without isolating or expanding a specific cell type. We successfully generated iPSCs from only 1x104 cells in a small cell culture plate.
Before reprogramming, blo...

Watch this Scientific Journal Video about Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation Video at JoVE.com

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation Video (Video) | JoVE

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

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