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 used for higher purity.</li>
		<li>Add 10 ml of density gradient media to a new 50-ml conical tube and carefully layer the diluted blood on top of the density gradient media. Centrifuge at 750 x g for 30 min at room temperature (RT) without a centrifugation brake.</li>
		<li>Carefully transfer the buffy layer to a new 50-ml conical tube, add 30 ml of PBS to the tube, and wash the cells.</li>
		<li>Centrifuge the cells at 515 x g for 5 min at RT.</li>
		<li>Discard the PBS and resuspend the cells in 0.5 ml of blood cell media.</li>
		<li>Count the cells and plate 1 x 106 cells per well of a 24-well plate. Add PBS to the surrounding wells to prevent evaporation.</li>
		<li>Stabilize the cells for 5 days at 37 &#176;C in 5% CO2 before transduction. Add an additional 0.5 ml of fresh blood cell media on days 3-4 without disturbing the cells.</li>
	</ol>
	</li>
</ol>
2. Transduction by Sendai Virus
<ol>
	<li>Transduction (Day 0)
	<ol>
		<li>Collect and transfer the blood cells to a 15-ml conical tube and count them using a hemocytometer.</li>
		<li>Prepare 3 x 105 cells per transduction and centrifuge the cells at 515 x g for 5 min at RT.</li>
		<li>Discard the supernatant by suction and resuspend the cells in 0.5 ml of blood cell media.</li>
		<li>Transfer the cells to a well of a non-coated 24-well plate.</li>
		<li>Thaw the Sendai virus mixture in ice and add it to the suspended cells. Add Sendai virus to the cells based on the manufacturer&#39;s recommendations.</li>
		<li>Seal the plate with a sealing film and centrifuge it at 1,150 x g for 30 min at 30 &#176;C.</li>
		<li>After centrifugation, incubate the cells at 37 &#176;C in 5% CO2 overnight (O/N).</li>
	</ol>
	</li>
	<li>Cell transfer to feeder matrix (Day 1)
	<ol>
		<li>The next day, coat a 24-well plate with vitronectin. Dilute the vitronectin solution in PBS for a final 5 &#181;g/ml concentration. Add 1 ml of vitronectin to a well of a 24-well plate and incubate it at RT for at least 1 hr. Remove the coating solution before use. Coated plates can be stored in RT for 3 days.</li>
		<li>Transfer all the media containing the cells and the virus to the coated well.</li>
		<li>Collect the remaining cells with an additional 0.5 ml of fresh blood cell media and add it to the cell-containing well.</li>
		<li>Centrifuge the plate at 1,150 x g for 10 min at 35 &#176;C.</li>
		<li>After centrifugation, remove the supernatant, add 1 ml of iPSC media, and maintain the cells at 37 &#176;C in 5% CO2 O/N.</li>
	</ol>
	</li>
	<li>Second cell transfer (Day 2)
	<ol>
		<li>Coat the wells of a new 24-well plate with 5 &#181;g/ml vitronectin, as described in step 2.2.1. Use one well of the plate for each transduction.</li>
		<li>Transfer the cell suspension from the first plate to the newly coated vitronectin plate. 
		&#8203;NOTE: If not needed, suspension cells can be discarded. The procedure mentioned in step 2.3 can be repeated 2-3 times with the suspension cells. If the steps will be repeated, harvest the cells.</li>
		<li>Meanwhile, add 1 ml of iPSC media to the well of the first plate, for maintenance, and incubate it at 37 &#176;C in 5% CO2 O/N.</li>
		<li>Maintain the attached cells at 37 &#176;C and 5% CO2 and perform a daily media change with fresh iPSC media. Colonies will appear on days 14-21 after transduction.</li>
		<li>Centrifuge the newly coated plate containing the suspension cells at 1,150 x g and 35 &#176;C for 10 min.</li>
		<li>After centrifugation, incubate the cells at 37 &#176;C in 5% CO2 O/N.</li>
		<li>The next day, remove the supernatant and replace it with fresh iPSC media. 
		&#8203;NOTE: The procedure mentioned in step 2.3 can be repeated 2-3 times with the suspension cells. If the steps will be repeated, harvest the cells in the supernatant and repeat step 2.3.</li>
		<li>Maintain the attached cells with daily media changes until 80% confluency is reached.</li>
	</ol>
	</li>
</ol>
3. Reprogrammed Cell Maintenance
<ol>
	<li>Early maintenance after the 24-well plate culture
	<ol>
		<li>7-10 days after transduction, once the cells are confluent, prepare a vitronectin-coated, 60-mm dish. Dilute vitronectin solution in PBS for a final 5 &#181;g/ml concentration. Add 1 ml of vitronectin to the dish and incubate it at RT for at least 1 hr.</li>
		<li>Wash the cells with PBS and add 1 ml of PBS/1 mM EDTA to detach the cells.</li>
		<li>Incubate them at 37 &#176;C and 5% CO2 for 2 min.</li>
		<li>Harvest the cells and centrifuge them at 250 x g and RT for 2 min. Remove the supernatant and resuspend the cells in 3 ml of fresh iPSC media.</li>
		<li>Plate all resuspended cells onto the newly coated 60-mm dish.</li>
		<li>Add 10 mM RHO kinase inhibitor to the cells and maintain them at 37 &#176;C in 5% CO2 until the cells are 80% confluent.</li>
	</ol>
	</li>
	<li>Split for colony appearance (Subcloning Preparation)
	<ol>
		<li>Prepare a vitronectin-coated, 100-mm dish, as described in step 3.1.2.</li>
		<li>Wash the cells with PBS and add 1 ml of PBS/1 mM EDTA to detach the cells.</li>
		<li>Incubate them at 37 &#176;C and 5% CO2 for 2 min.</li>
		<li>Harvest the cells and centrifuge them at 250 x g and RT for 2 min.</li>
		<li>Count the cells using a hemocytometer and prepare 1 x 104 cells per dish.</li>
		<li>Centrifuge the cells at 250 x g and RT for 2 min.</li>
		<li>Resuspend 1 x 104 cells in 6 ml of iPSC media, plate them onto the coated 100-mm dish, and add 10 mM RHO kinase inhibitor to the media.</li>
		<li>Incubate the cells at 37 &#176;C in 5% CO2 for a week until large colonies appear. 
		&#8203;NOTE: Maintain and expand the colonies for subcloning. Subcloning is usually done in under 5 passages.</li>
	</ol>
	</li>
	<li>Colony picking using iPSC colony detaching solution
	<ol>
		<li>A week before colony picking, seed 1 x 104 cells in a vitronectin-coated, 100-mm dish, as mentioned in step 3.2.7.</li>
		<li>Prepare a vitronectin-coated, 60-mm dish by adding 2 ml of vitronectin solution and incubating at RT for at least 1 hr.</li>
		<li>By observing through a microscope (40X or 100X magnification), mark colonies with clear boundaries using a marker pen. Remove the vitronectin solution from the new plate made in step 3.3.2 and add 6 ml of iPSC media supplemented with 10 mM RHO kinase.</li>
		<li>Remove the culture medium from the cells and wash them with 3 ml of PBS.</li>
		<li>Add 1 ml of iPSC colony detaching solution and incubate them for 30 s at RT.</li>
		<li>Remove the solution from the plate and incubate it at RT for additional 30 sec.</li>
		<li>Using a P200 pipette, draw 200 &#181;l of media from the plate and detach the targeted colonies by pipetting. Transfer the scattered colonies to the new 100-mm dish.</li>
		<li>Incubate and maintain the cells at 37 &#176;C in 5% CO2. 
		NOTE: After obtaining a pure iPSC colony, cells were maintained until they reached passage 10. Characterization was done after at least 10 passages. Antibody dilutions and primer information are shown in Tables 1 and 2.</li>
	</ol>
	</li>
</ol>

<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>

The authors have nothing to disclose.

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>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100mm Dish</td>
			<td>TPP&#160;</td>
			<td>93100</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Plate</td>
			<td>TPP</td>
			<td>92006</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Cornical Tube</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Cornical Tube</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Disposable Pipette</td>
			<td>Falcon</td>
			<td>7551</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Disposable Pipette</td>
			<td>Falcon</td>
			<td>7543</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well Plate</td>
			<td>TPP</td>
			<td>92012</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Plate</td>
			<td>TPP</td>
			<td>92024</td>
			<td></td>
		</tr>
		<tr>
			<td>PBMC Isolation Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>StemSpan</td>
			<td>STEMCELL Technologies</td>
			<td>9805</td>
			<td>Blood cell media</td>
		</tr>
		<tr>
			<td>CC110</td>
			<td>STEMCELL Technologies</td>
			<td>8697</td>
			<td>Blood cell media supplement (100x)</td>
		</tr>
		<tr>
			<td>iPSC Generation and Culture Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CytoTune-iPSC Sendai Reprogramming Kit</td>
			<td>Life Technologies</td>
			<td>A16518</td>
			<td></td>
		</tr>
		<tr>
			<td>TeSR-E8 Media</td>
			<td>STEMCELL Technologies</td>
			<td>5940</td>
			<td>iPSC media</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Life Technologies</td>
			<td>A14700</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor</td>
			<td>Sigma Aldrich</td>
			<td>Y0503</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE express (TrypLE)</td>
			<td>Life Technologies</td>
			<td>12604-039</td>
			<td></td>
		</tr>
		<tr>
			<td>ReleSR</td>
			<td>STEMCELL Technologies</td>
			<td>12604-039</td>
			<td>Colony detaching solution</td>
		</tr>
		<tr>
			<td>Quality Control Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm Cover Glass</td>
			<td>Superior</td>
			<td>HSU-0111580</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldyhyde</td>
			<td>Tech &#38; Innovation</td>
			<td>BPP-9004</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BIOSESANG</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</td>
			<td>Vector Lab</td>
			<td>SP-5050&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SSEA4 Antibody</td>
			<td>Millipore</td>
			<td>MAB4304</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Oct4 Antibody</td>
			<td>Santa Cruz</td>
			<td>SC9081</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TRA-1-60 Antibody</td>
			<td>Millipore</td>
			<td>MAB4360</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Sox2 Antibody</td>
			<td>Biolegend</td>
			<td>630801</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TRA-1-81 Antibody</td>
			<td>Millipore</td>
			<td>MAB4381</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Klf4 Antibody</td>
			<td>Abcam</td>
			<td>ab151733</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG (H+L) antibody</td>
			<td>Molecular Probe</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti-rabbit IgG (H+L) antibody</td>
			<td>Molecular Probe</td>
			<td>A11037</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Molecular Probe</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong gold antifade reagent</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Glass, Coated&#160;</td>
			<td>Hyun Il Lab-Mate</td>
			<td>HMA-S9914</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma Aldrich</td>
			<td>366919</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoprypylalcohol</td>
			<td>Millipore</td>
			<td>109634</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Duksan</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>RevertAid First Strand cDNA Synthesis kit</td>
			<td>Thermo Scientfic</td>
			<td>K1622</td>
			<td></td>
		</tr>
		<tr>
			<td>i-Taq DNA Polymerase</td>
			<td>iNtRON BIOTECH</td>
			<td>25021</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 10X TBE Buffer&#160;</td>
			<td>Life Technologies</td>
			<td>15581-044</td>
			<td></td>
		</tr>
		<tr>
			<td>loading star</td>
			<td>Dyne Bio</td>
			<td>A750</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>9012-36-6</td>
			<td></td>
		</tr>
		<tr>
			<td>1kb (+) DNA ladder marker</td>
			<td>Enzynomics</td>
			<td>DM003</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase</td>
			<td>Millipore</td>
			<td>SCR004</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Rinse Buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Duchefa Biochemie</td>
			<td>S0520.1000</td>
			<td>Rinse Buffer</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>BIOSESANG</td>
			<td>T1027</td>
			<td>Rinse Buffer</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Duksan</td>
			<td>1129</td>
			<td>Rinse Buffer</td>
		</tr>
	</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...

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

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

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

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14.1K Views.  The Catholic University of Korea. 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.

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

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...