The authors wish to acknowledge the following grants from the NIH for supporting this research: K08DK082783 (AR), P30DK56465 (BTS), U01HL099993 (BTS), T32HL00715036 (SKS), and K12HL0806406 (SKS); and the JP McCarthy Foundation (AR).

Written informed consent, as approved by the Institutional Review Boards of the Fred Hutchinson Cancer Research Center and the Children&#34;s Hospital of Philadelphia, was obtained from patients before collecting peripheral blood samples. All institutional guidelines were observed. All animal experiments including MEF generation and teratoma formation were approved by the Institutional Animal Care and Use Committee.
1. Ficoll Separation of PBMCS and Expansion of Erythroblasts &#8211; Day 0
<ol>
	<li>Dilute peripheral blood 1:1 with Dulbecco&#8217;s Phosphate Buffered Saline (DPBS). In a 15 ml round-bottom polystyrene tube, carefully layer 7 ml of diluted blood onto 3 ml of room temperature Ficoll-Hypaque.</li>
	<li>Centrifuge the sample at 400 x g for 30 min.</li>
	<li>The PBMCs will have settled to the interface between the plasma and the Ficoll-Hypaque, seen as a cloudy white layer. Using a sterile transfer pipette, collect the PBMCs into one 15 ml conical tube. Bring the volume to 10 ml using DPBS.</li>
	<li>Using a hemacytometer, count the PBMCs.</li>
	<li>Pipette 2 &#215; 106 PBMCs into a separate 15 ml conical tube and centrifuge at 300 x g for 5 min. Spin down the remaining cells and freeze at a concentration of 2 &#215; 106 PBMCs/ml in 90% fetal bovine serum (FBS)/10% dimethyl sulfoxide (DMSO).</li>
	<li>Prepare Expansion Medium: QBSF-60 serum free medium (protect from light) + penicillin/streptomycin (1%) + ascorbic acid (50 ng/ml) + stem cell factor (SCF, 50 ng/ml) + interleukin 3 (IL-3, 10 ng/ml) + erythropoietin (EPO, 2 U/ml) + insulin-like growth factor 1 (IGF-1, 40 ng/ml) + dexamethasone (1 &#956;M; protect from light; thaw a fresh aliquot each media change).</li>
	<li>Resuspend 2 &#215; 106 PBMCs in 2 ml of freshly made Expansion Medium and place into one well of a 12 well tissue culture plate and incubate in a 37 &#176;C humidified incubator with an atmosphere of 5% O2, 5% CO2, and 90% N2.</li>
	<li>On Day 3 and Day 6, replace media.
	<ol>
		<li>Collect the cells and wash the well with 2 ml of QBSF-60 to collect any remaining cells.</li>
		<li>Centrifuge the cell suspension at 300 x g for 5 min and aspirate the supernatant.</li>
		<li>Resuspend the cells in 2 ml of new Expansion Medium and return them to the same 12 well plate.</li>
	</ol>
	</li>
</ol>
2. Reprogramming Cells by Nucleofection &#8211; Day 9
<ol>
	<li>Pipette the reprogramming plasmids into a sterile 1.5 ml Eppendorf tube (Table 1).</li>
	<li>Mix the nucleofection solution with Supplement (supplied by manufacturer) and pipette into a sterile 1.5 ml Eppendorf tube.</li>
	<li>Pipette 2 ml of Expansion Medium into one well of a 12 well plate and place in a humidified 37 &#176;C incubator (5% O2, 5% CO2, and 90% N2) for equilibration prior to adding cells.</li>
	<li>Collect the cultured cells and wash the well with 2 ml of QBSF-60 to collect remaining cells.</li>
	<li>Centrifuge the cells at 300 x g for 5 min and aspirate the supernatant.</li>
	<li>Wash the cells by resuspending in 5 ml of DPBS and centrifuging at 300 x g for 5 min.</li>
	<li>Aspirate the supernatant.</li>
	<li>Resuspend the cell pellet in 100 &#956;l of nucleofection solution + Supplement, taking care to avoid generating bubbles.</li>
	<li>Pipette the cell suspension into the tube containing the reprogramming plasmids, pipette up and down once, and pipette the sample into the bottom of the nucleofection cuvette.</li>
	<li>Place the cuvette into the nucleofection machine and run Program T-019.</li>
	<li>Transfer 100 &#956;l of the prewarmed Expansion Medium (from the equilibrating plate in Step 2.3) into the nucleofection cuvette.</li>
	<li>Immediately collect the entire sample and deposit into the well of pre-warmed Expansion Medium. Avoid collecting the white, floating dead cell debris.</li>
	<li>Place the plate in a humidified 37 &#176;C incubator (5% O2, 5% CO2, and 90% N2) for growth.</li>
</ol>
3. Plating Feeder Cells &#8211; Day 10 or 11
<ol>
	<li>On Day 10, plate feeder cells.
	<ol>
		<li>Coat one 6 well tissue culture plate with gelatin.
		<ol>
			<li>Pipette 1 m&#160; of 0.1% gelatin in Hank&#8217;s Buffered Saline Solution (HBSS) into each well of the plate.</li>
			<li>Place the plate in a humidified 37 &#176;C incubator for at least 5 min.</li>
			<li>Immediately before use, aspirate the gelatin solution from the plate.</li>
		</ol>
		</li>
		<li>Thaw one vial of 2 &#215; 106 mouse embryonic fibroblasts irradiated at 3,000 cGy into 10 ml of prewarmed MEF Media (Dulbecco&#8217;s Modified Eagle Medium (DMEM) + FBS (10%) + penicillin/streptomycin (1%)).</li>
		<li>Centrifuge the cells at 300 x g for 5 min and aspirate the supernatant. Resuspend the cells in 12 ml of MEF Media and pipette 2 ml into each well of the gelatin-coated 6 well plate. Place the plate in a humidified 37 &#176;C incubator (5% O2, 5% CO2, and 90% N2) until Day 12.</li>
	</ol>
	</li>
</ol>
4. Plating Cells on Feeder Cells and Changing Medium &#8211; Day 12
<ol>
	<li>Collect the nucleofected cells into one 15 ml conical tube and collect any remaining cells by washing the well with 2 ml of QBSF-60. Centrifuge the sample at 300 x g for 5 min.</li>
	<li>Prepare Reprogramming Medium: Iscove&#8217;s Modified Dulbecco&#8217;s Medium (IMDM) + FBS (10%) + non-animal L-glutamine (1 mM) + non-essential amino acids (NEAA, 1%) + penicillin/streptomycin (1%) + &#946;-mercaptoethanol (0.1 mM) + basic fibroblast growth factor (bFGF, added fresh at feeding, 10 ng/ml) + ascorbic acid (added fresh at feeding, 50 &#956;g/ml).</li>
	<li>Aspirate the supernatant and resuspend the cells in 12 ml of Reprogramming Medium. Pipette 2 ml per well of the cell suspension into the 6 well plate of feeder cells (as prepared in Section 3). Centrifuge the plate at 50&#160;x g for 30 min at room temperature.</li>
	<li>Place the plate in a humidified 37 &#176;C incubator (5% O2, 5% CO2, and 90% N2) for growth. Change the Reprogramming Medium every other day until iPSC colonies begin appearing (1-2 weeks).</li>
	<li>Prepare hESC Medium: DMEM/F12 1:1 + Knockout Serum Replacement (20%) + penicillin/streptomycin (1%) + NEAA (1%) + non-animal L-glutamine (1 mM) + sodium pyruvate (1%) + sodium bicarbonate (0.12%) + &#946;-mercaptoethanol (0.1 mM) + bFGF (added fresh at feeding, 10 ng/ml)</li>
	<li>Prepare HDAC inhibitors: sodium butyrate (added fresh at feeding, 100 &#956;M), suberanilohydroxamic acid (SAHA, added fresh at feeding, 200 nM). Once iPSC colonies appear, begin feeding the cells with hESC Medium + HDAC inhibitors, changing medium every day. 
	NOTE: Sodium butyrate is a small molecule that is adsorbed to plastic tubes, therefore store it in a borosilicate glass tube at 4 &#176;C.</li>
</ol>
5. Isolating iPSC Clones
<ol>
	<li>Once iPSC colonies are large enough to be isolated (20-25 cells), pick them with a P20 pipette.</li>
	<li>Prepare HDAC inhibitors: sodium butyrate (added fresh at feeding, 100 &#956;M), SAHA (added fresh at feeding, 200 nM)</li>
	<li>Place the isolated colonies onto iMEFs in individual 35 mm dishes containing hESC Medium + HDAC inhibitors + Cloning &#38; Recovery Supplement.</li>
	<li>The day after picking the colonies, change the medium to hESC Medium + HDAC inhibitors.</li>
	<li>Change the medium every day until iPSC colonies are ready for passaging.</li>
	<li>Remove HDAC inhibitors from the medium once colonies stabilize at passage 2-3.</li>
</ol>

<ol>
	<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>Koche, R. P., 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+factor+expression+initiates+widespread+targeted+chromatin+remodeling.">Reprogramming factor expression initiates widespread targeted chromatin remodeling.</a> Cell Stem Cell. 8 (1), 96-105 (2011).</li><li>Baum, C., Kustikova, O., Modlich, U., Li, Z., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+and+oncogenesis+by+chromosomal+insertion+of+gene+transfer+vectors.">Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors.</a> Human Gene Therapy. 17 (3), 253-263 (2006).</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 (7151), 313-317 (2007).</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 (5928), 797-801 (2009).</li><li>Hong, 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=Suppression+of+induced+pluripotent+stem+cell+generation+by+the+p53-p21+pathway.">Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.</a> Nature. 460 (7259), 1132-1135 (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 (5), 409-412 (2011).</li><li>Yates, J. L., Warren, N., Sugden, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+replication+of+plasmids+derived+from+Epstein-Barr+virus+in+various+mammalian+cells.">Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells.</a> Nature. 313 (6005), 812-815 (1985).</li><li>Ware, C. B., 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=Histone+deacetylase+inhibition+elicits+an+evolutionarily+conserved+self-renewal+program+in+embryonic+stem+cells.">Histone deacetylase inhibition elicits an evolutionarily conserved self-renewal program in embryonic stem cells.</a> Cell Stem Cell. 4 (4), 359-369 (2009).</li><li>Mali, P., 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=Butyrate+greatly+enhances+derivation+of+human+induced+pluripotent+stem+cells+by+promoting+epigenetic+remodeling+and+the+expression+of+pluripotency-associated+genes.">Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.</a> Stem Cells. 28 (4), 713-720 (2010).</li><li>Azuara, V., 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=Chromatin+signatures+of+pluripotent+cell+lines.">Chromatin signatures of pluripotent cell lines.</a> Nature Cell Biology. 8 (5), 532-538 (2006).</li><li>Sommer, A. G., 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+from+peripheral+blood+using+the+STEMCCA+lentiviral+vector.">Generation of human induced pluripotent stem cells from peripheral blood using the STEMCCA lentiviral vector.</a> J. Vis. Exp. (68), e4327 (2012).</li><li>Yoshida, Y., Takahashi, K., 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=Hypoxia+enhances+the+generation+of+induced+pluripotent+stem+cells.">Hypoxia enhances the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 5, 237-241 (2009).</li><li>Abbondanzo, S. J., Gadi, I., Stewart, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+Embryonic+Stem+Cell+Lines.">Derivation of Embryonic Stem Cell Lines.</a> Methods in Enzymology. 225, 803-823 (1993).</li><li>Kim, H. J., Bae, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors:+molecular+mechanisms+of+action+and+clinical+trials+as+anti-cancer+drugs.">Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs.</a> American Journal of Translational Research. 3 (2), 166-179 (2011).</li></ol>

The authors declare that they have no competing financial interests.

For successful iPSC generation when using this protocol, there are several important caveats that should be considered. During the erythroblast expansion stage, the media change schedule should be strictly followed, as deviations may lead to inefficient stimulation of the target progenitor cell population and a lower efficiency of iPSC generation. It is important to make new expansion medium with fresh dexamethasone with each media change; and the QBSF-60 base medium and dexamethasone should be protected from light during storage. During nucleofection, the DPBS wash is critical to remove excess solutes from the cell suspension that may cause the electric current from the nucleofector to arc, resulting in widespread cell death. Approximately 10 days after nucleofection, the plate should be examined daily for iPSC colony appearance. Once several small colonies are present, the Reprogramming Medium should be changed to hESC Medium (with HDAC inhibitors) as prolonged exposure to serum may induce spontaneous differentiation.
Depending on laboratory set-up and available resources, modifications to the protocol may be considered. We prefer a low O2 incubator because hypoxic conditions have been shown to increase the efficiency of iPSC generation13; however, we have had success generating iPSCs from peripheral blood using normoxic conditions with 5% CO2. Therefore, a standard humidified CO2 incubator can also be used. The MEFs used in this protocol may be purchased from a life science vendor. However, due to commercial batch variability, we prefer to generate iMEFs from CF-1 mice following a published protocol for MEF isolation, culture, and irradiation14. The inclusion of HDAC inhibitors is a favorable aspect of this protocol as epigenetic remodeling is a crucial aspect of cellular reprogramming9,15. HDAC inhibitors may be omitted, however the number of fully reprogrammed iPSC colonies will be reduced. Lastly, we have generated iPSCs using this method with a bone marrow sample with no additional modifications needed.
In summary, this protocol uses a plasmid based reprogramming system that avoids some of the drawbacks of iPSC generation with integrating vectors, including random insertions into the host genome and safety concerns regarding the handling of recombinant virus. Additionally, small scale virus production and characterization is relatively labor intensive when compared to plasmid generation. Reprogramming efficiency can also vary significantly due to batch variability inherent to virus production, while one large scale production of quality plasmids can lead to consistent iPSC generation over time. Combined with HDAC inhibitors, this protocol offers an efficient method to generate integration free and footprint free iPSCs for stem cell research.

iPSCs are derived from somatic tissues via ectopic expression of a minimal set of pluripotency genes. This technique was initially demonstrated by retroviral transduction of human fibroblasts with OCT4, SOX2, KLF4, and cMYC, which are highly expressed in the pluripotent state1. These transiently expressed &#8220;reprogramming factors&#8221; alter the target cell&#8217;s epigenetic landscape and gene expression profile analogous to human embryonic stem cells2. Once created, iPSCs can potentially be differentiated into any tissue type for further investigation. Thus, they hold promise for use in regenerative medicine, disease modeling, and gene therapy applications. However, disrupting the genome with integrating viruses has the potential to alter endogenous gene expression, influence cellular phenotype, and ultimately bias scientific results. Furthermore, random viral integrations can lead to deleterious cellular effects, including the possibility of malignant transformation3 or re-expression of the oncogenic transgenes4. Future clinical applications will require non-integrating iPSC generation.
Episomes, which are extra chromosomal circular DNA molecules, offer a strategy to generate cost effective, integration-free iPSCs5. The combination of episomal vectors shown in Table 1 express the reprogramming factors OCT4, SOX2, KLF4, MYCL, and LIN28A. The pCXLE_hOCT3/4-shp53-F plasmid also contains a p53 shRNA for temporary suppression of TP53 to enhance cellular reprogramming6. The replication deficient pCXWB-EBNA1 vector promotes amplification of reprogramming factors and increased reprogramming efficiency by providing a transient increase in EBNA1 expression7. The pCXLE_EGFP plasmid can be added to the nucleofection mixture for the purpose of determining the transfection efficiency or for cell sorting applications. With the exception of pCXWB-EBNA1, the episomal plasmids used in this protocol contain the Epstein-Barr virus origin of viral replication and EBNA1 gene, which mediate replication and partition of the episome during division of the host cell8. The episomes are spontaneously lost with successive iPSC expansion7. Subcloning and characterization of iPSCs with episomal vector loss, which can be inferred from loss of eGFP expression, can lead to completely integration free iPSCs for future clinical applications.
Inherent to the process of iPSC generation is suppression of lineage specific genes and reactivation of pluripotency-associated genes. Regulation of gene expression occurs at multiple levels within the nucleus, including modifications to DNA and chromatin to allow transcription factors, regulatory DNA elements, and RNA polymerase access to target genes. Remodeling of the epigenetic landscape via global chromatin modifications is a key component to re-expression of the pluripotency genetic program. A specific chromatin modification that is important in regulation of gene expression is acetylation of histones at particular lysine residues, which allows access to target genes through decreased tension of the histone-DNA coil. HDAC inhibitors are small molecules that have been shown to enhance iPSC reprogramming and hESC self-renewal9,10, likely due to supporting the acetylated state11. The protocol described below, adapted from a prior publication using integrating lentiviruses12, provides a step-by-step method for optimized iPSC generation from peripheral blood using episomes and HDAC inhibitors. The HDAC inhibitor concentrations used here are half of those described by Ware, et al.9, and have routinely led to a 2 fold increase in fully reprogrammed iPSC colonies over standard episomal reprogramming protocols without HDAC inhibitors. This level of reprogramming is on par with the efficiency we observe with lentiviral methods. Using this protocol, we have efficiently generated iPSC from individuals as old as 87 years of age.

<table><tbody><tr><td>Ficoll-Paque PLUS</td><td>Fisher Scientific</td><td>45-001-750</td><td>Store at 4 &amp;deg;C. Warm to room temperature before use.</td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-250</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>11875-093</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>fetal bovine serum (FBS)</td><td>Life Technologies</td><td>10437028</td><td>Store at -20 &amp;deg;C until needed. Thaw, aliquot, store at 4 &amp;deg;C.</td></tr><tr><td>dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>154938</td><td>Store at room temperature.</td></tr><tr><td>QBSF-60 serum-free medium</td><td>Fisher Scientific</td><td>50-983-234</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>penicillin/streptomycin</td><td>Life Technologies</td><td>15140122</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Cell Line Nucleofector Kit V</td><td>Lonza</td><td>VCA-1003</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>2% gelatin solution</td><td>Sigma-Aldrich</td><td>G1393</td><td>Store at 4 &amp;deg;C, liquify in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Hank&#039;s Balanced Saline Solution (HBSS)</td><td>Life Technologies</td><td>14175-103</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>11965-092</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Life Technologies</td><td>12440-061</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>L-glutamine, 200 mM</td><td>Life Technologies</td><td>25030-081</td><td>Aliquot, freeze at -20 &amp;deg;C.</td></tr><tr><td>non-essential amino acids (NEAA), 100X</td><td>Life Technologies</td><td>11140-050</td><td>Store at 4 &amp;deg;C, away from light.</td></tr><tr><td>DMEM/Ham&#039;s F12, 1:1</td><td>Fisher Scientific</td><td>SH30023.02</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>KnockOut Serum Replacement</td><td>Life Technologies</td><td>10828-028</td><td>Aliquot, freeze at -20 &amp;deg;C.</td></tr><tr><td>sodium pyruvate, 100 mM</td><td>Life Technologies</td><td>11360-070</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>sodium bicarbonate, 7.5%</td><td>Life Technologies</td><td>25080094</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>basic fibroblast growth factor (bFGF)</td><td>Life Technologies</td><td>PHG0263</td><td>Make 10 mg/ml stock in DPBS, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>sodium butyrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B5887-1G</td><td>Make 2000x stock (400 mM) in DPBS, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>hES Cell Cloning &amp;amp; Recovery Supplement</td><td>Stemgent</td><td>01-0014-500</td><td>Store at -20 &amp;deg;C until needed. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>ESC-qualified BD matrigel</td><td>BD Biosciences</td><td>35-4277</td><td>Thaw overnight on ice at 4 &amp;deg;C, aliquot into pre-chilled tubes using pre-chilled pipette tips. Store at -20 &amp;deg;C until needed. Thaw at 4 &amp;deg;C, use immediately.</td></tr><tr><td>StemSpan SFEM&amp;nbsp;</td><td>STEMCELL Technologies</td><td>9650</td><td>Aliquot, freeze at -20 &amp;deg;C.</td></tr><tr><td>ascorbic acid, powdered</td><td>Sigma-Aldrich</td><td>A4403-100MG</td><td>Make 5 mg/ml stock in DPBS, sterile filter, store at 4 &amp;deg;C.</td></tr><tr><td>recombinant human stem cell factor (SCF)</td><td>R &amp;amp; D Systems</td><td>255-SC-010</td><td>Make 100 &amp;mu;g/m stock in SFEM, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>recombinant human interleukin 3 (IL-3)</td><td>R &amp;amp; D Systems</td><td>203-IL-010</td><td>Make 100 &amp;mu;g/ml stock in SFEM, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>erythropoietin (EPO)</td><td>R &amp;amp; D Systems</td><td>287-TC-500</td><td>Make 1000 U/ml stock in SFEM, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>recombinant human insulin-like growth factor 1 (IGF-1)</td><td>R &amp;amp; D Systems</td><td>291-G1-200</td><td>Make 100 &amp;mu;g/ml stock in SFEM, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M7522</td><td>Make 1000x stock (100 mM) in DPBS.</td></tr><tr><td>dexamethasone</td><td>Sigma-Aldrich</td><td>D4902-25MG</td><td>Make 50x stock (50 &amp;mu;M) in DPBS, sterile filter, store at 4 &amp;deg;C.</td></tr><tr><td>SAHA (vorinostat)</td><td>Cayman Chemical</td><td>149647-78-9</td><td>Make 2,000x stock (400 mM) in DPBS, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>12 well tissue culture plate</td><td>Fisher Scientific</td><td>08-772-29</td><td></td></tr><tr><td>15 ml conical tube</td><td>Sarstedt</td><td>62553002</td><td></td></tr><tr><td>1.5 ml Eppendorf tube</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>6 well tissue culture plate</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>35 mm tisue culture plates</td><td>BD Biosciences</td><td>353001</td><td></td></tr><tr><td>10 ml disposable serological pipettes</td><td>Fisher Scientific</td><td>13-675-20</td><td></td></tr><tr><td>5 ml disposable serological pipettes</td><td>Fisher Scientific</td><td>13-675-22</td><td></td></tr><tr><td>2 ml disposable serological pipettes</td><td>Fisher Scientific</td><td>13-675-17</td><td></td></tr><tr><td>20 &amp;mu;l pipette tips, barrier tips</td><td>Genessee</td><td>24-404</td><td></td></tr><tr><td>glass Pasteur pipettes</td><td>Fisher Scientific</td><td>13-678-20D</td><td></td></tr><tr><td>pipette aid</td><td>Fisher Scientific</td><td>13-681-15</td><td></td></tr><tr><td>pCXLE-hOCT3/4-shp53-F</td><td>Addgene</td><td>27077</td><td></td></tr><tr><td>pCXLE-hSK</td><td>Addgene</td><td>27078</td><td></td></tr><tr><td>pCXLE-hUL</td><td>Addgene</td><td>27080</td><td></td></tr><tr><td>pCXLE-EGFP</td><td>Addgene</td><td>27082</td><td></td></tr><tr><td>pCXWB-EBNA1</td><td>Addgene</td><td>37624</td><td></td></tr></tbody></table>

efficient ips cell generation from blood using episomes and hdac inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Three days after nucleofection and before plating the nucleofected cells onto iMEFs, the efficiency of successful nucleofection should be estimated by fluorescence microscopy for eGFP. Figure 1 shows a typical nucleofection experiment with approximately 5-10% of the total cell population expressing eGFP.
Reprogrammed iPSC colonies will begin to appear approximately two weeks after nucleofection. The colonies are generally circular with well-defined borders and may be identified by characteristic hESC morphology including (1) small, tightly packed cells with a high nuclear-cytoplasmic ratio and (2) visible nucleoli (Figure 2, Bright field). Incompletely reprogrammed cells will either deteriorate or form heterogeneous cell masses that are easily distinguished from true iPSC colonies.
Of the representative lines that we have chosen to characterize completely, all express the standard pluripotency markers (Figure 2), reactivate endogenous pluripotency genes (Figure 2), and form teratomas when injected into NOD-SCID mice.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/52009/52009fig1highres.jpg" width="500" />
Figure 1: Nucleofected Cells. Stimulated PBMCs three days after nucleofection with plasmids containing OCT4, SOX2, KLF4, MYCL, EBNA1, p53 shRNA, and eGFP (Scale bar is 100 &#956;m).
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52009/52009fig2highres.jpg" width="500" /> 
Figure 2: IPSC Characterization. IPSC generated using this method exhibit hESC-like morphology when grown on iMEF (Bright field), test positive for alkaline phosphatase activity (AP), and are positive by immunohistochemistry for pluripotency markers TRA-1-60, TRA-1-81, and SSEA4. Additionally, endogenous expression of the pluripotency genes Oct4 (O), Sox2 (S), Klf4 (K), and cMyc (M) is detectable by RT-PCR. (All scale bars are 100 &#956;m). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52009/52009fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="275">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:132px;">Episome</td>
			<td style="width:143px;">Mass per reaction (ng)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pCXLE-hOct3/4-shp53-F</td>
			<td align="right">830</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pCXLE-hSK</td>
			<td align="right">830</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pCXLE-hUL</td>
			<td align="right">830</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pCXLE-EGFP</td>
			<td align="right">500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pCXWB-EBNA1</td>
			<td align="right">500</td>
		</tr>
	</tbody>
</table>
Table 1: Optimal Ratio of Reprogramming Plasmids6. These plasmids are available to order through Addgene.&#160;

Watch this Scientific Journal Video about Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors at JoVE.com

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

efficient-ips-cell-generation-from-blood-using-episomes-hdac

Nanyang Technological University

Research

JoVE Journal

Biology

12.7K Views.  Fred Hutchinson Cancer Research Center. Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

Video: Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

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, ...

Developmental Biology

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 ...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

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.

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 ...

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 ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Bioengineering

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, ...