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 durin...

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 identifie...

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 ...

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Research

JoVE Journal

Biology

12.7K Views.  Fred Hutchinson Cancer Research Center.  Hier beschrijven we een protocol voor het genereren van humane geïnduceerde pluripotente stamcellen uit perifeer bloed met behulp van een episoom gebaseerd herprogrammering strategie en histondeacetylaseremmers. 

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

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

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 ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

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 Mice Derived from Induced Pluripotent Stem Cells

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

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

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

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

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