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>
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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. 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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...

iPSCs는 만능 유전자의 최소한의 자궁외 표현을 통해 체세포 조직에서 파생됩니다. 이 기술은 처음에 매우 만능 상태 1로 표현된다 OCT4, SOX2, KLF4, 및 cMYC 인간 섬유 아세포의 레트로 바이러스 전달에 의해 입증되었다. 이러한 일시적 발현 &quot;리 프로그래밍 인자&quot;는 인간 배아 줄기 세포 (2) 표적 세포의 후성 프리 및 유전자 발현 프로파일이 유사한 변경. 생성 한 후에, iPSCs는 잠재적으로 추가 조사에 대한 조직 형으로 구분 될 수있다. 따라서, 그들은 재생 의학, 질병 모델링, 유전자 치료의 용도로 약속을 누르고 있습니다. 그러나, 바이러스 통합과 함께 게놈을 방해하는 내인성 유전자 발현, 영향 세포 표현형을 변화시킬 가능성이 있고, 궁극적으로 과학적 결과를 편향. 또한, 임의의 바이러스 통합은 해로운 세포 전자로 이어질 수악성화 3 발암 유전자 (4)의 재 발현의 가능성을 포함 ffects. 미래의 임상 적용은 비 통합 IPSC 생성을 필요로 할 것이다. 여분의 염색체 원형 DNA 분자이다 에피 좀은 비용 효율적인 통합없는 iPSCs 5를 생성하기위한 전략을 제공한다. 표 1에 나타내는 에피 솜 벡터의 조합은 리 프로그래밍이 OCT4, SOX2, KLF4, MYCL 및 LIN28A 인자 표현한다. pCXLE_hOCT3 / 4 shp53-F 플라스미드는 세포 재 프로그래밍 (6)을 향상시키기 위해 TP53의 일시적인 억제를위한 p53의 shRNA를 포함되어 있습니다. 복제 - 결핍 pCXWB EBNA1 벡터가 EBNA1 발현 7 일시적인 증가를 제공함으로써 리 프로그래밍 인자의 증폭과 증가 된 리 프로그래밍 효율을 촉진한다. pCXLE_EGFP 플라스미드 dete의 목적 nucleofection 혼합물에 첨가 될 수있다셀 정렬 응용 프로그램에 대한 형질 전환 효율 또는 rmining. P는 CXWB-EBNA1을 제외하고,이 프로토콜에서 사용하는 에피 솜 플라스미드는 숙주 세포 (8)의 분할 중에 복제 및 에피 솜의 파티션을 매개 바이러스 복제 및 EBNA1 유전자의 엡스타인 바 바이러스 원점을 함유한다. 에피 솜은 자발적으로 연속 IPSC 확장 7 손실됩니다. 서브 클로닝 및 eGFP는 표현의 손실을 추측 할 수있다 에피 솜 벡터 손실과 iPSCs의 특성은 미래의 임상 응용 프로그램에 완전히 통합 무료 iPSCs로 이어질 수 있습니다. IPSC 생성의 과정에 고유 혈통 특정 유전자의 억제 및 분화능 관련 유전자의 활성화입니다. 유전자 발현의 규제 대상으로 전사 인자를 허용하는 DNA 및 염색질 변형을 규제 DNA 요​​소, 및 RNA 폴리머 라제 이용과 같은 핵 내부의 여러 레벨에서 일어난다유전자. 글로벌 염색질 수정을 통해 후성 유전 적 풍경의 리모델링을 다시 표현하는 핵심 요소 다 능성 유전 적 프로그램입니다. 유전자 발현의 조절에 중요​​한 특정 염색질 변형 히스톤-DNA 코일 장력의 감소를 통해 유전자를 타겟팅 액세스를 허용하는 특정 리신 잔기에서 히스톤의 아세틸 화이다. HDAC 억제제 인해 아세틸 상태 (11)를 지원하는 것으로, IPSC 리 프로그래밍 및 hESC의자가 재생 (self-renewal) 9,10 향상하는 것으로 나타났다 작은 분자이다. 통합 렌티 12을 사용 전에 공보에서 적응 후술 프로토콜은 말초 혈액 이용한 에피 솜 및 HDAC 억제제에서 최적화 IPSC 생성을위한 단계적인 방법을 제공한다. 여기에 사용되는 HDAC 억제제의 농도는 도자기에 의해 설명 된의 절반, 등. 9, 일상적으로 표준을 통해 완벽하게 재 프로그램 IPSC 식민지에서 2 배 증가 주도HDAC 억제제없이 에피 솜 재 프로그래밍 프로토콜. 재 프로그래밍의 수준은 우리가 렌티 바이러스 방법과 관찰 효율성과 비슷한 수준이다. 이 프로토콜을 사용하여, 우리는 효율적으로 나이 팔십칠년만큼 오래된 개인에서 IPSC를 생성 한.

<table border="0" cellpadding="0" cellspacing="0" width="1100">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name of Reagent/ Equipment</td>
			<td style="width:151px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:503px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ficoll-Paque PLUS</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">45-001-750</td>
			<td>Store at 4&#176;C. Warm to room temperature before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DPBS</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14190-250</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">RPMI 1640</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11875-093</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">fetal bovine serum (FBS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10437028</td>
			<td>Store at -20&#176;C until needed. Thaw, aliquot, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dimethyl sulfoxide (DMSO)</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">154938</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">QBSF-60 serum-free medium</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">50-983-234</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">penicillin/streptomycin</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">15140122</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Cell Line Nucleofector Kit V</td>
			<td style="width:151px;">Lonza</td>
			<td style="width:119px;">VCA-1003</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2% gelatin solution</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1393</td>
			<td>Store at 4&#176;C, liquify in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Hank&#39;s Balanced Saline Solution (HBSS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14175-103</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:329px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">12440-061</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">L-glutamine, 200 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">non-essential amino acids (NEAA), 100X</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td>Store at 4&#176;C, away from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DMEM/Ham&#39;s F12, 1:1</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">SH30023.02</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">KnockOut Serum Replacement</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium pyruvate, 100 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11360-070</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium bicarbonate, 7.5%</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25080094</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">basic fibroblast growth factor (bFGF)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">PHG0263</td>
			<td>Make 10 mg/mL stock in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium butyrate&#160;</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887-1G</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">hES Cell Cloning &#38; Recovery Supplement</td>
			<td style="width:151px;">Stemgent</td>
			<td style="width:119px;">01-0014-500</td>
			<td>Store at -20&#176;C until needed. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ESC-qualified BD matrigel</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">35-4277</td>
			<td>Thaw overnight on ice at 4&#176;C, aliquot into pre-chilled tubes using pre-chilled pipette tips. Store at -20&#176;C until needed. Thaw at 4&#176;C, use immediately.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">StemSpan SFEM&#160;</td>
			<td style="width:151px;">STEMCELL Technologies</td>
			<td style="width:119px;">9650</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ascorbic acid, powdered</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4403-100MG</td>
			<td>Make 5 mg/mL stock in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human stem cell factor (SCF)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">255-SC-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human interleukin 3 (IL-3)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">203-IL-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">erythropoietin (EPO)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">287-TC-500</td>
			<td>Make 1000 U/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">recombinant human insulin-like growth factor 1 (IGF-1)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">291-G1-200</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">&#946;-mercaptoethanol</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7522</td>
			<td>Make 1000X stock (100 mM) in DPBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dexamethasone</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4902-25MG</td>
			<td>Make 50X stock (50 &#956;M) in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">SAHA (vorinostat)</td>
			<td style="width:151px;">Cayman Chemical</td>
			<td style="width:119px;">149647-78-9</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">12 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">15 mL conical tube</td>
			<td style="width:151px;">Sarstedt</td>
			<td style="width:119px;">62553002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">1.5 mL Eppendorf tube</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">6 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">35 mm tisue culture plates</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">10 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">5 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">20 &#956;L pipette tips, barrier tips</td>
			<td style="width:151px;">Genessee</td>
			<td style="width:119px;">24-404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">glass Pasteur pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pipette aid</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-681-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hOCT3/4-shp53-F</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hSK</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hUL</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-EGFP</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXWB-EBNA1</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">37624</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient ips cell generation from blood using episomes and hdac inhibitors

이 원고는 효율적으로 에피 솜 플라스미드와 히스톤 탈 아세틸 화 효소 (HDAC) 억제제를 사용하여 말초 혈액에서 통합이없는 인간 유도 만능 줄기 세포 (iPSCs)를 생성하기위한 프로토콜을 보여줍니다. 이 방법의 장점은 : (1) 원료로서 말초 혈액의 최소량의 사용; (2) 프로그램을 다시 벡터를 nonintegrating; (3) 자유 iPSCs 벡터를 생성하는 비용 효과적인 방법; (4) 단일 트랜; (5) 작은 분자의 사용은 후생 유전 학적 리 프로그래밍을 용이하게한다. 리 프로그래밍 현저 의무가 높은 증식 적혈구 전구 세포 집단을 수득 요약하면, 말초 혈 단핵 세포 (PBMC)는 루틴 채혈 샘플로부터 분리되어 정의 된 증식 인자에서 배양 하였다. Nonintegrating, OCT4, SOX2, KLF4, MYCL, LIN28A 및 p53의 짧은 머리 핀의 자형 (SH) RNA를 발현하는 nontransmissible 에피 솜 플라스미드는 introdu 있습니다하나의 nucleofection를 통해 도출 된 적혈구 모세포에 세드릭. 녹색 형광 단백질을 강화 표현한다 에피 솜 (eGFP는) cotransfection 된 세포는 형질 전환 된 세포를 쉽게 식별 할 수 있습니다. 엡스타인 - 바르 핵 항원 1 (EBNA1)를 별도의 발현 플라스미드의 복제 - 결함은 에피 솜 단백질의 발현 증가에 대한 반응 혼합물에 첨가된다. 형질 감염된 세포는 계속 재 프로그래밍에 대한 조사 마우스 배아 섬유 아세포 (iMEFs) 층에 도금되어있다. 즉시 IPSC 같은 식민지 nucleofection 후 약 십이일에 나타나는대로, HDAC 억제제는 후생 유전 학적 리모델링을 용이하게하기 위해 중간에 추가됩니다. 우리는 HDAC 억제제를 포함 정기적으로 2 배 완전히 재 프로그래밍 IPSC 식민지의 생성을 증가 시킨다는 것을 발견했다. IPSC 식민지 일반적인 인간 배아 줄기 세포 (hESC의) 형태를 전시되면, 그들은 부드럽게 지속적인 성장과 확장을위한 개별 IMEF 코팅 조직 배양 플레이트로 전송됩니다.

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.

에피 솜과 HDAC 억제제를 사용하여 혈액에서 효율적인 유도 만능 줄기 세포 생성

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

Research

JoVE Journal

Biology

12.6K 조회수.  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.