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>

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	<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. 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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 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.

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 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.remotexs.ntu.edu.sg/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.

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

Nanyang Technological University

Research

JoVE Journal

Biology

12.7K 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.

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

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

미국 호르세쇼 게, Limulus Polyphemus의 혈액 수집

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

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생물학

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

마우스 신경 조직에서 단일 셀 Suspensions의 생성

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

신생아 마우스의 폐 내피 세포의 분리 및 문화

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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.

유도 만능의 줄기 세포에서 파생 마우스의 생성

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.

STEMCCA Lentiviral 벡터를 사용하여 말초 혈에서 인간 유도 만능 줄기 세포의 생성

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

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Multititre 판 형식으로 인간 만능의 줄기 세포에서 뉴런의 신속하고 효율적인 생성

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

골격 근육의 Isometric 나기도하고 강제로 생성 평가는 절연 근육질 Dystrophies의 손쥐 모델에서

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

고해상도 용융 분석과 PCR을 이용하여 신속하고 효율적인 Zebrafish의 유전자형

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

인간의 골격 근육에서 혈관 유래 다 능성 전구체의 분리

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

센다이 바이러스와 인간의 체세포의 효율적인 생성 인간 유도 만능 줄기 세포

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...