Name: generation of induced pluripotent stem cells from human peripheral t cells using sendai virus in feeder-free conditions
Uploaded: 2015-11-11T15:00:00.000+00:00
Duration: 9 min 32 s
Description: Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

우리는 기술 지원을 의학의 게이오 대학에서 요시코 미야케, 사야카 Kanaami, Chihana 후지타, 미호 야마구치, 나츠코 Henmi 및 레이 오노 감사합니다. 이 작품은 부분적으로 건강 연구 성과의 실용화를 촉진하기 위해 R &amp; D 시스템 지원 프로그램, 및 재생 의학의 실현을위한 고속도로 프로그램에 의해 투자되었다.

1. 활성화 된 인간 T 세포를 준비 <ol><li> 정맥 천자에 의한 기증자 전체 혈액을 헤파린 10 ㎖를 수집합니다. <ol><li> 정맥 천자의 사전에 기증자의 동의를 얻습니다. 정맥 천자는 자격을 갖춘 숙련 된 기술자에 의해 수행되어야합니다. </li><li> 멸균 장비와 다음 적절한 바이오 안전성 가이드 라인을 얻은 혈액 샘플을 처리합니다. </li></ol></li><li> 10 ml의 D-PBS로 전혈 헤파린 10 ml에 희석 (-). </li><li> 새로운 50 ML 원뿔 튜브에 15 ml의 피콜 용액 (피콜 - 페이크 PREMIUM)를 넣습니다. </li><li> 단계 1.3에서 피콜 용액에 1.2 단계에서 제조 20 mL의 혈액 용액을 층을 포함한다. 전기 피펫을 사용하여, 혈액 솔루션은 튜브의 내부 벽을 따라 천천히 단계에서 1.2 실행을 준비 할 수 있습니다. 혈액 솔루션 및 피콜 용액을 혼합하지 않도록주의하십시오. </li><li> RT에서 30 분 동안 400 × g에서 원심 분리한다. &quot;느린&quot;할 수있는 원심 감속 속도를 설정합니다. 주 : 원심 분리 후, 흰색 얇은상부 층은 유백색 플라즈마 층과 하부 맑은 피콜 층 사이에 나타난다. 이 흰색 얇은 층은 단핵 세포가 포함되어 있습니다. </li><li> 1 ml의 피펫 새로운 50ml의 원뿔형 튜브에 단핵 세포층을 전송. 피콜 솔루션을 포함하지 않도록주의하십시오. </li><li> D-PBS를 추가 (-) RT에서 5 분 동안 200 × g에서 10 ㎖ 총 부피의 원심 분리에 단핵 세포. </li><li> 상층 액을 버린다. 단핵 세포 및 실온에서 5 분간 200 × g에서 원심 분리기 (-) D-PBS의 10 ML을 추가합니다. </li><li> 상청액을 폐기 회수 단핵 세포 KBM502 배지 1 mL를 넣어 혈구를 이용한 세포를 카운트. × 106 5 000 단핵구 적절한 분리를 Ficoll을 사용하여 전혈 헤파린을 10 ㎖로부터 얻을 수있다. </li><li> D-PBS 중의 10 ㎍ / ml의 농도로 항 - 인간 CD3 항체 용액을 제조 (-). 서핑을 담가, 6 웰 플레이트에 항 인간 CD3 항체 용액을 첨가각각의 ACE 아니라, 적어도 30 분 동안 5 % CO2 인큐베이터에서 37 ℃에서 배양 접시. </li><li> 각 웰로부터의 항 인간 CD3 항체 용액을 제거하고, D-PBS로 한번 세척 (-) 세포를 파종 직전. </li><li> KBM502 매체에 항 인간 CD3 항체 - 코팅 된 6- 웰 플레이트에 2.5 × 105 세포 / ㎠의 밀도로 190 단계에서 제조 된 시드 단핵 세포. T 세포가 포화 상태에 도달 할 때까지 변화없이 배지를 3-7 일 동안 5 % -CO 2 인큐베이터에서 37 ℃에서 배양한다. 이 기간에있어서 T 세포가 모두 부착 된 현탁 세포이다. </li></ol> SEV 벡터 2.을 감염 인간 T 세포 <ol><li> 3-7일 배양 한 후, 피펫으로 매체를 기존에 활성화 된 T 세포를 수집합니다. 실온에서 5 분 동안 200 × g에서 15 ML 원뿔 튜브와 원심 분리기로 전송합니다. </li><li> 상층 액을 제거하고 1 ml의에게 신선한 KBM502 매체를 추가 할 수 있습니다. 세포를 세어 1 접시입니다.5 × 106 세포를 항 CD3 항체 - 코팅 된 6- 웰 플레이트의 각 웰에 (2 ㎖ KBM502 신선한 배지에서). </li><li> 얼음에 OCT3 / 4-SEV / TSΔF, SOX2-SEV / TSΔF, KLF4-SEV / TSΔF 및 c-MYC (HNL) -SeV / TS15ΔF의 SEV 솔루션을 녹여. </li><li> OCT3 / 4-SEV / TSΔF, SOX2-SEV / TSΔF, KLF4-SEV / TSΔF 및 c-MYC (HNL)를 포함 SEV 솔루션을 추가 -SeV / TS15ΔF 개별적으로 우물에 감염 다수의 각 (MOI) 추가로 24 시간 동안 5 %-CO 2 배양기에서 37 ° C에서 10 품어의. </li></ol> 3. 인간 T 세포에서 SEV 벡터를 제거 <ol><li> 24 시간 감염 후, 피펫에 감염된 세포를 수집합니다. 부착 된 세포가있는 경우, 그들은 쉽게 반복 상하 피펫 팅 그들을 분리 할 수​​있다. 실온에서 5 분 동안 200 × g에서 15 ML 원뿔 튜브와 원심 분리기로 전송합니다. </li><li> SEV 벡터를 포함하는 상층 액을 제거하고, 2 ml의에게 신선한 KBM502 매체를 추가 할 수 있습니다. EAC에서 Replate 세포우물의 시간. 추가로 24 시간 동안 5 % -CO 2 인큐베이터에서 37 ℃에서 배양한다. </li></ol> 마트 리겔 계층 4. 시드 세포 <ol><li> 4 ℃에서 (본 연구에 사용 된 특정 브랜드 자재 목록 참조) 및 DMEM / F12 배지 10 ㎖에 0.2 ㎖에 용해 해동 행렬 겔 기저막 매트릭스 용액을 제조 하였다. 참고 : 매트릭스 겔 (이 연구에서 사용 된 특정 브랜드에 대한 자료 목록을 참조하십시오 것은) 4 ° C에서 완전히 해동하는 O / N을 해동 할 필요가있다. 사용 직전까지 얼음에 보관하십시오. </li><li> 100-mm 접시에 접시 당 5 ㎖ 접시 기저막 매트릭스 용액과 세포를 파종 전에 적어도 30 분 동안 RT에서 떠난다. 적어도 두 개의 100-mm의 요리는 하나의 실험이 필요하다. </li><li> , 피펫 팅에 의해 SEV 감염된 세포를 수집 실온에서 5 분 동안 200 × g에서 15 ML 원뿔 튜브와 원심 분리기로 전송. </li><li> 상층 액을 제거하고 1 ml의에게 신선한 KBM502 매체를 추가 할 수 있습니다. CE의 수를 계산혈구를 사용 LLS, 단계 4.2 및 플레이트 제조 100-mm 접시에서 기저막 매트릭스 용액을 제거 1 × 105 1 × 106 세포를 100-mm 기저막 매트릭스 피복에 (10 mL의 신선한 mTeSR 매체) 그릇. 마지막으로, 식민지 쉽게 포착되는 플레이트를 사용합니다. </li><li> 5 %-CO 2 배양기의 O / N 37 ° C에서 접시를 품어. </li><li> 10 매체를 변경 ml의 매일 mTeSR 매체를 살. 참고 : 감염 후 약 20-30일 밀접 배아 줄기 세포 (는 ESC)를 닮은 식민지를 나타납니다. </li></ol> 5. T 세포에서 파생 된 iPSCs를 확장 <ol><li> 4 ℃에서 (본 연구에 사용 된 특정 브랜드 자재 목록 참조) 및 DMEM / F12 배지 10 ㎖에 0.2 ㎖에 용해 해동 행렬 겔 기저막 매트릭스 용액을 제조 하였다. </li><li> 플레이트 (1) 6 웰 플레이트에 웰 당 기저막 매트릭스 ml의 용액에 실온에서 떠나식민지를 따기 전에 적어도 30 분. </li><li> mTeSR 매체 20 μL로 96- 웰 플레이트를 웰 당 채우기. </li><li> 20 μL 피펫을 사용하여 실체 현미경 아래는 ESC를 닮은 식민지를 선택합니다. 참고 : 그림 1B와 같이는 ESC와 iPSCs의 식민지는 원형과 평면이다. </li><li> 단계 5.3 mTeSR 매체로 채워진 96 웰 플레이트에 전송 선택 식민지. </li><li> 200 μL 피펫을 사용하여 실체 현미경 아래에 작은 덩어​​리로 식민지를 깨고 아래로 피펫 조심스럽게 각 웰에 mTeSR 매체의 200 μl를 추가합니다. 5.2에 잘 준비에서 기저막 매트릭스 솔루션을 제거하고 mTeSR 매체 2 ㎖와 기저막 매트릭스 코팅 6 웰 플레이트 잘 하나에 각 식민지를 넣어 당 잘. </li><li> 2 매체를 변경 ml의 매일 mTeSR 매체를 살. </li></ol> 6. T 세포에서 파생 된 iPSCs 유지 T 세포 유래 iPSCs은 유지 될 수있다인간 및 인간 iPSCs는 ESC와 동일한 기술을 사용하여 저장된다. IPSC 식민지가 성장하면, 동일한 절차를 반복하여 큰 요리로를 확장합니다. <ol><li> 모든 1-2일 mTeSR 매체를 변경합니다. </li><li> 식민지가 합류 될 때마다 5~7일, 인간 iPSCs 및 제조업체의 지침에 따라 인간는 ESC를 위해 분리 솔루션을 사용하여 통로 세포. </li></ol>

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C., 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+by+a+nonintegrating+RNA+Sendai+virus+vector+in+feeder-free+or+xeno-free+conditions.">Generation of human-induced pluripotent stem cells by a nonintegrating RNA Sendai virus vector in feeder-free or xeno-free conditions.</a> Stem cells Int. , 564612 (2012).</li><li>Iida, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sendai+virus+vector:+vector+development+and+its+application+to+health+care+and+biotechnology.">Sendai virus vector: vector development and its application to health care and biotechnology.</a> Uirusu. 57, 29-36 (2007).</li><li>Yoshida, 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=In+vivo+repopulation+of+cytoplasmically+gene+transferred+hematopoietic+cells+by+temperature-sensitive+mutant+of+recombinant+Sendai+viral+vector.">In vivo repopulation of cytoplasmically gene transferred hematopoietic cells by temperature-sensitive mutant of recombinant Sendai viral vector.</a> Biochem Biophys Res Comm. 361, 811-816 (2007).</li><li>Bitzer, M., Armeanu, S., Lauer, U. M., Neubert, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sendai+virus+vectors+as+an+emerging+negative-strand+RNA+viral+vector+system.">Sendai virus vectors as an emerging negative-strand RNA viral vector system.</a> J Gene Med. 5, 543-553 (2003).</li><li>Seki, T., Yuasa, S., Fukuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+from+a+small+amount+of+human+peripheral+blood+using+a+combination+of+activated+T+cells+and+Sendai+virus.">Generation of induced pluripotent stem cells from a small amount of human peripheral blood using a combination of activated T cells and Sendai virus.</a> Nat Protoc. 7, 718-728 (2012).</li><li>Okano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+Sendai+virus+vectors+for+activated+T+lymphocytes.">Recombinant Sendai virus vectors for activated T lymphocytes.</a> Gene Ther. 10, 1381-1391 (2003).</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=An+efficient+nonviral+method+to+generate+integration-free+human-induced+pluripotent+stem+cells+from+cord+blood+and+peripheral+blood+cells.">An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.</a> Stem Cells. 31 (3), 458-466 (2013).</li><li>Zhang, X. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+reprogramming+of+human+peripheral+blood+cells.">Cellular reprogramming of human peripheral blood cells.</a> Genom Proteom Bioinform. 11 (5), 264-274 (2013).</li><li>Su, R. J., Neises, A., Zhang, X. B. <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+iPS+Cells+from+Human+Peripheral+Blood+Mononuclear+Cells+Using+Episomal+Vectors.">Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors.</a> Methods Mol Biol. , (2014).</li></ol>

저자는 더 경쟁 또는 공개의 이익 충돌이 없습니다.

We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation. 
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger. 
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.

iPSCs는 재생 의학 1-3 세포의 획기적인 소스로 주목을 받고있다. 현재까지 iPSCs를 생성 다양한 방법들이 4,5보고되었다. 이들 중에서, 인간 T 세포로부터 생성되는 세포 iPSCs 때문에 샘플링 6-8의 덜 침습적 방법의 특별한 관심왔다. 또한 T 세포로부터 유래 iPSCs는 T 세포 수용체 (TCR) 유전자의 재 배열을 포함하며, 따라서 항원 특이 적 T 세포 9,10의 근원이다. 따라서, T 세포 유래 iPSCs를 생성 안전하게 재생 의학을 진행하는 데 유용합니다. 이 방법은 예측 불가능한 병원성의 위험을 감소시키는 개념에 기초한다. iPSCs의 안전한 임상 응용을 위해 그것을 11은 병원균에 대한 노출의 위험을 줄이는 것이 중요하다. 이미 다 능성 줄기 세포의 배양 많은 시스템에서, 소 태아 혈청과 피더 세포를 필수적 시약으로서 사용되어왔다12이야. IPSC 생성 예측할 병원성의 위험을 감소시키기 위해 그러나, 배양 시스템에서 이러한 두 시약의 제거가 바람직하다. 또한,이 방법은 환자와 피더 세포의 제조에서 힘드는 침습적 셀 샘플링을 피할 수있는 장점이있다. T 세포 유래 iPSCs 이미 질병 연구 (13, 14)에 성공적으로 사용 되었기 때문에,이 방법은 환자의 질환 별 iPSCs를 생성 적용 가능하고 유용하다. 유전자 차량으로 센다이 바이러스 (SEV) 벡터를 사용하여 T 세포의 리 프로그래밍 방법 중 고효율 7,16과 iPSCs를 생성 할 수있는 방법이다. SEV는 단일 - 가닥 RNA 바이러스이고, DNA 복제 단계가 필요하지 않기 때문에 또한, IPSC 세대에서의 사용은 숙주 게놈 17-19 속보 피한다. 따라서 혈청 FR에서 인간 말초 T 세포로부터 iPSCs를 생성하기위한 프로토콜을 확립EE 및 마트 리겔, mTeSR 매체의 조합을 이용하여 피더가없는 조건, SEV 벡터.

<table><tbody><tr><td>Ficoll-Paque PREMIUM</td><td>GE Healthcare</td><td>17-5442-02</td><td></td></tr><tr><td>Purified NA/LE mouse anti-human CD3</td><td>BD Pharmingen</td><td>555336</td><td></td></tr><tr><td>Bovine albumin fraction V solution</td><td>Gibco</td><td>15260-037</td><td></td></tr><tr><td>mTeSR1 medium kit</td><td>STEM CELL</td><td>5850</td><td>Warm at room temperature before use</td></tr><tr><td>Dissociation Solution</td><td>ReproCELL</td><td>RCHETP002</td><td></td></tr><tr><td>D-PBS(&amp;ndash;)</td><td>Wako</td><td>045-29795</td><td></td></tr><tr><td>SeV Vector kit CytoTune-iPS ver.1.0</td><td>DNAVEC</td><td>DV-0303c</td><td>Thaw on ice before use</td></tr><tr><td>100-mm tissue culture dish</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>96-well tissue culture plate</td><td>Falcon</td><td>353078</td><td></td></tr><tr><td>6-well tissue culture plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>15&amp;#160;ml Centrifuge Tube</td><td>Greiner Bio-One</td><td>188271</td><td></td></tr><tr><td>50&amp;#160;ml Centrifuge Tube</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr></tbody></table>

generation of induced pluripotent stem cells from human peripheral t cells using sendai virus in feeder-free conditions

최근 iPSCs 회생 치료를위한 세포를 새로운 소스로서 주목 받고있다. 생성 iPSCs의 초기 방법은 침습적 조직 검사와 유전자의 레트로 바이러스 게놈에 삽입하여 얻은 피부 섬유 아세포에 의존하지만, 이러한 단점을 피하기에 많은 노력을 기울이고있다. 인간 말초 T 세포를 생성 iPSCs 고유 셀 소스이다. T 세포로부터 유래 iPSCs는 T 세포 수용체 (TCR) 유전자의 재 배열을 포함하고 항원 특이 적 T 세포의 근원이다. 또한, 게놈의 T 세포 수용체 재배치 개별 세포주 라벨 및 이식 공여자 세포를 구별 할 수있는 잠재력을 갖는다. iPSCs의 안전한 임상 적용의 경우, 유해한 제제에 새롭게 생성 iPSCs 노출의 위험을 최소화하는 것이 중요하다. 소 태아 혈청 및 피더 세포가 다 능성 줄기 세포 배양에 필수적인 하였지만, 이것은 위험을 줄이기 위해 배양 시스템에서 제거하는 것이 바람직하다예측할 수없는 병원성의. 이 문제를 해결하기 위해, 우리는 정의되지 않은 병원체에 iPSCs 노출의 위험을 줄이기 위해 센다이 바이러스를 사용하여 인간 말초 T 세포로부터 iPSCs를 생성하기위한 프로토콜을 확립했다. 센다이 바이러스를 처리하는 것이 적절한 바이오 레벨 장비가 필요하지만, 센다이 바이러스가 감염 게놈 삽입없이 T 세포를 활성화하면서도 고효율. 이 프로토콜에서는 활성화 된 T 세포 배양 및 센다이 바이러스의 조합을 이용하여 피더가없는 조건에서 인간 말초 T 세포로부터 iPSCs의 생성을 보여준다.

<html> 이 프로토콜을 이용하여, 사용자가 안정되게 인간 말초 T 세포로부터 iPSCs를 생성 할 수있다. . 몇 구절 후 검출되지 않았다 여러 기증자의 경우와 SEV 사이에 세포 재 프로그래밍의 효율성의 0.005 % (16) 그림 1A는 T의 생성을위한 프로토콜의 개략적 인 세포가 유래 보여줍니다 - SEV 및 마트 리겔와 T 세포에서 IPSC 세대는 약 0.002이 %가 나타났다 마트 리겔과 mTeSR 매체를 이용하여 공급 장치가없는 상태에서 iPSCs. 약 20~30일 SEV와 감염 후, IPSC 식민지들은 ESC 식민지와 같은 형태 (그림 1B)에 의해 인식됩니다. 면역 형광 염색법은 전형적인 만능 세포 마커의 발현 공개 (NANOG, OCT3 / 4, SSEA4는, TRA-1-60 및 TRA-1-81) T 세포 유래 iPSCs에서이 피더없는 조건 (그림 1C)에서 생성됩니다. &quot;폭 =&quot;700 &quot;/&gt; 도 1 (A) : 본 연구에서 피더가없는 조건 하에서 재 프로그래밍 T 세포에 대한 프로토콜의 개략도. (B) : 공급 장치가없는 상태에서 채혈 후 하루 27 전형적인 ESC와 같은 IPSC 식민지. (C). T 세포 유래 iPSCs에서 ALP 염색과 분화능 및 표면 마커에 대한 면역 형광 염색 (NANOG, OCT3 / 4, SSEA4, TRA-1-60 및 TRA-1-81)의 공급이없는 조건에서 생성 <a href="https://www.jove.com/files/ftp_upload/53225/53225fig1highres.jpg" target="_blank">하십시오 이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

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

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

피더가없는 조건에 센다이 바이러스를 사용하여 인간 말초 T 세포로부터 유도 된 다 능성 줄기 세포의 생성

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

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Developmental Biology

11.9K 조회수.  Keio University School of Medicine. This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

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

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

말초 혈액 조혈 전구 세포에서 인간 신경 줄기 세포의 직접 유도

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

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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.

비 통합 에피 솜 플라스미드를 사용하여 냉동 버피 코트에서 유도 만능 줄기 세포의 생성

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

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

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

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

인간의 흑색 종 종양 침윤 림프구에서 유도 만능 줄기 세포의 생성

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

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.

센다이 바이러스 원심 분리를 이용하여 혈액 세포로부터 유도 만능 줄기 세포의 생성

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.

에피 솜 벡터를 이용하여 인간 말초 혈액 단핵 세포로부터의 통합없이 유도 다 능성 줄기 세포의 생성

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

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.

Stemgent 인간 TF의 Lentivirus와 인간의 섬유아 세포를 프로그래밍하여 유도 Pluripotent 줄기 세포의 생성 세트

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

생물학

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

말초 단핵 세포에서 유도 된 신경 줄기 세포의 생성 및 이식 연구에 대 한 도파민성 신경 전구체로 분화

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

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, ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

인간의 Cardiomyocytes의 생성 : 피더없는 인간 유도 만능 줄기 세포에서 분화 프로토콜

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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 ...