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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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 border="0">
	<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>KBM502 medium</td>
			<td>KOHJIN BIO</td>
			<td>16025020</td>
			<td>Warm in 37 &#8451; water bath before use</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V solution</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td>Thaw completely at 4&#8451; overnight and dilute it 50 times with Dulbecco&#39;s Modified Eagle&#39;s Medium before coating culture dishes</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(&#8211;)</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>15ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</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

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, ...

연구

발생학

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

헤어 유래 각질 세포를 사용하여 통합없는 인간 유도 만능 줄기 세포의 생성

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

인간 다 능성 줄기 세포에서 멜라닌 세포의 공급이없는 유도

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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 ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

정의 및 간세포와 같은 세포의 확장 성 생성 인간 다 능성 줄기 세포에서

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

말초 혈액 유래 인간 유래 다 능성 줄기 세포에서 연골 세포 분화

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

인간에서 표준화 되 고 재현 가능한 개 타입 대뇌 Organoids의 생성 유도 만능 줄기 세포

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

코드 혈액 파생에서 3D 피부 Organoid의 생성 유도 만능 줄기 세포

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...