이 작품은 Friedreich 운동 실조증 연구 얼라이언스과 아놀드 가족 재단과 줄기 세포 및 MD 앤더슨 암 센터에서 발달 생물학을위한 센터에서 시범 교부금에 의해 지원되었다.

1. 바이러스 생산과 형질 도입 <ol><li> DMEM 배지 (DMEM 높은 포도당, inactivated 10% FBS 열, 2 개의 MM L-글루타민, 아니 항생제)의 10 ML에서 ~ 7-8x10과 6 당 10cm 판의 밀도에 플레이트 피닉스 Ampho 세포. 37 야간 보육 및 문화 플레이스 ° C에서 5 % CO 2. </li><li> 12 벡터 인코딩 μg 중 Oct3 / 4, Sox2, Klf4, C-myc, 또는 GFP 유전자 (Addgene plasmids 17,217, 17,218, 17,219, 17,220) 35 μl Fugene 6.를 사용하여 다음 날 transfect 피닉스 세포 DMEM 배지 (DMEM 높은 포도당, 2 밀리미터 L-글루타민, 아니 FBS와 항생제) 500 μl에 transfection 믹스를 준비합니다. 20 분 알을 품다. 부드럽게 70-80% 합류 피닉스 세포를 포함 10cm 플레이트로의 DNA 단지를 피펫. </li><li> 8 H 페니실린과 스트렙토 마이신을 포함한 DMEM 배지 (DMEM 높은 포도당, inactivated 10% FBS 열, 2 개의 MM L-글루타민)과 사후 transfection - 미디어 6 바꿉니다. </li><li> 그 후 수집, 바이러스를 포함하는 12 미디어 4 번 H간격은 모든 부분을 하나로 합치는 분리된 세포 파편 (바이러스 입자를 포함하는 미디어가 그러나 동결을 권장하지 않습니다, 전염성 활동을 잃지 않고 2 주 동안 냉장고에 보관하실 수 있습니다) 제거 필터 0.45 μm의를 사용하여 필터링된. </li><li> retroviral 미디어 컬렉션의 마지막 날 이전 냉동 주식 24 H에서 retroviral 형질 도입, 플레이트 인간 성인 섬유아 세포 (통로 10, Coriell 연구소)의 경우. 종자는 DMEM 높은 포도당에 잘 당 밀도를 1x10 5 세포에서 6 - 잘 젤라틴 덮여 접시에 세포를 10 % FBS, 2 개의 MM L-글루타민, 페니실린, 스트렙토 마이신 및 비 필수 아미노산. </li><li> 피닉스 세포에서 얻은 바이러스 입자를 포함하는 매체와 다음 날 대신할 DMEM 미디어. 위해 1천6백그램에서 fibroblast의 문화와 원심 분리기의 6 잘 플레이트의 각도에 6 μg / polybrene의 ML과 보완 바이러스 미디어를 (1 각 Oct4의 ML, Sox2, Klf4, 그리고 C-myc 미디어, 4 ML 총) 추가 20 1H ° C. 약 12 H AFter 형질 도입은 fibroblast 문화 매체와 입소문 미디어를 대체합니다. 24 H 간격의 바이러스성 감염과 원심 분리 3 회를 수행합니다. </li><li> 마지막으로 감염 후 48 H에 대한 DMEM 미디어 문화 섬유아 세포. GFP를 표현 retroviral 매체와 병렬 transductions에 의해 감염의 효율성을 확인합니다. 그림 1은 인간 fibroblast의 원심 분리-촉진 retroviral 감염의 효율성을 보여줍니다. </li></ol> 2. 프로그래밍 <ol><li> 당 잘 젤라틴 ~ 2x10 5 세포 밀도에 MEF 세포을 퍼뜨리고하여 γ-조사 MEF 피더 레이어 6 잘 접시를 준비는 fibroblast 성장 매체 6 잘 접시를 취급. 다음날은 섬유아 세포 성장 매체에 당 잘 ~의 밀도에 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)과 종자들을 1.2x10 4 0.05 %를 사용하여 감염된 인간의 섬유아 세포를 분리. </li><li> 다음날 hES 미디어 (DMEM/F12, 20 % 마네의 세럼의 교환이 아닌 필수 아미노산, penicilli로 미디어 교체N / 스트렙토 마이신, 2 개의 MM L-글루타민, 0.1 MM β-메르 캅 토 에탄올, 20 NG / ML 기저 Fibroblast의 성장 인자 (bFGF), 언어학 재활 치료의 초기 7 일간 0.5 밀리미터 나트륨 butyrate와 보충. 매일 미디어를 변경합니다. </li><li> 일일 transduced 세포의 형태학의 변화를 모니터링합니다. 엉덩이와 같은 세포의 콜로니는 약 5 반발을 시작합니다 - 십일 피더 세포 진입 transduced 섬유아 세포를 전송 후. hiPSC 식민지는 약 14 격리 준비가 - 28 일 MEF 피더 세포에서 그들을 도금 후. </li></ol> 3. hiPSC 식민지의 분리 <ol><li> hiPSC 식민지를 따기 전에 하루 섬유아 세포 성장 매체의 γ-조사 MEF 피더 세포 (물론 당 ~ 4x10 4 세포)와 함께 24 잘 접시를 준비합니다. 이 단계에서는 hiPSC의 식민지도 Matrigel과 mTeSR1 매체를 사용하여 피더 무료 문화에 전송할 수 있습니다. </li><li> hiPSC 콜로니를 분리하기 전에는 섬유아 세포 성장 mediu을 제거하여 MEF 플레이트를 준비M 및 FBS의 흔적을 제거하는 PBS로 MEFs을 rinsing. 그 후 각 잘 7,8에 10 μm의 록 억제제 Y27632와 0.5 ML hES 매체 (혹은 Matrigel 코팅 우물의 경우 mTeSR1 중간)에 추가합니다. </li><li> 필요한 경우, 층류 후드 (- C 그림 2A)에 마운트 현미경으로 21 게이지 바늘로 hiPSC 식민지를 둘러싼 섬유아 세포를 제거합니다. PBS로 번호판을 씻어하고 Tra-1-81 StainAlive 특정 항체 (1:200, Stemgent)를 포함하는 신선한 hES 미디어를 추가합니다. 후 30 분., 10 μm의 바위 억제제 보충 신선한 hES 미디어와 항체를 포함하는 미디어를 대체합니다. 형광 현미경으로 접시를 검사 마크 Tra-1-81 객관적인 마커 (그림 2D)를 사용하여 긍정적인 식민지. </li><li> 그림 2E와 F. 같이 21 게이지 바늘을 사용하여 여러 개의 작은 조각으로 판상 후드의 현미경 Tra-1-81 긍정 hiPSC 식민지를 잘라내기 </li><li> 공업로 hiPSC 식민지의 자동 피펫 (P200) 전송 조각을 사용하여MEFs 혹은 Matrigel과 24 잘 접시의 ividual 우물. 비 허리 세포를 전송하지 마십시오. 37 ° C 5 % CO 2 배양기에 플레이트를 삽입하고 hiPSC 식민지가 24-36 H 위해 연결할 수 있습니다. </li><li> 변경 hES 또는 mTeSR1 (Matrigel에 성장 식민지 용) 미디어 일상. 올바른 hES 같은 형태로 hiPSC 식민지는 24도 판 진입 초기 전송 후 48 H 표시됩니다. </li><li> 수동으로 통과 hiPSC의 식민지 매 6 - 12 잘하고 이후 6 - 잘 접시를 향해 팔일. </li><li> clonal 확장과 hiPSC 라인을 맺은 후 그림 3과 같이 immunocytochemistry를 사용 pluripotency 마커 TRA-1-60, SSEA-4, Oct3 / 4, Sox2와 Nanog의 표현을 분석합니다. 게놈 무결성 및 핵형 및 teratoma 형성 분석 6,9를 사용하여 얻을 라인의 분화 잠재력을 평가하십시오. </li></ol> 4. 대표 결과 레트로 바이러스-포함된 미디어를 내가 가진 효율적인 형질 도입의 성공적인 언어학 재활 치료에 중요합니다. 그것은 그림 1과 같이 효율성을 모니터링하는 바이러스마다 하나의 프로그래밍을 실험을 표현 GFP를 사용하여 전체 transfection / 감​​염 절차를 수행하는 것이 좋습니다. 일반적으로 0.5의 범위에 비 집중 바이러스 미디어를 사용하는 인간 섬유아 세포의 형질 도입을 통해, 10에서 설명한대로 GFP 표현 바이러스의 titer는 결정 - 5 × 10 ML (VP / ML) 당 7 바이러스성 입자. 섬유아 세포는 지난 감염 후 빠르면 이일과 같은 형태로 변경합니다. Trypsinized 인간 섬유아 세포는 신중하게 MEF 피더 세포에 그들을 퍼뜨리고하기 전에 계산해야합니다. 각 세포주 서로 다른 성장 특성을 보여주고 시딩 밀도 프로그래밍의 효율성에 중요한지 3 가지 밀도 (6x10 3, 1.2x10 4, 6 잘 접시의 잘 단일 당 2.5x10 4)에서 씨앗 세포를 권장합니다. 에 사용되는 나트륨 butyrate초기 7 - 프로그래밍 14 일 hiPSC 형성 약 5 배의 효율성을 증가시킵니다. 자주, 감염된 섬유아 세포가 고밀도로 놓는되었다 특히, fibroblast 같은 세포는 배양 접시를 자라다와 그림 2A와 같이 hiPSC 식민지를 커버하실 수 있습니다. 이 경우, fibroblast 계층은 신중하게 드러내기 시작하고 hiPSC의 식민지 (그림 2B)을 밝히기 위해서 제거할 수 있습니다. 그 후, hiPSC의 식민지 hES 미디어 씻어서 Tra-1-81 항체 물들일한다. fibroblast 세포에 따라 약 20 - iPSC 같은 형태로 보여주는 식민지의 40 %는 Tra-1-81 항체와 착색하지 않습니다. 24 - 확인된 hiPSC 식민지는 앞으로 12 시간 이내 접시에서 전송할 수 있습니다. 장기 배양은 hiPSCs의 급속한 분화집니다. 식민지는 수동 MEF 피더 세포 혹은 Matrigel - 코팅 접시 하나에 passaged 수 있습니다. 확장 설립 후 hiPSC의 클론은 pluripoten의 표현을 위해 immunocytochemistry (ICC)를 사용하여 테스트해야그림 3에서 보여준대로 싸이가 마커. 또한, 생성된 IPS 세포 라인에 대한 자세한 분자 특성을 포함한다 : RT-PCR, 9 입을 transgenes의 pluripotency 유전자와 분석의 발기인의 DNA의 demethylation의 데모를 사용 pluripotency 유전자 발현의 분석. <img alt="그림 1" src="/files/ftp_upload/3416/3416fig1.jpg" /> 1 그림. GFP retroviral 매체와 연속 2 감염 후 GFP 표현 레트로 바이러스. (A, B) Friedreich 운동 실조증 환자에서 파생된 인간 성인 섬유아 세포 (GM03665, Coriell 저장소)는 시각있었습니다을 사용하여 결정 바이러스 형질 도입의 효율. (C, D) 인간 섬유아 세포를 직접 1천6백g에서 1 시간을위한 6 자 접시에있는 세포를 원심 분리에 의해 다음 GFP retroviral 미디어 동일한 일괄 처리에 감염되었다. 이미지는 감염 이후에 48h를 점령했다. <img alt= &quot;그림 2&quot;SRC = &quot;/ files/ftp_upload/3416/3416fig2.jpg&quot;/&gt; 그림 2. hiPSC 식민지의 식별 및 분리. () 섬유아 세포에 둘러싸여 hiPSCs를 포함하는 접시의 위상 콘트라스트 이미지입니다. 세포는 hES 미디어 21 일간 배양해되었다. 주변 fibroblast 계층의 제거 후 (B, C) 같은 hiPSC 식민지. (D) 올바르게 다시 프로그램 hiPSC 식민지가 Tra-1-81 표면 마커 항체 라이브 염색법에 의해 식별되며, 무균 바늘 (E, F)을 사용하여 절단 및 24도 접시의 별도의 우물로 전학 왔어. <img alt="그림 3" src="/files/ftp_upload/3416/3416fig3.jpg" /> 그림 3. pluripotency 특정 마커 Oct3 / 4, Nanog, Sox2, SSEA4 및 hiPSCs의 Tra-1-60 표현은 immunocytochemistry에 의해 결정되었다.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+gene+delivery+can+reprogram+human+fibroblasts+to+induced+pluripotent+stem+cells.">Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells.</a> Stem Cells. 27, 2667-2674 (2009).</li><li>Warren, L., Manos, P. D., Ahfeldt, T., Loh, Y. H., Li, H., Lau, F., Ebina, W., Mandal, P. K., Smith, Z. D., Meissner, A., Daley, G. Q., Brack, A. S., Collins, J. J., Cowan, C., Schlaeger, T. M., Rossi, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+reprogramming+to+pluripotency+and+directed+differentiation+of+human+cells+with+synthetic+modified+mRNA.">Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.</a> Cell Stem Cell. 7, 618-630 (2010).</li><li>Kim, D., Kim, C. H., Moon, J. I., Chung, Y. G., Chang, M. Y., Han, B. S., Ko, S., Yang, E., Cha, K. Y., Lanza, R., Kim, K. 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+human+induced+pluripotent+stem+cells+by+direct+delivery+of+reprogramming+proteins.">Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.</a> Cell Stem Cell. 4, 472-476 (2009).</li><li>Han, S. S., Williams, L. A., Eggan, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+and+deconstructing+stem+cell+models+of+neurological+disease.">Constructing and deconstructing stem cell models of neurological disease.</a> Neuron. 70, 626-644 (2011).</li></ol>

저자가 공개하는 게 없다.

특히 신경 및 neurodegenerative 인간의 질병, 유학, 특히 충분한 사람 세포 모델의 어려움으로 인해 도전되었습니다. 다양한 세포 유형으로 그들을 차별 유도된 pluripotent 줄기 세포의 가능성에 쉽게 얻을 체세포를 재설 정할 능력은 유전 질병 세포 모델을 만들 수있는 가능성을 열었습니다. 또한, iPSCs은 재생 의료의 미래에 큰 약속을 누르고 있습니다. 따라서 pluripotency로 체세포를 프로그래밍의 안정적인 효율적이고 안전한 방법을 개발하고 최적화하기 위해 필수적입니다. 치료적 응용 프로그램의 관점에서 그것은 호스트 게놈에 풋프린트 변이 부족 iPSC 세대의 안전한 접근법을 개발하는 것이 매우 중요합니다. 이러한 episomal 벡터의 transfection 등 다시 프로그램 세포의 게놈에 영구적인 변경을 도입하지 않고 프로그래밍을 체세포의 방법은 excisable tra의 사용nsposons, adenoviral 감염, 그리고 mRNA 또는 단백질의 직접적인 전달은 이미 11-15 설립되었습니다. 지금까지 이러한 transgene없는 방법을 사용하여 프로그래밍 효율이 낮습니다 그리고 다시 프로그램 체세포 세포의 문자 유형과 연령에 따라 달라집니다. 따라서 이러한 프로토콜을 늦게 통과를 프로그래밍에 적합하지, 성인 체세포 자주 세포 저장소로 입금. 또한 여러 iPSC 라인의 세부적인 특성을 활성화하고 인간의 질병의 새로운 모델을 확립하고 전사 요인 바이러스 전송을 사용하여 체세포의 프로그래밍, 높은 처리량 약물 화면을 진행하는 것은 적절한 전략이다. 20 개 이상의 연구와 데이트를하기 위해서는 모델이 인간의 신경계 및 신경근육학 질환 ​​환자의 특정 iPSCs의 생성을보고했다. 하나를 제외한 모든 경우에 iPSCs는 lentiviral 또는 retroviral 형질 도입 16를 사용하여 생성되었습니다. 당사의 데이터를 보여주는 그 일을 기준으로 간단한 프로토콜OSKM 전사 요인 전자 retroviral 전달이 효율적으로 심지어 높은 통과 성인 인간의 섬유아 세포를 재설정하는 데 사용할 수 있습니다. 10cm 플레이트에서 피닉스 세포의 단일 transfection에서 얻은 바이러스의 금액은 10 개 이상의 프로그래밍을 실험을하기에 충분 해요. 성인 섬유아 세포 우리의 프로그래밍 프로토콜 세 중요한 단계가 있습니다 : 극적으로 형질 도입의 효율을 증가 형질 도입하는 동안 추가로 원심 분리 단계에 의해 촉진은 (i) 고효율 바이러스 형질 도입, (ii) 본 심는 MEFs쪽으로 transduced fibroblast의 밀도와 ( 3) 직접 피더없는 문화로 전송하실 수 있습니다 잠재적으로 완전하게 다시 프로그램 식민지의 선택을 촉진하기 Tra-1-81 표지 항체와 라이브 염색법을 사용하지 않습니다. 프로그래밍의 효율성이 크게 언어학 재활 치료의 두 ​​번째 주 동안 나트륨 butyrate를 사용하여 개선되었습니다. 더욱이, 우리는 iPSC 식민지의 큰 분수가에서 배양해 것을 관찰약물의 존재는 확대하고 완전히 다시 프로그램 iP​​SC 라인으로 작성할 수도 있습니다.

<table border="1"><tbody><tr><td> 시약 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11,965 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11,330 </td></tr><tr><td> KSR </td><td> Invitrogen </td><td> 10,828 </td></tr><tr><td> 중요하지 않은 aminoacids </td><td> Invitrogen </td><td> 11,140 </td></tr><tr><td> 나트륨 butyrate </td><td> 시그마 </td><td> B5887 </td></tr><tr><td> Y27632 </td><td> Stemgent </td><td> 04-0012 </td></tr><tr><td> bFGF </td><td> Stemgent </td><td> 03-0002 </td></tr><tr><td> Tra-1-81 항체 </td><td> Stemgent </td><td> 09-0069 </td></tr><tr><td> Oct3 / 4 항체 </td><td> 산타 크루즈 </td><td> SC-8628 </td></tr><tr><td> Nanog 항체 </td><td> 세포 신호 전달 기술 </td><td> 4903S &lt;/ TD&gt; </tr><tr><td> Tra-1-60 항체 </td><td> Millipore </td><td> MAB4360 </td></tr><tr><td> Sox2 항체 </td><td> 세포 신호 전달 기술 </td><td> 3579S </td></tr><tr><td> SSEA4 </td><td> Millipore </td><td> MAB4304 </td></tr><tr><td> CF1 MEFs </td><td> Globalstem </td><td> GSC-6201G </td></tr><tr><td> 목표 마커 </td><td> 니콘 </td><td> MBW10010 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354,277 </td></tr><tr><td> mTeSR1 </td><td> StemCell 기술 </td><td> 05,850 </td></tr><tr><td> β-메르 캅 토 에탄올 </td><td> 시그마 </td><td> M7522 </td></tr><tr><td> Fugene 6 </td><td> 로슈 </td><td> 11814443001 </td></tr><tr><td> polybrene </td><td> 시그마 </td><td> H9268 </td></tr><tr><td> 객체 마커 </td><td> 니콘 </td><td> MBW10010 </td></tr></tbody></table>

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

여기에 우리가 1-3 나트륨 butyrate의 면전에서 Oct3 / 4, Sox2, Klf4과 c-myc (OSKM)를 인코딩 retroviral 벡터를 사용하여 인간의 유도된 pluripotent 줄기 세포 (hiPSC)로 인간 성인 섬유아 세포를 프로그래밍의 프로토콜을 제시한다. 우리는 늦게까지 통로를 재설정하는 데이 방법을 사용 (&gt; p10) Friedreich 운동 실조증 환자에서 파생된 인간의 성인 섬유아 세포 (GM03665, Coriell 리포지 토리). 프로그래밍 접근 방식은 바이러스를 함유 미디어의 면전에서 섬유아 세포의 반복적인 원심 분리를 사용하여 고효율 형질 도입 프로토콜을 포함한다. 다시 프로그램 hiPSC의 식민지가 Tra-1-81 실시간으로 immunostaining를 사용하여 확인되었다 pluripotent 세포 표면 마커가 아닌 다시 프로그램 섬유아 세포에서 분리하고 수동 4,5 passaged. 이러한 hiPSC 그런 다음 Matrigel 접시에 양도하고 직접 프로그래밍을 접시에서, 피더없는 조건에서 재배되었다. 첫 번째 통로부터는 hiPSC의 식민지는 hES - 내가 특성 입증IKE 형태. 이 프로토콜을 사용하여 선택한 식민지 이상의 70 %는 성공적으로 확장과 셀 라인으로 작성할 수도 있습니다. 설립 hiPSC 라인 표면 마커 TRA-1-60 SSEA-4뿐만 아니라 핵 마커 Oct3 / 4, Sox2와 Nanog 포함하여 특색있는 pluripotency 마커를 표시합니다. 여기에 제시된 프로토콜은 설립 Friedreich 운동 실조증 환자 및 제어 개인 6, 인간의 신생아 섬유아 세포뿐만 아니라, 인간 keratinocytes에서 얻은 성인 섬유아 세포를 사용하여 테스트되었습니다.

Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

우리는 함께 retroviral Oct3 / 4, Sox2, Klf4과 c-myc (OSKM)를 인코딩 벡터, 라이브 염색법에 의해 정확하게 다시 프로그램 hiPSC의 신분을 이용하여 인간의 유도된 pluripotent 줄기 세포 (hiPSC) Tra-로 인간의 체세포의 효율적인 언어학 재활을위한 프로토콜을 제시 1-81 항체.

인간 유도 Pluripotent 줄기 세포의 선택 및 분리 식민지는 성인 섬유아 세포에서 다시 프로그램

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

selecting-isolating-colonies-human-induced-pluripotent-stem-cells

Nanyang Technological University

Research

JoVE Journal

Biology

19.8K 조회수.  University of Texas M.D. Anderson Cancer Center. 우리는 함께 retroviral Oct3 / 4, Sox2, Klf4과 c-myc (OSKM)를 인코딩 벡터, 라이브 염색법에 의해 정확하게 다시 프로그램 hiPSC의 신분을 이용하여 인간의 유도된 pluripotent 줄기 세포 (hiPSC) Tra-로 인간의 체세포의 효율적인 언어학 재활을위한 프로토콜을 제시 1-81 항체.

비디오: Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

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 ...

연구

생물학

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

소프트 Musculoskeletal 조직에서 줄기 세포를 분리

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

인간 유도 만능의 줄기 세포를 Transfecting 및 Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFP와 Retroviral 벡터를 사용하여 유도 Pluripotent 줄기 세포 (iPSCs)로 인간 체세포를 프로그래밍

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Culturing 인간의 배아와 유도 Pluripotent 줄기 세포에 적합 마우스 배아 Fibroblast 세포의 준비

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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, ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...