이 원고에서 설명한 작업하는 것은 UCR의 줄기 세포 핵심에 대한 재생 의료에 대한 캘리포니아 연구소 (CIRM)에서 기금에 의해 가능하게되었다.

우리 프로토콜은 GeneJuice (EMD)와 AMAXA nuclefector 장치를 사용하여 인간의 iPSCs의 nucleofection를 사용하여 인간의 iPSCs를 transfecting을위한 프로토콜 다음 피더 - 무료 문화에 인간의 iPSCs을 적용 할 수있는 방법으로 시작합니다. 참고 : 다음 절차는 무균 층류 후드에서 수행됩니다. 모든 미디어 및 솔루션은 37 ° C 또는 별도로 명시하지 않는 한 시작하기 전에 실내 온도에 equilibrated 있습니다. 1. 피더 - 무료 시스템에서 인간의 iPSCs 구축 이전 피더 세포에서 유지 관리 인력 iPSCs는 Geltrex - 코팅 접시에 양도 및 이전 피더 - 무료 transfection 두 구절에 대한 유지, 분할 할 수 있습니다. <ol><li> 4 ° C.에서 밤새 Geltrex을 해동 Geltrex 코팅을 준비하는, 차가운 DMEM에 defrosted Geltrex 1시 50분을 희석. 부드럽게 솔루션을 섞는다. </li></ol> 참고 : Geltrex, Matrigel 같은이 지하 막 matr의 용해 형태손쥐에서 IX 정화는 Engelbreth-Holm이 - 떼 (EHS) 종양 세포. 또한, Matrigel는 피더 - 무료 인간 iPSC 문화를 구축 할 수있는 세포 외 기질로 사용하실 수 있습니다. <ol start="2"><li> Geltrex 솔루션 (35 mm 잘 1 ML)와 문화 우물의 전체 표면을 커버. 37 번 Geltrex있는 코트는 우물 ° 1 시간 C 보육. </li><li> 통로 인간의 iPSCs에, 당 잘 accutase 1 ML을 추가하고 대부분의 세포가 분리되기 전에는 ° C 1 분에 37 품다. </li></ol> 통로 인간의 iPSCs에, 당 잘 accutase 1 ML을 추가하고 대부분의 세포가 분리되기 전에는 ° C 1 분에 37 품다. <ol start="4"><li> 세포와 부드럽게 소용돌이 판에 10-15 유리 구슬을 추가합니다. 넉 아웃 DMEM / F12의 2 ML을 추가하고 부드럽게 가루로 빻다. 10 ML의 원심 분리기 튜브에 세포 현탁액을 전송합니다. </li><li> 상온에서 3 분 800 rpm으로 세포를 봐. </li><li> 인간 iPSC 펠렛은 그대로두고, 관에서 표면에 뜨는을 대기음. 부드럽게에 관을 버려야지세포 펠렛을 분산. </li><li> 코팅 우물에서 Geltrex를 제거합니다. </li><li> 부드럽게 STEMPRO의 적절한 볼륨으로 인간 iPSC 펠렛을 resuspend. ) 확산 속도에 따라 피더의 우물 사이에 배포합니다. 인간 iPSCs는 1시 2분에서 1시 6분까지의 분할 비율에 passaged 할 수 있습니다. </li><li> 조심스럽게 장소 5% CO 2 인큐베이터 신중하게 소용돌이 판 우물에서 세포도 분포를 보장하기에. </li><li> 세포까지 매일 피드 세포가 다시 분할 할 준비가되어 (세포가 80 % confluency에 도달했을 때). </li><li> 1시 2분의 분할 비율에 새로운 Geltrex 코팅도로 항로 인간 iPSCs (단계 1.3-1.9). 작은 식민지가 형성하고 잘 Geltrex 코팅 transfection 이전에 균일하게 분포되어야한다. </li></ol> 2. GeneJuice과 인간 iPSCs의 Transfection 셀 (6 잘 접시에 성장) 최적의 transfection 효율을 달성 할 수 transfection의 날에 합류 약 40 -50 %이어야합니다. 그것은이다하지 다음 날까지 셀 매체를 변경하는 데 필요한. <ol><li> 멸균 1.5 ML eppendorf 튜브에 100 μl KO-DMEM/F12를 준비합니다. 27 μl GeneJuice의 transfection 시약을 추가합니다. 잘 섞는다. 5 분 동안 실온에서 알을 품다. </li><li> 4 μg 플라스미드 DNA를 추가합니다. 15 분 동안 실온에서 튜브를 품다. 플라스미드의 선택은 최적의 transfection 효율을위한 중요합니다. 우리는 발기인 5 (pCAG-eGFP)를 전투 편대에 의해 구동 강화 된 녹색 형광 단백질 (eGFP)와 플라스미드를 사용합니다. 발기인을 전투 편대 인간 iPSCs의 transcriptionally 적극적이고 따라서 이러한 세포의 transgene 표현을 유도하는 데 사용할 수있는 강력한 발기인이다. 손에서 플라스미드의 선형화는 transfection 효율에 영향을 보이지 않았다. </li><li> 세포와 소용돌이 판에 GeneJuice-DNA 혼합물을 추가합니다. 잘에 인간의 iPSCs를 transfection 혼합물의 접촉을 높이기 위해 5 분 1,200 RPM ( &#39;spinoculation&#39;방법)에서 판을 봐. </li><li> 37 &amp;에있는 세포를 품다℃ 일, C 하룻밤. </li><li> 다음 날 transfection 효율 모니터링합니다. </li><li> 안정적인 transfection를 들어, 신선한 STEMPRO으로 다음날 매체를 변경합니다. 48시간 - 24 후 적절한 항생제 선택을 추가합니다. </li></ol> 3. 인간 iPSCs의 Nucleofection 모이통 또는 Geltrex에 성장 인간 iPSCs는 nucleofection 사용할 수 있습니다. 그러나, 우리는 강력 마우스 배아 섬유 아세포 (MEF) 또는 인간의 포피의 섬유 아세포 (HFF) 높은 세포 생존과 복구를 보장하기 위해 피더에 nucleofected 인간 iPSCs를 replating하시기 바랍니다. 적어도 2,000,000 세포는 nucleofection에 따라 높은 세포 생존을 달성하기 위해 사용되어야합니다. <ol><li> 박 피더 세포와 세럼 교체 (KOSR) 미디어를 인간의 iPSC 넉 아웃을 잠복기에 의해 피더 셀이 설치된 미디어 (CM)를 준비합니다. CM에게 24 시간마다 수집합니다. </li></ol> 참고 : 피더 레이어 (예 : BLK6, CF1과 MF1 등) 준비에 사용되는 마우스 긴장은 구경이 사용하기에 적합합니다R은 CM를 갖추고 있습니다. <ol start="2"><li> nucleofection의 일에 최소 1 시간 10 μM ROCK 억제제와 인간 iPSCs를 미리 취급합니다. </li><li> 멸균 1.5 ML eppendorf 튜브에 인간 줄기 세포 nucleofector 솔루션의 82 μl를 준비합니다. 18 μl 보충 추가 1. 잘 섞는다. 5 분에 37 ° C에서 솔루션을 품다. </li><li> 사전 따뜻한 피더 셀이 설치된 미디어 (CM)와 0.25 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) 37 ° C. 후드 아래 prelabel 한 멸균 15 ML 원뿔 튜브. </li><li> 조심스럽게 nucleofected 할 인간 iPSC 문화에서 미디어를 제거합니다. 부드럽게 잘 당 2 ML 1X 인산 버퍼 용액 (PBS)으로 세포를 씻어. PBS를 대기음. 물론 당 0.25 %의 트립신의 1 ML을 추가합니다. 3 분에 37 ° C에서 세포를 배양. </li><li> 부드럽게 1000 ML 피펫 팁으로 세포를 씹다. 모든 인간의 iPSCs가 완전히 분리되어 있는지 확인 할 수있는 우물의 바닥을 씻으십시오. 분류 15 ML 원뿔 튜브에 세포 현탁액을 전송합니다. </li></ol> 참고 : 하나의 C에 Trypsinizationells는 엄격히 피해야한다. 세포의 약 세 쌍둥이의 구성 세포는 세포의 작은 clumps에 dislodged해야합니다. 세포의 작은 clumps (셀 트리플)는 세포의 후속 처리하는 동안 하나의 셀에 dissociated 될 것입니다. <ol start="7"><li> 트립신을 비활성화 9 ML MEF 미디어를 추가 할 수 있습니다. 3 분에 800 rpm으로 세포를 봐. 조심스럽게 표면에 뜨는을 기음과 셀 펠렛 그대로 둡니다. </li><li> 단계 3.3에서 인간 줄기 세포 nucleofector 솔루션의 prewarmed 100 μl에 세포를 Resuspend. </li><li> 1 ML 피펫 팁을 사용하여 nucleofector 큐벳에 세포를 전송합니다. </li><li> 큐벳의 세포 현탁액에 플라스미드 DNA의 4 μg을 추가합니다. 부드러운 소용돌이에 의해 세포와 DNA를 섞는다. 후드 표면에 두 번 큐벳을 누르세요. </li><li> nucleofector 홀더에 큐벳을 삽입합니다. 프로그램 B-016을 사용합니다. 버튼 X를 눌러 Nucleofect 세포 </li></ol> &lt; <ok> nucleofection 프로세스가 complet 때&gt; 표시됩니다에드 (보통 1-5초 소요 만 해당). 다른 nucleofection 프로그램 사용 (A-023, A-033와 U-023)은 인간의 iPSCs에서 eGFP 양성 세포의 10 % 미만이 나왔고. </ok><ol start="12"><li> 제공된 파스퇴르 (Pasteur) 플라스틱 피펫을 사용하여 큐벳에서 nucleofected 세포를 검색합니다. 멸균 1.5 ML eppendorf 튜브에 prewarmed CM과 ROCK 억제제에서 그들을 resuspending하여 세포를 복구 할 수 있습니다. 세포가 복구하도록 10 분에 37 ° C에서 세포를 배양. </li><li> 한 ML 피펫 팁을 사용하여 피더 레이어로 드롭 현명한 전송 세포. 37 ° C 하룻밤에 세포를 배양. </li><li> 다음 날 transfection 효율 모니터링합니다. 유전자 변형 인간 iPSCs의 안정적인 클론을 도출 해당 안정적으로 CM과 ROCK 억제제로 다음 날 중간을 변경할 수 있으며, transgene 표현. 48시간 - 24 후 적절한 항생제 선택을 추가합니다. </li></ol> 4. 대표 결과 : <img alt="그림 1" src="/files/ftp_upload/3110/3110fig1.jpg" /> <st롱&gt; 그림 1. pCAG - eGFP와 transfected Riv1 인간 iPSCs의 Photomicrographs. (A) eGFP-표현 Riv1 세포 것이 Geltrex 12시간 포스트 transfection에 GeneJuice를 사용하여 transfected. Riv1 인간 iPSCs의 식민지는 도금 및 B-016 (B)와 A-023 (C) 프로그램을 사용하여 nucleofection에 따라 피더에 형성 될 수있다. (D) 유비쿼터스 eGFP 식으로 안정적으로 eGFP - 표현 인간 iPSC 식민지가 GeneJuice에서 파생. (E) 안정적-transfected Riv1 인간 iPSCs는 embryoid 바디 차별화 동안 제정 eGFP 식을 유지.

<ol>
	<li>Liew, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells:+possibilities+for+human+cell+transplantation.">Human embryonic stem cells: possibilities for human cell transplantation.</a> Ann Med. 37, 521-532 (2005).</li><li>Eiges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Zwaka, T. P., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogous+recombination+in+human+embryonic+stem+cells.">Homogous recombination in human embryonic stem cells.</a> Nat Biotechnol. 21, 319-321 (2003).</li><li>Siemen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Liew, C. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25, 1521-1528 (2007).</li><li>Xia, X., Ayala, M., Thiede, B. R., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-+and+in+vivo-induced+transgene+expression+in+human+embryonic+stem+cells+and+derivatives.">In vitro- and in vivo-induced transgene expression in human embryonic stem cells and derivatives.</a> Stem Cells. 26, 525-533 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+genetic+manipulation+of+human+embryonic+stem+cells.">Improved genetic manipulation of human embryonic stem cells.</a> Nat Methods. 5, 389-392 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+culture+of+human+embryonic+stem+cells+in+conditioned+medium+for+efficient+genetic+modification.">Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.</a> Nat Protoc. 3, 1435-1443 (2008).</li><li>Hohenstein, K. A., Pyle, A. D., Chern, J. Y., Lock, L. F., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+mediates+high-efficiency+stable+gene+knockdown+and+transgene+expression+in+human+embryonic+stem+cells.">Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells.</a> Stem Cells. 26, 1436-1443 (2008).</li><li>Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenesis+in+human+embryonic+stem+cells+as+a+novel+tool+to+define+the+human+neural+lineage.">BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.</a> Stem Cells. 27, 521-532 (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

간단 견고하고 높은 재현 기술에있는 우리 프로토콜 결과는 눈에 잘 띄는 독성 효과 세포 사망하지 않고 인간 iPSCs에 transgenes을 소개합니다. 인간 iPSCs는 세포의 작은 clumps (5-10 세포)에 passaged과 수많은 작은 식민지에 최적의 transfection 효율을 보장하기 위해 높은 밀도 (1시 2분)에 Geltrex에 도금해야합니다. 차별화 및 셀 죽음에 더 경향이 인간의 iPSC 라인의 경우, 인간 iPSCs 높은 번호 (최대 4 X 10 6 세포) 하나의 nucleofection 실험에 사용되어야합니다. 과도 transfection 분석은 1 일 이내 transgene - 표현 인간 iPSCs 많은 수의를 생성합니다. 안정적으로 transfected iPSC 클론은 보통 7 일 이내 표시, 이러한 유전자 변형 식민지은 3 주 내에 수령 할 준비가되어 있어야합니다. 편대의 편대장 발기인의 사용은 eGFP 기자의 유비쿼터스 표현을 보장 여기에 설명했다. 이러한 개선에서 우리의 프로토콜은 overexpression, C 등 다른 응용 프로그램에 공급 될 수onditional 유도, 혈통 별 기자 라인, shRNA 또는 siRNA의 최저의 유도, 유전자 타겟팅 및 동종 재조합.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산)와 0.25 %의 트립신 </td><td> Invitrogen </td><td> 25200056 </td><td></td></tr><tr><td> 2 - 메르 캅토 에탄올 </td><td> Invitrogen </td><td> 21985-023 </td><td></td></tr><tr><td> 3mm 유리 구슬 </td><td> 피셔 과학 </td><td> 11-312A </td><td> 유리 구슬은 수산화 나트륨 (NaOH)와 증류수와 도망을 헹구어 박 염산 산 (HCL)로 세척하고 사용 전 압력솥으로 소독해야합니다. </td></tr><tr><td> Accutase 세포 분리 시약 </td><td> Invitrogen </td><td> A11105-01 </td><td></td></tr><tr><td> 기본 섬유 아세포 성장 인자 (bFGF) </td><td> Invitrogen </td><td> PHG0263 </td><td></td></tr><tr><td> DMEM (높은 혈당) </td&gt; <td> Lonza </td><td> 12-741 F </td><td></td></tr><tr><td> 태아 송아지 혈청 </td><td> Invitrogen </td><td> 16000044 </td><td></td></tr><tr><td> Geltrex </td><td> Invitrogen </td><td> 12760013 </td><td> 성장 인자가 감소 </td></tr><tr><td> GeneJuice의 transfection 시약 </td><td> EMD Biosciences </td><td> 70,967 </td><td></td></tr><tr><td> Glutamax-I (100X) </td><td> Invitrogen </td><td> 35050061 </td><td> L-글루타민 (Invitrogen, 25030081)도 대신 사용할 수 있습니다. </td></tr><tr><td> 인간 iPSC KOSR 미디어 </td><td></td><td></td><td> 500 ML 인간 ES 미디어는 390 ML의 넉 아웃 DMEM / F12, 100 ML의 넉 아웃 세럼의 Replacer, 5ml Glutamax-I (100X), 5ml NEAA (100X), 500 μl 2 메르 캅토 에탄올 (55mM)로 구성되어 있으며, 10 NG / ML bFGF와 보충 . </td></tr><tr><td> 녹아웃 DMEM/F12 </td><td> Invitrogen </td><td> 12660-012 </td><td></td></tr><tr><td>넉 아웃 세럼 교체 (KOSR) </td><td> Invitrogen </td><td> 10828-028 </td><td></td></tr><tr><td> 마우스 태아의 섬유 아세포 미디어 </td><td></td><td></td><td> MEF 미디어의 90 % FBS, 1X NEAA, 1X Glutamax-I과 DMEM의 1X 나트륨 pyruvate (높은 혈당)로 구성 </td></tr><tr><td> 비 필수 아미노산, NEAA? (100X) </td><td> Invitrogen </td><td> 11140050 </td><td></td></tr><tr><td> 인간 줄기 세포 nucleofector 솔루션을 하나 추가 비용 </td><td> Lonza </td><td> VAPH-5012 </td><td></td></tr><tr><td> 인산은 칼슘이없이 +와 MG 2 식염 버퍼 + </td><td> Invitrogen </td><td> 10010023 </td><td></td></tr><tr><td> 나트륨 pyruvate (100X) </td><td> Invitrogen </td><td> 11360070 </td><td></td></tr><tr><td> STEMPRO 매체 키트 </td><td> Invitrogen </td><td> A1000701 </td><td> 500 ML STEMPRO 미디어는 454 ML 노크로 구성되어 있습니다아웃 DMEM / F12, 10 ML STEMPRO 혈청이없는 성장 보충 (50X), 5ml Glutamax-I (100X), 5ml NEAA (100X), 36 ML BSA (소 혈청 알부민, 25 %), 909 μl 2 메르 캅토 에탄올 (55mM ) 10 NG / ML bFGF와 보충. </td></tr></tbody></table>

transfecting and nucleofecting human induced pluripotent stem cells

유전자 변형은 줄기 세포 생물학을 연구와 인간 배아 줄기 세포 (HESCs) 1의 잠재적 인 임상 응용 프로그램을 명시 설정에 필수적인 도구가 될 계속하고 있습니다. 여러 유전자 ​​전달 방법의 개선이 2-9에 설명되어 있지만, transfection은 HESCs의 변덕 과정 그대로 남아 있으며, 아직 인간 유도 만능 줄기 세포 (iPSCs)에보고되지 않았습니다. 이 동영상에서는, 우리는 우리 연구소가 정기적으로 transfects 및 강화 된 녹색 형광 단백질 (eGFP) 기자와 플라스미드를 사용 인간 iPSCs를 nucleofects 방법을 보여줍니다. 인간 iPSCs는 적응과 피더 세포 transfection의 가능성을 제거하고 transfection에 따라 안정적으로 유전자 변형 iPSC의 클론의 효율적인 선택을 허용하도록 피더 - 무료 문화로 유지됩니다. nucleofection를 들어, 인간의 iPSCs는 피더 셀이 설치된에 모이통에 nucleofected과 replated 세포의 작은 clumps로 trypsinized ROCK 억제제를 11으로 미리 처리됩니다셀 복구를 향상 할 매체입니다. Transgene - 표현 인간 iPSCs은 6 시간 후 얻을 수 있습니다. 항생제 선택은 24 시간 안정적으로 유전자 변형 라인이 1 주일 이내에 게재 된 후 적용됩니다. 우리 프로토콜은 견고하고 이러한 세포의 pluripotency를 변경하지 않고 인간의 iPSC 라인에 재현합니다.

Watch this Scientific Journal Video about Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells at JoVE.com

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

유전자 변형의 최근 발전에도 불구하고, 인간 배아 줄기 세포 (HESCs)의 transfection은 변덕 과정 남아있다. 우리의 지식, 인간 유도 만능 줄기 세포를 (iPSCs) transfect하는 체계적이고 효율적인 방법은보고되지 않았습니다. 여기, 우리는 효율적으로 transfect과 인간 iPSCs를 nucleofect하는 강력한 프로토콜을 설명합니다.

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 ...

transfecting-and-nucleofecting-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

21.3K 조회수.  University of California Riverside. 유전자 변형의 최근 발전에도 불구하고, 인간 배아 줄기 세포 (HESCs)의 transfection은 변덕 과정 남아있다. 우리의 지식, 인간 유도 만능 줄기 세포를 (iPSCs) transfect하는 체계적이고 효율적인 방법은보고되지 않았습니다. 여기, 우리는 효율적으로 transfect과 인간 iPSCs를 nucleofect하는 강력한 프로토콜을 설명합니다.

비디오: Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Amaxa Nucleofector과 인간 신경 줄기 세포를 Transfecting

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

연구

생물학

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 ...

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

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

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 ...

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, ...

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 ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.