참고 : 무균 문화 조건을 유지하기 위해서는이 프로토콜의 절차를 모두 무균 실험실 방법을 사용하여 수행하고 층류 후드 아래에 실시하고 있습니다. 시작하기 전에, 모든 미디어는 37 equilibrated되도록 ° C 적절하게 기름. Geltrex - 코팅 문화 요리를 준비 참고 : CELLstart 코팅 문화 요리의 사용을 위해 부록을 참조하십시오. <ol><li> 해동 한 천천히 2-8에서 Geltrex (1 ML) ° C의 튜브 및 넉아웃 D-MEM/F-12 99 ML에 1:100 그것을 희석. 부드럽게 솔루션을 섞는다. 참고 : 일부 IPS 세포 라인은 최적의 성장을 위해 다른 Geltrex 희석이 필요할 수 있습니다. 대체 dilutions에 대한 부록을 참조하십시오. </li><li> Geltrex 솔루션 (35 - mm 요리 1 ML, 60 mm 요리 1.5 ML) 각 문화 요리의 전체 표면을 커버. </li><li> 건조를 방지하고 37에서 1 시간 동안 요리를 부화 Parafilm 각 접시 ° C. 봉쇄해 참고 : 최대 1 개월이 지점에서 4 ° C에서 Geltrex - 코팅 문화 요리를 저장할 수 있습니다. 밖으로 건조에서 Geltrex을 방지하기 위해 Parafilm 각 요리를 밀봉합니다. </li><li> 이전 사용하기 층류 후드에 Geltrex - 코팅 요리를 전송하고는 실온 (약 1 시간)에 평형 수 있습니다. </li></ol> 완료 녹아웃의 SR 공급기 - 무료 매체를 준비 <ol><li> 10 μg / ML 기본 FGF 솔루션 1 ML를 준비하려면, 무균 아래에 나열된 구성 요소를 섞는다. -20 ° C에서 나누어지는 솔루션 및 저장 최대 6 개월까지. <table border="0"><tbody><tr><td> 기본 FGF </td><td> 10 μg </td></tr><tr><td> D - PBS </td><td> 990μL </td></tr><tr><td> 10% BSA </td><td> 10 μL </td></tr></tbody></table> 참고 : BSA는 같은 농도에서 HSA 또는 넉아웃의 SR로 대체 수 있습니다. </li><li> 2 MG / ML Dispase 솔루션의 50 ML를 준비하려면, 무균 아래에 나열된 구성 요소를 섞는다. 4 솔루션 및 저장 ° C 최대 2 주 동안을 소독 필터. <table border="0"><tbody><tr><td> Dispase </td><td> 100 MG </td></tr><tr><td> D - PBS </td><td> 50 ML </td></tr></tbody></table></li><li> 전체 녹아웃의 SR 100 ML를 준비하는 피더 - 무료 (KSR - FF)는 무균 아래 표에 나와있는 구성 요소를 결합. <table border="1"><tbody><tr><td> 구성 요소 </td><td> 주식 농도 </td><td> 최종 농도 </td><td> 음량 </td></tr><tr><td> 마네 DMEM/F12 (Cat. 번호. 12660-012) </td><td> - </td><td> 1X </td><td> 76.8 ML </td></tr><tr><td> GlutaMAX - I (Cat. 번호 35050-061) </td><td> 200 MM </td><td> 2 MM </td><td> 1 ML </td></tr><tr><td> 녹아웃 SR (Cat. 번호. 10828-028) </td><td> - </td><td> 20% </td><td> 20 ML </td></tr><tr><td> 녹아웃 SR - GFC (Cat. 번호. A10580 - 01) </td><td> 50X </td><td> 1X </td><td> 2 ML </td></tr><tr><td> bFGF (Cat. 번호. PHG0024). </td><td> 10 μg / ML </td><td> 20 NG / ML </td><td> 200 μL </td></tr></tbody></table> 최대 일주일을 위해 당신은 2-8 ° C에서 KSR - FF 매체를 저장할 수 있습니다. </li><li> 온도와 가스의 전체 매체를 미리 equilibrating 바로 전에 무균 0.1 MM 최종 농도 2 메르 캅 토 에탄올의 필요한 볼륨 (55 MM 주식 농도)을 추가합니다. 예를 들어, KSR - F 100 ML를 준비하는F 매체 2 - 메르 캅 토 에탄올 (1:550 희석) 또는 55 mm의 182 μL를 추가, 2 - 메르 캅 토 에탄올은 1X 완료 매체에 추가 및 최대 일주일 동안 2-8 ° C에 저장된 수 있습니다. </li></ol> 냉동 / Cryopreservation 중간 준비 cryopreservation 매체는 두 미디어로 구성되어, 냉동 매체는 A와 동결 매체 B.는 그들은 다른 시간에 세포에 추가되고 남아 분리해야합니다. 그들은 각 Cryopreservation 매체의 전체 볼륨의 50 %가 필요합니다. 필요한 Cryopreservation 매체의 총 부피는 냉동으로 각 유리병 1 ML입니다. hESC의 90% 합류 60mm 요리는 cryopreservation 미디어에 hESC 은행이 유리병하는 데 사용할 수 있습니다. 없더군요을 보장하기 위해 추가 2 5mL 각 냉동 매체의 충분한 볼륨을 준비합니다. <table border="1"><tbody><tr><td> 냉동 중간 (최종 볼륨의 50 %) </td><td> 50% DMEM/F12 </td><td> 50 % KSR </td></tr><tr><td> 냉동 중간 B (최종 부피의 50 %) </td><td> 80% DMEM/F12 </td><td> 20% DMSO </td></tr></tbody></table> 예 : 이 세포의 20 튜브를 동결하는 경우 Cryopreservation 매체 20mL가 필요합니다. 24mL Cryopreservation 매체의 총 없더군요에 대한 4mL를 추가합니다. 당신은 냉동 매체의 12mL가 필요합니다 즉 A (24mL의 50 %)과 동결 매체 B (24mL의 50 %)의 12mL. <table border="1"><tbody><tr><td> 냉동 중간 (12 ML) : </td><td> 6 ML DMEM/F12 </td><td> 6 ML KSR </td></tr><tr><td> 냉동 중간 B (12 ML) : </td><td> 9.6 ML DMEM/F12 </td><td> 2.4 ML DMSO </td></tr></tbody></table> Cryopreservation 매체의 최종 구성 65 % DMEM/F12, 25 % KSR하고, 10 % DMSO입니다. Cryopreserving iPSCs <ol><li> 37 ° C의 물을 욕조에 Dispase 사전 따뜻한 필요한 볼륨. 필요한 볼륨에 대한 자세한 내용은 아래의 표 1을 참조하십시오. </li><li> 37 ° C의 물 목욕 for15 분에 KSR - FF의 필요한 볼륨을 사전에이 - 평형. 필요한 볼륨에 대한 자세한 내용은 테이블 1below을 참조하십시오. </li><li> 피펫을 사용하여 문화 선박에서 보낸 매체를 대기음하고, D - PBS로 두 번 세포를 씻어. </li><li> 부드럽게 문화 선박 (예, 60 mm 문화 요리 당 Dispase 솔루션 1 ML)로 사전 예열 Dispase 솔루션을 추가합니다. 코트 전체 세포 표면에 소용돌이 문화 우주선합니다. </li><li> 3 분 37 ° C에서 문화 함선을 품어. </li><li> 인큐베이터에서 혈관을 제거 Dispase 솔루션을 기음, 부드럽게 D - PBS로 세포를 씻어. </li><li> 부드럽게 세포 스크레이퍼를 사용하여 문화 접시의 표면에서 세포를 긁어 및 살균 15mL 원심 튜브에 세포를 전송합니다. </li><li> 부드럽게 분리하지 않은 세포를 &quot;해제 분사.&quot;, KSR - FF로 두 번 문화 접시를 씻어 수영장은 15mL 튜브에 세포와 매체를 씻어. </li><li> 상온에서 펠릿 세포 5 분 200 XG에서 튜브를 원심 분리기. </li><li> 조심스럽게 세포 펠렛을 방해하지 않고 표면에 뜨는를 대기음하고 폐기. </li><li> cryovials의 충분한 수량을 준비합니다. 세포 DMSO와 연락되면, 그들은 aliquoted 후 2-3 분 이내에 냉동해야합니다. </li><li> 천천히 완벽하게 [부드럽게까지 pipetting 아래 5 ML serological 피펫을 사용하여 소리] 튜브 바닥에서 세포 펠렛을 이동시키다 및 냉동 매체 A.에있는 세포를 다시 정지, 튜브를 버려. 부드럽게 혼합에 세포 현탁액을 소용돌이 치는 동안 대단히 짧은 시간의 균일한 현탁액에 따라, 한 방울 현명한 방식으로, 그것 동결 매체 B의 같은 볼륨을 추가합니다. 참고 :이 시점에서, 세포가 DMSO와 연락하고, 작업이 효율적으로 수행되어야합니다. 세포 DMSO와 연락되면, 그들은 aliquoted 후 2-3 분 이내에 냉동해야합니다. </li><li> 각 cryovial에 세포 현탁액의 나누어지는 1 ML. </li><li> 신속 [급속 동결] 미스터 프로 스티에 튜브를 넣고 ° C -80 하룻밤에 전송할 수 있습니다. </li><li> -80에서 하룻밤 보관하면 ° C는 장기 저장을위한 액체 질소 탱크에 세포를 전송합니다. </li></ol> iPSC의 유리 해동 및 세포 복구 참고 : KSR - FF는 KSR 함유 MEF - 시설 중간, 또는 피더와 함께 사용하기위한 KSR 매체로 대체될 수 있습니다. 미디어 준비하기위한 지침은 부록을 참조하십시오. 50 ML 튜브에 KSR - FF 매체 사전 따뜻한 10mL. <ol><li> 당신을 도금하기 전에 실내 온도에 Geltrex - 코팅 요리의 적절한 수량 평형과 요리로부터 액체를 기음R 세포. </li><li> 조심스럽게 hESC (또는 iPSC) 액체 질소 저장 탱크에서 약병을 제거하십시오. </li><li> 단지 작은 냉동 덩어리가 유리병에 남아 때까지 빠르게, 37 ° C의 물을 욕조에 세포의 냉동 병을 해동. </li><li> 그것을 오염을 제거하다하고 멸균 조직 문화 후드로 전송하는 이소프로판올 70%로 병을 스프레이. </li><li> 무균 5 ML의 피펫을 사용하여 15 ML 원뿔 관에 유리병의 전체 내용을 전송합니다. </li><li> KSR - FF 1 ML로 병을 씻어 15 ML의 원심 관에 이것을 추가합니다. </li><li> 천천히, KSR - FF 4 ML 15mL 원뿔 튜브에 세포 드롭 현명한를 추가합니다. 미디어를 추가하는 동안 부드럽게 세포를 섞어 앞뒤로 튜브를 이동합니다. (이것은 세포 삼투 충격을 줄일 수). </li><li> 15 ML 튜브 실온에서 2 분 동안 1000rpm (200 X G)의 세포 현탁액을 원심 분리기로. </li><li> 조심스럽게 기음과 세포 펠렛을 방해하지 않고 뜨는 폐기하십시오. </li><li> 사전 예열 KSR - FF의 세포 펠릿 (예 4mL/60 mm 요리) 5 ML 피펫을 사용하고 세포 펠릿가 완전히 분산 때까지 부드럽게 세포를 피펫 아래 다시 일시 중지합니다. 생존 적은 70 %면 이상 70 % 생존이 예상된다, 그러나, 그것은 합류에 도달하는 세포를 위해 더 많은 시간이 소요될 수 있습니다. </li><li> 같은 피펫을 사용하여 세포 현탁액 실온에 미리 equilibrated Geltrex - 코팅 문화 요리 드롭 현명한를 전송합니다. </li><li> 공기 4-6%의 CO 2의 humidified 분위기, 37 ° C 배양기에서 배양 접시를 놓습니다. 고르게 세포를 배포 서부 패턴 남쪽, 동쪽 북쪽 신중하게 소용돌이 모양의 용기. </li><li> 부드럽게 문화 접시를 유체 - 변경 24 시간 이후 해동와 세포 부스러기를 제거하고 요리는 약 70-80% 합류하기 전까지 신선한 영양소를 제공하기 위해 이후 매일.이 귀하의 IPS 세포의 지속적인 문화와 passaging 들어, &quot;완료 마네 세럼 교체 사용하는 인간의 IPS 세포 피더 - 무료 문화 Passaging 피더를 무료 매체&quot;라는 제목의 프로토콜을 참조하십시오 </li></ol> 예상 결과 세포 전에 cryopreservation에 합류 약 70~80%되어야합니다. 최대 컬링과 식민지 마진 따라 식민지의 접이식의 소화 결과를 Dispase. 세포를 수확 후 그들은 cryopreservation 미디어의 작은 대단히 짧은 시간으로 냉동 있습니다. 유리병가 해동되면 세포가 회복 작은 대단히 짧은 시간으로 미리 코팅 요리에 첨부합니다. 그들은 급속하게 5-6일에 합류 접시에 선도적인 성장. 표 1 - 추천 볼륨 <table border="1"><tbody><tr><td> 구성 요소 </td><td> 35mm 디시 </td><td> 60mm 디시 </td><td> 100mm 디시 </td></tr><tr><td> 완료 녹아웃 SR 미디어 </td><td> 2 ML </td><td> 4 ML </td><td> 10 ML </td></tr><tr><td> Geltrex 솔루션 </td><td> 1 ML </td><td> 1.5 ML </td><td> 4-5 ML </td></tr><tr><td> Dispase </td><td> 0.5 ML </td><td> 1 ML </td><td> 3-4 ML </td></tr><tr><td> rinsing을위한 D - PBS </td><td> 2 ML </td><td> 4 ML </td><td> 10 ML </td></tr></tbody></table> 주십시오 <a href="https://www.jove.com/files/ftp_upload/2237/2237_appendix.pdf">부록을 보시려면 여기를 클릭하십시오</a> .

<ol><li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li>
<li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li>
<li>Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li>
<li>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li>
<li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li>
<li>Liao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+induced+pluripotent+stem+cell+lines+from+adult+rat+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Generation of induced pluripotent stem cell lines from adult rat cells.</a> Cell Stem Cell. 4, 11-15 (2009).</li>
<li>Dimos, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li>
<li>Aasen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes%5BTitle%5D)+AND+%22Nat+Biotechnli%22%5BJournal%5D)">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnli. 26, 1276-1284 (2008).</li>
<li>Park, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+human-induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Generation of human-induced pluripotent stem cells.</a> Nat Protoc. 3, 1180-1186 (2008).</li>
</ol>

이 문서의 저자는이 문서에서 사용되는 시약 및 악기를 생산하는 생명 기술에 의해 고용됩니다.

<table><tbody><tr><td>Knockout DMEM/F12</td><td></td><td>12660-012</td><td>Note: see appendix for the use of alternative DMEM products GlutaMAX-I (Cat. No. 35050-061)</td></tr><tr><td>KnockOut Serum Replacement</td><td></td><td>10828-028</td><td></td></tr><tr><td>KnockOut Serum Replacement Growth Factor Cocktail</td><td></td><td>A10580-01</td><td></td></tr><tr><td>FGF-basic, human recombinant protein, 10 &amp;mu;g</td><td></td><td>PHG0024</td><td>Note: see appendix for alternative bFGF pack sizes.</td></tr><tr><td>2-Mercapt&amp;#8211;thanol</td><td></td><td>21985-023</td><td></td></tr><tr><td>Dispase</td><td></td><td>17105-041</td><td>Note: see appendix for alternative passaging methods</td></tr><tr><td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td><td></td><td>A10480-01</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium</td><td></td><td>14190-144</td><td></td></tr><tr><td>DMSO, 500mL</td><td>JT Baker</td><td>JT9224-1</td><td></td></tr><tr><td>Sterile Tissue Culture Hood</td><td></td><td></td><td></td></tr><tr><td>Incubator set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Pipette-Aid</td><td></td><td></td><td></td></tr><tr><td>Water Bath set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Sterile serological pipettes (5 mL, 10 mL)</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>60 mm Tissue Culture treated dishes</td><td></td><td></td><td></td></tr><tr><td>Liquid nitrogen storage tank</td><td></td><td></td><td></td></tr><tr><td>Isopropanol freezing containers</td><td></td><td></td><td></td></tr><tr><td>1.5 mL Cryovials</td><td></td><td></td><td></td></tr></tbody></table>

cryopreserving and recovering of human ips cells using complete knockout serum replacement feeder-free medium

인간과 마우스 섬유아 세포가 배아 줄기 세포에 상당히 유사 자질을 가진 1-3 IPS 세포를 생성하는 재설정 될 수 있다고 2006 년에 발견은 약물 발견, 세포 치료와 기초 연구를위한 pluripotent 세포의 중요한 새로운 소스를 만들었습니다. GIBCO 미디어 및 시약 년 동안 pluripotent 줄기 세포 연구의 선두에있다. DMEM가 마네 세럼 교체와 보충 마네는 배아 줄기 세포의 성장과 지금은 IPS 세포 배양 3-9위한 최고의 매체입니다. 이 황금 표준 미디어 시스템은 이제 마네의 SR 성장 팩터 칵테일의 추가와 함께 피더 무료 문화 사용할 수 있습니다. 전통적인 인간의 ES와 IPS의 세포 배양 방법은 마우 스나 인간 fibroblast 피더 레이어의 사용 또는 피더 - 에어컨 매체를 필요로합니다. 이러한 문화 방법은 규모에 노동 집약, 하드 있으며, 그것은 정의되지 않은 상황으로 인해 undifferentiated 허리의 세포를 유지하기 어렵습니다. Invitrogen은 여전히​​ 마네의 SR의 사용을 유지하면서 쉽게 전환하여 엉덩이와 hES 세포 문화를 피더없는 수 있도록 마네의 SR 성장 팩터 칵테일을 개발했습니다.

Watch this Scientific Journal Video about Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

이 프로토콜은 녹아웃 SR의 cryopreservation 매체 인간의 IPS 세포를 cryopreserving 완벽한 마네 SR 공급기 무료 (KSR - FF) 중간 또는 피더 기반 마네의 SR 매체에서 이러한 세포를 복구하는 자세한 절차를 설명합니다.

Cryopreserving와 인간 IPS 세포 복구 완료 마네에게 세럼 교체 피더 - 무료 매체를 사용하여

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

cryopreserving-recovering-human-ips-cells-using-complete-knockout

Research

JoVE Journal

Biology

18.2K 조회수.  Life Technologies.  中文 日本語 

비디오: Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

삭스 - 2의 강제 표현을 통해 MEFS에서 IPS 세포를 생성, 10 - 4, C - Myc, 그리고 Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

연구

생물학

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

일상적인 면역 세포 화학 특성에 대한 혈청이없는 조건에서 인간 iPSCs의 소규모 전파

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

발생학

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

정의 된 이종 무료 및 급지대 무료 문화 세대의 인간 iPSC 파생 된 망막 세포 모델에 대 한

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

완료 녹아웃 세럼 교체 피더 - 무료 매체를 사용하여 인간의 IPS 세포 피더 - 자유 적응력, 문화와 Passaging

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 ...

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 ...

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 ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

재 프로그래밍 가능한 마우스 모델에서 유도 만능 줄기 세포 중간체의 세포 표면 마커 중재 정화

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

에피 솜과 HDAC 억제제를 사용하여 혈액에서 효율적인 유도 만능 줄기 세포 생성

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

인간 유도 만능 줄기 세포 유래 키메라 항원 수용체 발현 자연 살해 세포의 발전

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...