이러한 연구 GJM와 GM에 대한 NIH UO1HL107443-01 상에 의해 부분적으로 기금을했다.

1. 말초 혈 Mononuclear 세포 (PBMCs)의 절연 및 확장 DAY 0 <ol><li> 나트륨 시트르산과 BD Vacutainer CPT 셀 준비 튜브에 말초 혈 4 ML을 그립니다. 관에게 8-10 번 반전하고 실온에서 30 분에 1,800 XG에 원심 분리기. 이상적으로,이 단계는 컬렉션의 2 시간 이내에 완료해야합니다. </li><li> 멸균 15 ML 원뿔 원심 분리기 튜브에 버피 코트 (겔 장벽과 플라즈마 사이의 세포 층)을 pipetting하여 mononuclear 세포 (MCS)를 수집합니다. 15 분 300 XG에 여러 시간과 원심 분리기 반전, 멸균 인산 버퍼 생리 (PBS)로 10 ML에 총 볼륨을 가져와. </li><li> 멸균 PBS의 10 ML에있는 세포를 Resuspend과 셀 카운트를 수행합니다. 10 분 300 XG에서 멸균 15 ML 원뿔 원심 분리기 튜브와 원심 분리기로 1 2x10 6 세포를 전송합니다. </li><li> 확장 매체 2 ML (EM) (에서 세포를 Resuspend 50 μg / ML Asco을 포함 QBSF-60 줄기 세포 중rbic 산성, 50 NG / ML SCF, 10 NG / ML IL-3, 2 U / ML EPO는 40 NG / ML IGF-1, 1 μM의 Dexamethasone 및 100 μg / ML primocin 또는 1 %의 펜 / 패 혈성)과에 이전 일 잘 12 잘 접시의. 5% CO 2 인큐베이터, 37 ° C에서 세포를 배양. </li><li> 300 x에 남아있는 세포를 원심 분리기 10 % DMSO를 포함하는 FBS에서 10 분, 동결 ~ 2x10 6 셀 / 유리 병에 g. </li><li> 냉동 PBMCs를 사용하여 프로토콜을 시작하려면 10 분 300 XG에 QBSF 매체와 원심 분리기 10 ML로 세포의 1 병을 해동. EM 2 ML에있는 세포를 Resuspend과 12 잘 판 중 하나를 잘에 전송할 수 있습니다. 5% CO 2 인큐베이터, 37 ° C에서 세포를 배양. </li></ol> DAY 3 DAY 6 <ol start="17"><li> 멸균 15 ML 원뿔 튜브에 세포를 전송하고 자기편 세포를 수집하는 QBSF-60 줄기 세포 중의 1 ML과 함께 한 번 잘 씻어. </li><li> 10 분 300 XG에서 세포를 원심 분리기. </li><li> EM 2 ML에있는 세포를 Resuspend하고 12의 잘 하나 전송- 잘 판. 5% CO 2 인큐베이터, 37 ° C에서 세포를 배양. </li></ol> 2. STEMCCA Lentivirus과 PBMCs의 전달 9 일 <ol><li> 멸균 15 ML 원뿔 튜브에 세포를 전송하고 자기편 세포를 수집하는 QBSF-60 줄기 세포 중의 1 ML과 함께 한 번 잘 씻어. </li><li> 10 분 300 XG에서 세포를 원심 분리기. </li><li> 5 μg / polybrene 및 STEMCCA lentivirus의 ML을 포함하는 신선한 EM의 1 ML에있는 세포를 Resuspend (전부다 = 1 10)과 12 잘 접시의 잘 하나 전송됩니다. (lentiviral 입자의 생산을 설명하는 프로토콜은에서 찾을 수 있습니다 <a href="http://www.bumc.bu.edu/stemcells/protocols/" target="_blank">http://www.bumc.bu.edu/stemcells/protocols/</a> ) </li><li> 90 분에 25 ° C에서 2,250 rpm으로 판을 봐. </li><li> 스핀 후, 신선한 EM의 추가 한 ML을 추가 매체 2 ML의 총 5 μg / polybrene의 ML을 포함하고 37 ° C에서 판을 품다,5% CO 2 인큐베이터. </li></ol> 10 일 <ol start="16"><li> 멸균 15 ML 원뿔 튜브에 세포를 전송하고 자기편 세포를 수집하는 QBSF-60 줄기 세포 중의 1 ML과 함께 한 번 잘 씻어. </li><li> 10 분 300 XG에서 세포를 원심 분리기. </li><li> EM 2 ML에있는 세포를 Resuspend과 12 잘 접시의 한 우물 전송할 수 있습니다. 5% CO 2 인큐베이터, 37 ° C에서 세포를 배양. </li></ol> 3. 도금은 MEFs에 셀을 Transduced DAY 11 <ol><li> 코트 2x10 5 세포에서 0.1 % 젤라틴과 판 inactivated 마우스 배아 섬유 아세포 (MEFs)와 / 잘 MEF 매체 (IMDM 10 % FBS, 1 % 비 필수 아미노산을 포함, 100 μM의 β-에서 6 잘 접시의 우물 메르 캅토 에탄올, 2 MM L-글루타민, 100 μg / ML primocin 또는 1 %의 펜 / 패 혈성). 감염 당 3 우물을 준비합니다. </li></ol> 하루에 12 <ol start="2"><li> 멸균 15 ML 원뿔 튜브에 세포를 전송<html> 그리고 자기편 세포를 수집하는 QBSF-60 줄기 세포 중의 1 ML과 함께 한 번 잘 씻는다. </li><li> 10 NG / ML bFGF와 아스코르브 산 및 EM에 사용되는 것과 동일한 농도의 성장 요소를 포함하는 MEF 매체의 3 ML에있는 세포를 Resuspend 중간 (50 μg / ML 아스코르브 산, 50 NG / ML SCF, 10 NG / ML IL -3, 2 U / ML EPO, 40 NG / ML IGF-1, 1 μM Dexamethasone). </li><li> 플레이트 (1) 세포의 ML 당 잘 MEFs를 포함하는 6 잘 접시의. / 잘 매체의 2.​​5 ML의 총 bFGF, 아스코르브 산, 및 성장 요인과 MEF 미디어의 1.5 ML를 추가합니다. </li><li> 25 500 rpm으로 판을 돌려 ° C를 30 분에. 37 ° C, 5 % CO 2 인큐베이터에서 판을 품다. </li></ol> DAY 14 <ol start="6"><li> 10 NG / ML bFGF와 50 μg / ML 아스코르브 산 (NO 성장 요소)를 포함 MEF 미디어 2.5 ML로 피드 전지는 매일. 기음과 각 피드와 부동 셀을 폐기합니다. </li><li> 로 (보통 일주일에 한 번) 필요한 추가 MEFs을 추가합니다. </li></ol>P &quot;&gt; ~ DAY 20 <ol start="8"><li> 일단 작은 식민지 피드 세포는 인간 배아 줄기 세포 (hESC) 매체 (DMEM/F12가 포함 된 20 %의 넉 아웃 세럼 교체, 1 % 비 필수 아미노산, 100 μM의 β-메르 캅토 에탄올, 2 MM L-글루타민 2 ML과 매일 표시 , 100 μg / ML primocin 또는 1 %의 펜 / 패 혈성, 10 NG / ML bFGF). </li></ol> 4. 따기 및 iPSC 클론의 확장 ~ DAY 30-40 <ol><li> 12 잘 판 inactivated MEFs가 / 잘 hESC 매체 1 ML 플러스 Stemolecule Y27632 (ROCK 저해제) 10 μM을 포함하는으로 미리 놓는의 개별 우물에 각 식민지를 선택합니다. </li><li> 만 hESC 매체 (NO ROCK 억제제) 1 ML과 이후 매일 세포를 피드. </li></ol> 5. Pluripotency 마커에 대한 Immunofluorescence 착색 <ol><li> 착색은 Millipore에서 키트 &#39;ES 세포 마커 샘플 키트 &quot;를 사용하고 제조업체의 프로토콜에 따라 수행되었다. </li></ol> 6. Integ의 절단정격 STEMCCA 벡터 <ol><li> 카세트를 재 프로그램의 절단은 Cre-IRES-Puro 표현 벡터에 따라 transfection 및 6에 설명 된대로 간단한 Puromycin 선택을 활용 이루어집니다. </li></ol>

<ol>
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관심 없음 충돌이 선언 없습니다.

우리는 여기에서 갓 수집 말초 혈의 몇 밀리리터 격리 mononuclear 세포에서 인간 iPSCs를 생성하는 STEMCCA lentiviral 벡터의 사용을 설명합니다. 프로토콜은 먼 위치에서 획득 한 기증자 세포를 활용하면 냉동 PBMCs (버피 코트에서 직접 취득), 상당한 실질적인 의미의 상세를 재설정하는 데 사용할 수 있습니다. 재 프로그램의 유도하기 전에, 절연 PBMCs는 핵 재활 더받을 수있는 세포의 건강한 proliferating 인...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> BD Vacutainer CPT 셀 준비 튜브 나트륨 구연산과 </td><td> BD Biosciences </td><td> 362760 </td><td></td></tr><tr><td> QBSF-60 줄기 세포 중 </td><td> 품질 생물 </td><td> 160-204-101 </td><td></td></tr><tr><td> IMDM </td><td> Invitrogen </td><td> 12,440 </td><td></td></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 11,330 </td><td></td></tr><tr><td> FBS </td><td> 애틀랜타 체액 </td><td> S10250 </td><td></td></tr><tr><td> 넉 아웃 세럼 교체 </td><td> Invitrogen </td><td> 10,828 </td><td></td></tr><tr><td> Primocin </td><td> Invivogen </td><td> 개미-PM-2 </td><td></td></tr><tr><td> 펜 / 패 혈성 </td><td> Invitrogen </td><td> 15,140 </td><td></td></tr><tr><td> L-글루타민 </td><td> Invitrogen </td><td> 25,030 </td><td></td></tr><tr><td> 비 필수 아미노산 </td><td> Invitrogen </td><td> 11,140 </td><td></td></tr><tr><td> β-메르 캅토 에탄올 </td><td> MP의 Biomedicals </td><td> 190242 </td><td></td></tr><tr><td> 아스 코르 비 산 </td><td> 시그마 </td><td> A4544 </td><td></td></tr><tr><td> IGF-1 </td><td> R &amp; D 시스템 </td><td> 291-G1 </td><td></td></tr><tr><td> IL-3 </td><td> R &amp; D 시스템 </td><td> 203-IL </td><td></td></tr><tr><td> SCF </td><td> R &amp; D 시스템 </td><td> 255-SC </td><td></td></tr><tr><td> EPO </td><td> R &amp; D 시스템 </td><td> 286-EP </td><td></td></tr><tr><td> Dexamethasone </td><td> 시그마 </td><td> D4902 </td><td></td></tr><tr><td> Polybrene </td><td> 시그마 </td><td> H-9268 </td><td></td></tr><tr><td> bFGF </td><td> R &amp; D 시스템 </td><td> 233-FB </td><td></td></tr><tr><td> Stemolecule Y27632 </td><td> Stemgent </td><td> 04-0012 </td><td></td></tr><tr><td> ES 세포 마커 샘플 키트 </td><td> Millipore </td><td> SCR002 </td><td></td></tr></tbody></table>

generation of human induced pluripotent stem cells from peripheral blood using the stemcca lentiviral vector

네 전사 인자, Oct4, Klf4의 이소성 표현을 통해 Sox2와 cMyc는 인간의 체세포의 세포는 소위 유도 만능 줄기 세포 (iPSCs) 1-4 발생 만능 상태로 변환 할 수 있습니다. 환자 별 iPSCs는 배아 줄기 세포 (ESCs)를 둘러싸고 가능한 면역 거부를 무시할 것이다 윤리적 문제를 부족합니다. 따라서, iPSCs는 질병 모델링 연구, 약리 화합물의 심사 및 재생 치료 5에 대한 상당한 주목을 받고있다. 우리는 야마나카의 요인 6 인코딩 한 excisable polycistronic lentiviral의 줄기 세포 카세트 (STEMCCA)를 사용하여 서로 다른 폐 질환 환자에서 transgene 무료 인간 iPSCs의 생성을 보여 주었다. 이 iPSC 라인은 피부 섬유 아세포, 재 프로그램에 사용되는 가장 일반적인 세​​포 유형에서 생성되었습니다. 일반적으로 섬유 아세포를 취득하면 세포의 확장에 의한 펀치 생검은 다음 피부가 필요합니다몇 구절에 대한 문화에 s입니다. 중요한 것은 그룹의 수는 iPSCs 7-9으로 인간의 말초 혈액 세포의 재 프로그래밍을보고했습니다. 한 연구에서, STEMCCA 벡터의 Tet의 inducible 버전은 혈액 세포가 동시에 반대 테트라 사이클린 transactivator 인코딩 constitutively 활성화 lentivirus에 감염 될 필요하는 9 고용되었다. 섬유 아세포는 대조적으로, 말초 혈 세포는 크게 환자의 불편 함과 고통을 감소 최소한 침략 절차를 통해 수령하실 수 있습니다. 제정 한 excisable 벡터를 사용하여 혈액 세포를 재 프로그램에 대한 간단하고 효과적인 프로토콜은 광범위한 연구 공동체에 액세스하여 iPSC 기술의 응용을 촉진 할 수 있습니다. 또한, 주변 혈액 세포의 재 프로그래밍 피부 biopsies을 피할 수 (예. 탈선은 흉터) 또는 인해 기존의 질병 상태 preventi에해야하는 개인의 iPSCs의 생성을 허용펀치 biopsies에 NG 액세스 할 수 있습니다. 여기 constitutively 4 요소를 표현하는 하나의 floxed - excisable lentiviral 벡터를 사용하여 말초 혈 mononuclear 세포 (PBMCs)에서 인간 iPSCs의 생성을위한 프로토콜을 보여줍니다. 갓 수집하거나 해동 PBMCs은 STEMCCA의 lentivirus와 transduced되기 전에 같은 매체를 포함하는 아스코르브 산에 10,11 설명 구일, SCF, IGF-1, IL-3와 EPO에 대해 확장됩니다. 셀은 다음 MEFs에 도금되어 있으며 ESC와 같은 식민지 두 주 감염 후 시각화 할 수 있습니다. 마지막으로, 선택한 클론을 확장하고 pluripotency 마커 SSEA-4, Tra-1-60 Tra-1-81 표현으로 테스트됩니다. 이 프로토콜은 혈액의 쉽게 접근 할 4 ML에서 인간 iPSCs의 생성을위한 신뢰할 수있는 방법론을 제공, 간단한 튼튼하고 매우 일치합니다.

우리는 하나의 lentiviral 벡터를 사용하여 PBMCs에서 인간 iPSCs의 생성을위한 간단하고 효과적인 프로토콜을 보여줍니다. 그림 1A는 프로토콜의 개략도를 보여줍니다. 피가 BD Vacutainer CPT 셀 준비 튜브 나트륨 구연산과로 수집하고 있으며, 원심 분리 후, mononuclear 세포는 폴리 에스터 젤과 플라즈마 (버피 코트) (그림 1b) 사이의 인터페이스에서 수령하실 수 있습니다. 절연 PBMCs ...

Watch this Scientific Journal Video about Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector at JoVE.com

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

여기 하나의 lentiviral 재활 벡터를 사용하여 말초 혈 3-4 ML에서 인간 iPSCs의 생성을위한 간단하고 효과적인 프로토콜을 보여줍니다. 즉시 사용할 수 혈액 세포의 재 프로그래밍하면 폭 넓은 연구 공동체에 액세스하여 iPSC 기술의 활용을 촉진 것을 약속드립니다.

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, ...

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26.4K 조회수.  Boston University School of Medicine. 여기 하나의 lentiviral 재활 벡터를 사용하여 말초 혈 3-4 ML에서 인간 iPSCs의 생성을위한 간단하고 효과적인 프로토콜을 보여줍니다. 즉시 사용할 수 혈액 세포의 재 프로그래밍하면 폭 넓은 연구 공동체에 액세스하여 iPSC 기술의 활용을 촉진 것을 약속드립니다.

비디오: Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

4 전사 인자와 마우스 배아 섬유아 세포를 프로그래밍하여 유도 Pluripotent 줄기 세포의 생성, Doxycycline는 Lentiviral 전달 시스템 Inducible

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

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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 ...

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 ...

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 ...

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 ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Multititre 판 형식으로 인간 만능의 줄기 세포에서 뉴런의 신속하고 효율적인 생성

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.