1. 프로그래밍 <ol><li> 6 잘 플레이트 잘 당 1 × 10 5 세포의 밀도에 시드 BJ 세포. 문화 성장 매체 2 ML (450 ML EMEM 50 ML ES 자격 FBS, 5 ML 10 MM이 아닌 필수 아미노산, 5 ML 페니실린 / 스트렙토 마이신, 5 ML 200 MM의 L - 글루타민과 0.9 ML 55 MM의 β와 보충의 세포 37 하루 - 메르 캅 토 에탄올) ° C와 5% CO 2. </li><li> 같은 날, 6 잘 접시에 물에 희석 젤라틴 0.1 %를 추가하고 37 ° C 야간 및 5% CO 2 잠복기하여 MEF 피더 플레이트를 준비하고 시작합니다. </li><li> 부화 후, BJ 세포에서 매체를 제거하고 각 잘하는 polybrene 6 μg / ML과 보완이 ML 성장 매체, 500 μl hOct4 - lentivirus, 50 μl hSox2 - lentivirus, 50 μl hNanog - lentivirus 50 μl hLin28 - lentivirus을 추가 . 부드럽게 매체도 코팅을 보장하기 위해 접시를 바위. 37 밤새 품어 ° C와 5% CO 2. </li><li> 같은 날, 액체 질소와 해동에서 CF - 1 MEF 세포 1 병을 제거합니다. 젤라틴 코팅 우물 (단계 1.2 참조)에서 액체를 제거하고 0.2 × 10 5 세포 / 음의 밀도에 MEF 피더 세포를 추가합니다. 물론 당 전체 볼륨은 MEF 성장 매체 (450 ML DMEM 50 ML FBS 및 5 ML 아닌 필수 아미노산과 보충)에서 세포의 2ml해야합니다. </li><li> 다음날 5 분 200 XG에서 0.05 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)과 원심 분리기와 BJ 세포를 분리합니다. 성장 매체 매체와 resuspend을 대기음. MEF 피더 플레이트 (단계 1.4 참조)에서 미디어를 제거하고 / 잘 BJ 세포 현탁액 2 ML를 추가합니다. 세포의 농도는 약 5 X 10 4 셀 / 잘해야합니다. 37 밤새 품어 ° C와 5% CO 2. </li><li> 인간의 ES / IPS 세포 배양 매체 (400 ML DMEM/F12 100 ML의 넉아웃 세럼 교체, 5 ML 10 MM이 아닌 필수 아미노산, 5 ML 200 MM L - 글루타민, 0.9 ML로 보충과 함께 중간 24시간 다시 시딩 이후를 교체 55 음 β - 메르 캅 토 에탄올 20 ML / NG 인간 재조합 bFGF). 7 일 동안 24 시간마다 중간 변경합니다. </li><li> 7 일 후에, (섹션 2 참조) MEF 시설 중간 중간 변경합니다. </li></ol> 2. MEF 에어컨 매체의 준비 <ol><li> 시드 CF - 1 2 × 10 5 세포에서 MEF 피더 세포 / 음 2 ML MEF 성장 매체에서 6 잘 접시에. 37 밤새 품어 ° C와 5% CO 2. </li><li> 인간의 ES / IPS 세포 배양 매체 매체를 변경합니다. 37 밤새 품어 ° C와 5% CO 2. </li><li> 나흘 동안 24 시간마다 뜨는 수집합니다. 0.22 μm의 필터를 통해 뜨는을 필터링합니다. </li><li> bFGF 50 NG / ML로 뜨는 보충. </li></ol> 3. IPS 콜로니 선택과 Passaging <ol><li> 세포 배양 접시 CF - 1 MEF의 피더 세포와 함께 미리 씨앗에 Stemolecule Y27632 10 μm의 보충과 함께 인간의 ES / IPS 문화 매체 ES 같은 식민지 다시 씨앗을 선택합니다. 37 밤새 품어 ° C와 5% CO 2. </li><li> 처음 7 일 (Stemolecule Y27632과 보완하지 않고)을 24 시간마다 중간 문화를 변경합니다. 7 일 후에 (준비 섹션 2 참조) MEF 시설 중간 중간 변경합니다. 그들은 일반적인 인간 ES의 형태를 보여주 때까지 우리는 세포를 passaging 계속했다. </li></ol> 4. Pluripotency 마커 Immunocytochemical 시험 <ol><li> 세포에게 PBS로 가볍게 세 번 씻으십시오. </li><li> 실온에서 20 분 정착액 500 μl와 세포를 수정. </li><li> 세포에게 PBS로 가볍게 세 번 씻으십시오. </li><li> 상온에 한 시간 동안 500 μl 차단 버퍼가 아닌 특정 바인딩을 차단합니다. </li><li> 4 하룻밤의 특정 기본 항체 250 μl와 세포를 품어 ° C (우리는 1:100에서 트라 - 1 - 81, 트라 - 1 - 60, SSEA - 4, SSEA - 3, Oct4, Sox2, Nanog와 Lin28을 사용 dilutions). </li><li> 세포에게 PBS로 가볍게 세 번 씻으십시오. </li><li> (우리가 안티 마우스 IgM Cy3 공액, 염소 안티 - 마우스 IgG Cy3 공액, 염소 방지 랫 IgM Cy3 공액 및 염소 안티 - 염소를 사용하여 빛으로부터 멀리 보관, 실온에서 1 시간 동안 이차 항체 250 μl와 세포를 품어 1:300 dilutions에서 래빗 ​​Cy3 공액). </li><li> 세포에게 PBS로 가볍게 세 번 씻으십시오. </li><li> 마지막으로 씻어 DAPI를 (최종 농도가 1 μg / ML) 추가하고 핵을 시각화하기 위해 5 분 알을 품다. </li><li> 확대에 따라 세포를 분석할 수 있습니다. </li></ol> 제 5 부 : 대표 결과 1. 형태 결과 : 인간 포피의 fibroblast (BJ) 세포는, Sox2, Nanog, Lin28과 Oct4와 함께 공동 transduced되었습니다. 형태학의 변경 사항은 4 일 후 전달 빠르면 관찰하고, 세포의 클러스터보다 밀접하게 일 17 일 (그림 1)에서 포장되었다되었습니다. 식민지는 수동 일 25 들어서 CF - 1 MEF의 피더 세포에 교양되었습니다. 프로그래밍 후 IPS 세포 콜로니 형성을 촉진하기 위해, 우리는 Stemolecule Y27632을 사용각 통과하는 동안 초기 밤새 시딩의 바위 억제제. 좋은 형태로 IPS 세포 식민지는 식민지 세 연속 라운드가 따기 passaging 후 관찰했다. 2. Pluripotency 마커 표현 : 더욱 고립 IPS 세포 식민지를 특성화하기 위해, 우리는 ES 세포에서 표현 일반 pluripotency 마커의 존재를 보았다. 식민지는 강한 알칼리성 인산 가수 분해 효소 (AP) 활동 (그림 2)을 전시. 또한, immunocytochemistry (ICC)는 표면 마커 트라 - 1 - 81, 트라 - 1 - 60, SSEA - 4 및 SSEA - 3뿐 아니라 핵 마커를 포함하여, pluripotency 마커 특정 항체의 패널과 IPS 세포 식민지에서 수행되었다 Oct4, Nanog Sox2 및. 절연 IPS의 식민지 모든 마커 (그림 3)에 대한 긍정했다. ICC의 결과는 IPS 세포가 이러한 IPS 세포가 밀접하게 undifferentiated 인간의 ES 세포를 닮은 것을 보여주는, 적절한 pluripotency 표시자 표현식 패턴을 전시 것을 보여줍니다. <img src="/files/ftp_upload/1553/1553fig1.jpg" alt="그림 1" /> 그림 1 :의 형태학의 변화 BJ 세포를 transduced. 일반적인 IPS 세포 콜로니의 명시야 이미지 (A) 4 일 (B) 17 일 후 전달에서 형성. <img src="/files/ftp_upload/1553/1553fig2.jpg" alt="그림 2" /> 그림 2 : 다시 프로그램 BJ 세포의 AP 활동. 세 가지 식민지는 Stemgent 알칼리성 인산 가수 분해 효소의 스테 이닝 키트 물들다. <img src="/files/ftp_upload/1553/1553fig3.jpg" alt="그림 3" /> 그림 3 : 표면 마커 트라 - 1 - 81, 트라 - 1 - 60, SSEA - 4 및 SSEA - 3, 핵 마커 10월 4일, Nanog와 Sox2 : 인간 IPS 세포는 다음과 ES 세포 특정 마커의 높은 수준을 표현.

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">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., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., Thompson, 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=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (1917).</li><li>Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Koyanagi, M., Takahashi, J., Arakawa, Y., Doi, D., Fukuda, H., Hayashi, H., Narumiya, S., Hashimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+Rho/ROCK+pathway+reduces+apoptosis+during+transplantation+of+embryonic+stem+cell-derived+neural+precursors.">Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors.</a> J. Neurosci. Res. 86, 270-280 (2008).</li></ol>

이 문서의 저자는이 문서에서 사용되는 시약 및 악기를 생산 Stemgent 고용하고 있습니다.

이러한 결과는 인간의 섬유아 세포에서 전사 요소를 transduced Stemgent 인간 TF의 Lentivirus 세트가 효율적으로의 이소성 표현을 유도하여 IPS의 식민지를 생성하는 데 사용할 수있는 것을 보여줍니다. 프로그래밍 실험을 설계할 때, 몇 가지 변수는 프로그래밍의 효율성을 최적화하는 것으로 간주되어야합니다. 첫째, 활성 바이러스에 대 목표 비율 (감염 뫄의 다중성)은 최적의 전달 효율을 달성하기 위해 기본 전달 단계에서 수정해야 할 수 있습니다. 둘째, 대상 세포의 성장 상태는 프로그래밍 영향을 줄 수 있습니다. 건강하고 proliferative 세포 프로그래밍을 더 의무가 있습니다. 셋째, 서로 다른 세포 숫자에 대한 프로토콜을 수정할 때, 그것은 대상 셀 번호가 문화 요리의 표면 영역에 비례 조정하는 것이 좋습니다. 마지막으로, 같은 Y27632으로 적용 록 저해제는 최근 연구에서 향상 hES 콜로니 생존에의 유틸리티를 보여준 것처럼 성공적인 프로그래밍을 보장하기 위해 고려되어야합니다. 4,5

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Human TF Lentivirus Set</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>00-0005</td>
<td>&nbsp;</td>
<tr>
<td>Stemolecule Y27632</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>04-0012</td>
<td>&nbsp;</td>
<tr>
<td>TRA-1-81 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0012</td>
<td>&nbsp;</td>
<tr>
<td>TRA-1-60 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0009</td>
<td>&nbsp;</td>
<tr>
<td>SSEA-4 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0003</td>
<td>&nbsp;</td>
<tr>
<td>SSEA-3 antibody</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>09-0014</td>
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generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human tf lentivirus set

2006 년, 야마 나카와 동료 먼저 Oct4의 레트로 바이러스 - 중재 전달과 표현, Sox2, C - Myc와 Klf4는 마우스 섬유아 세포에서 pluripotent 상태를 유도 할 수 있다고 보여주었다. 1 같은 그룹은 또한 인간의 체세포의 성공적인 프로그래밍에보고 retroviral 벡터에 의해 전달 같은 전사 요소의 인간 버전을 사용하여 유도된 pluripotent 줄기 (IPS) 전지 2. 또한, 제임스 톰슨 외가. 보고한 요인의 다른 세트의 lentivirus - 중재 공동 표현 (Oct4, Sox2, Nanog와 Lin28 ) IPS 세포에 인간의 체세포를 프로그래밍 가능했습니다 3. IPS 세포는 형태, 증식 및 신체의 모든 조직 유형으로 차별화할 수있는 능력의 ES 세포와 비슷합니다. 그들은 배아 파괴의 윤리적 딜레마없이 ES 세포의 주요 특성을 전시 등 인간 IPS 세포는 ES 세포에 비해 뚜렷한 장점이 있습니다. 환자 특정 IPS 세포의 생성이 아닌 autologous 이식 세포의 면역 거부의 가능성을 제거하여 맞춤형 재생 의료의 치료에 중요한 장애물을 circumvents. 여기서 우리는 Stemgent 인간 TF의 Lentivirus 세트를 사용하여 인간 fibroblast 세포를 프로그래밍을위한 프로토콜을 보여줍니다. 우리는 또한이 세트로 다시 프로그램 세포 형태 사일 후 전달 IPS를 표시하기 시작 것을 보여줍니다. Stemolecule Y27632를 사용하여, 우리는 식민지 세 연속 라운드가 따기 passaging 후 IPS 세포와 관찰 올바른 형태를 위해 선택했습니다. 우리는 또한 입증되는 프로그래밍 세포 pluripotency 마커 AP, 표면 마커 트라 - 1 - 81, 트라 - 1 - 60, SSEA - 4, SSEA - 3와, 그리고 핵 마커 Oct4, Nanog Sox2 및 표시 후.

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set at JoVE.com

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

우리는 인간의 요인 Oct4, Sox2, Nanog, 그리고 Lin28의 lentivirus - 중재 전송을 사용하여 인간의 체세포에서 유도된 pluripotent 줄기 세포의 생성에 대한 프로토콜을 보여줍니다. Pluripotency는 형태학 및 배아 줄기 (ES) 세포 특정 마커의 존재에 의해 확인되었다.

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 ...

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Embryonic and Induced Pluripotent Stem Cells

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29.7K 조회수.  Stemgent. 우리는 인간의 요인 Oct4, Sox2, Nanog, 그리고 Lin28의 lentivirus - 중재 전송을 사용하여 인간의 체세포에서 유도된 pluripotent 줄기 세포의 생성에 대한 프로토콜을 보여줍니다. Pluripotency는 형태학 및 배아 줄기 (ES) 세포 특정 마커의 존재에 의해 확인되었다.

비디오: Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

인간 배아 줄기 세포의 문화 및 유지 관리

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

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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 ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

인간 배아 줄기 세포를 동결 융해

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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 ...

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 ...