우리는 원고에 대한 가치있는 토론을위한 이명박 실험실의 구성원을 감사드립니다. 이명박 실험실에서 작업 세포 연구 기금 (TEDCO를) 줄기 뉴욕 줄기 세포 재단의 로버트슨 조사자 포상에서 메릴랜드에서 교부금에 의해 지원되었다.

1. 셀의 제조 및 미디어 (주 1) <ol><li> 문화 DMEM 매체는 10 % FBS가 포함 된 인간의 섬유 아세포를 확장합니다. </li><li> 플레이트 인간의 섬유 아 세포 전달 전날에 잘 당 적절한 밀도로 24 웰 플레이트 상에 (그림 1). 참고 : 다른 종류의 세포가 다른 첨부 파일 기능을 가지고 있기 때문에 다음과 같은 일련의 희석액 (200K, 100K, 50K, 25K, 12.5K 및 6.25K)을 권장합니다. </li><li> 세포가 완전히 준수 및 확장이 보장, 37 ° C, 5 % CO 2 배양기에서 하루 더의 세포를 품어. </li></ol> 2. 형질을 수행합니다 (2 일) <ol><li> 형질 도입의 날에, 세포 밀도를 확인하고 가장 효율적인 밀도 웰 (도 2)을 선택했다. 높은 (80 ~ 90 %), 중 (50 ~ 70 %)과 낮은 (20 ~ 40 %)의 세 가지 다른 밀도를 선택하는 것이 좋습니다. </li><li> 1 시간 이상 전달하기 전에, fibrobl를 대기음AST의 세포에서 미디어와 섬유 아세포 매체의 새로운 300 μl를 변경합니다. </li><li> -80 ° C 저장에서 4 개의 다른 센다이 바이러스 튜브의 한 세트를 제거합니다. 몇 초 동안 37 ° C의 물 배스 동시에 각 튜브를 해동하고 수욕으로부터 튜브를 가지고. 그 후, 실온까지 이들을 해동; 원심 분리기 10 초 동안 6,000 XG에 튜브 및 사용까지 얼음에 배치합니다. 동결을 다시 및 역가가 유지되지 않으므로 바이러스를 해동하지 마십시오. </li><li> 마이크로 원심 분리기 튜브에 네 센다이 바이러스 튜브 (10 월 - 4, KLF-4, C-Myc와와 삭스-2)의 각각의 지정된 볼륨을 추가합니다. 용액을 부드럽게 피펫 팅하여 잘 혼합되어 있는지 확인합니다. 예를 들어, 전달 24 - 웰 플레이트 lookwell의 50K 세포 / 잘 하나 : (생활 기술에서 분석의 인증서를 기반으로 역가가 배치에서 배치에 차이가있을 수 있습니다) 튜브 hOct-4 = 6.0 × 10 7 CIU, 3 MOI 5 μl를 = 튜브 hSox-2 = 6.5 × 10 7 CIU, 3 MOI는 = 4.6 μ; L 튜브 hKlf-4 = 6.3 × 10 7 CIU, 3 MOI = 4.8 μL 튜브 HC-Myc와 = 7.8 × 10 7 CIU, 3 MOI = 3.8 μL 총 = 24 - 웰 플레이트의 네 가지 바이러스 요인 혼합물 / 잘 하나 (50K 세포)의 18.2 μL </li><li> 세포 밀도에 따라; 2X, 1X, 그리고 잘에 바이러스 혼합물의 0.5X 볼륨을 추가합니다. 부드럽게, 플레이트 앞뒤 흔들어 좌우 (2X : 바이러스 혼합물, 1X의 36.4 μL : 18.2 μL와 0.5X : 9.1 μL). </li><li> 37 ° C, 5 % CO 2 배양기에서 세포를 놓고 밤새 품어. </li></ol> 3. 문화 매체의 교체 (3 일 · 5 일) <ol><li> 24 시간 전달 후, 500 ㎕의 신선한 섬유 아세포 매체와 매체를 교체하십시오. </li><li> 5 일에, 신선한 섬유 아세포 미디어와 중간 변경합니다. </li></ol> 4. 공동 문화 MEF 요리를 준비 (8 일) <ol><li> 10 % 60mm 배양 접시에 MEF 세포를 준비 FBS는 DMEM 매체 하나 다 포함MEF 피더 세포에 형질 도입 된 섬유 아 세포를 계대 전에 Y. 참고 : 우리는 각 60mm 접시에 MEF의 5 × 5를 사용하고 있습니다. </li></ol> 5. 시작 공동 문화 MEF 피더 세포 (9 일)으로 세포를 형질 도입 <ol><li> 형질 세포를 도금하기 전에 대기음 10 % FBS는 MEF 피더 요리에서 DMEM 매체를 포함하고 FBS는 DMEM 매체를 포함 신선한 10 %를 추가합니다. </li><li> 24 - 웰 플레이트에 있고 D-PBS로 한 번 세포를 씻어 형질 섬유 아 세포에서 매체를 제거합니다. 24 - 웰 플레이트 기계가 섬유 아세포의 세포에 0.25 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) 200 μl를 추가합니다. 세포가 플레이트에서 분리하기 시작할 때 트립신 / EDTA (에틸렌 다이아 민 테트라 초산), 미만 5 분 배양 후, 15 ML 원뿔 튜브에 성장 매체와 세포를 수집합니다. 그런 다음 4 분 6,000 XG에서 그들을 원심 분리기. 참고 : 2X, 1X, 0.5X 및 바이러스를 풀링은 형질 섬유 아세포를 권장합니다. </li><li> 뜨는 매체를 제거하고 다시 중단에 씻어트립신의 중화를위한 신선한 섬유 아세포 배지 중에서 4-5 ㎖를 가진 세포 펠렛. 그런 다음 4 분 동안 135 XG에이를 원심 분리. </li><li> 예 : 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 및 백이십팔분의 일 60 당 : 시리얼 희석 밀도 및 MEF 상에 섬유 아세포 미디어와 공동 문화에 시작 플레이트 세포 mm 접시. 전달의 첫 주 동안 세포 사멸 속도에 따라, 하나는 1 / 2 또는 128분의 1 밀도에 희석 할 필요가 있습니다. 형질 전환 유전자 RT-PCR에 대한 긍정적 인 제어로 총 RNA를 추출하기 위해 옆에 남아있는 세포를 넣어. </li><li> 5 % CO 2 배양기 하룻밤, 37 ° C에서 60mm 요리를 놓습니다. </li></ol> 6. 인간 배아 줄기 세포의 중간 피드와 셀 (주 10) 모니터 <ol><li> 다음 날, 10 μM Y-27632 인간의 ES 미디어에 섬유 아세포 미디어를 변경합니다. 그 후, 인간 ES 매체 매일 매체 변경 : 800 ml의 DMEM/F12 + 200 ㎖의 녹아웃 혈청 교체 + 10 ml의 L-글루타민 + 10 10 mM의 MEM 최소 비 필수 아미노산의 ML기본적인 섬유 아세포 성장 인자의 2 - 머 캅토 에탄올 + 10 겨 / ㎖의 + 1 ㎖. </li><li> 신선한 인간의 ES의 미디어와 매일 미디어 변경 작업을 수행합니다. 일주일 후, ES 식민지와 같은 세포 덩어리의 형성을 현미경으로 한 번에 2 ~ 3 일이다 요리를 확인합니다. 콜로니가 발견되기 전에 세포의 종류에 따라, 배양은 더 많거나 적은 시간이 걸릴 것이다. </li></ol> 7. 유도 다 능성이 세포 식민지 줄기 및 셀 확장 따기 (일 20 ~) <ol><li> 3 주 전달 후, 식민지 따기 제대로 성장 나타납니다. </li><li> 식민지를 따기 전날 24 - 웰 플레이트에 MEF 공급 장치를 준비합니다. 참고 : 우리는 하나 24 - 웰 플레이트에 MEF 12.5 × 10 5를 사용하고 있습니다. </li><li> 다음 날, 10 μM Y-27632 인간의 ES의 미디어에 섬유 아세포 미디어에서 MEF 요리의 매체를 변경합니다. 수동 따기 후드에서 현미경으로 각각 한 번에 하나의 식민지를 선택하고 피펫으로 작은 덩어​​리를 만들어 전송할새로 개의 클론을 선택하는 MEF dishes.Try 준비에. </li><li> 통로 처음 6 웰 플레이트에 24 웰 플레이트에서 각 웰 확장합니다. 그런 다음 6 잘 플레이트에서 더 전파하기 전에 60mm 요리에. </li></ol> (10 구절 후) 인간 유도 만능의 8. 특성 <ol><li> 면역 형광 <ol><li> 매일 미디어 변화 4-5 일 도중 24 웰 플레이트와 문화 플레이트 인간 유도 만능. </li><li> 면역 형광 분석을 시작, 한 번 D-PBS로 접시를 씻으십시오. 그런 다음 30 분 동안 0.4 % 파라 포름 알데히드 즉시 세포를 고정합니다. 그 후 5 분 동안 D-PBS로 세 번 씻어. </li><li> 실온에서 15 분 동안 PBS에서 0.1 % 트리톤 X-100 용액으로 고정 된 세포를 치료. </li><li> permeablization 용액 (0.1 % 트리톤 X-100 용액) 대기음 다음, 상온에서 1 시간 동안 0.5 % BSA 블로킹 용액을 추가한다. </li><li> Nanog를, 10 월 - 4, SSEA (무대 특정 배아 antig의 검출을 수행EN) 및 TRA-1-81 0.5 % BSA 용액에 항 인간 항체를 사용하여 (종양 제거 항원) (항체의 희석 비율에 대한 자료 표를 확인하시기 바랍니다.) 실온에서 2 시간 동안 차 항체를 품어. 그런 다음 5 분마다 D-PBS로 세 번 씻어. </li><li> 실온에서 0.5 % BSA 용액에 1:2,000 희석하는 2 차 항체와 같은 알렉사 플 루어 488 염소 항 - 마우스 IgG 항체와 배양 1 시간 후 형광 현미경을 사용하여 확인합니다. DAPI 각 MEF 피더 세포와 hiPSCs의 모든 핵을 얼룩. </li></ol></li><li> 형질 전환 유전자 확인의 RT-PCR <ol><li> RNA 추출 시약을 사용하여 세포 펠렛에서 총 mRNA의 압축을 풉니 다. </li><li> 역전사하여 PCR 증폭을위한 총 RNA와 그 결과의 cDNA의 분취에 역전사 (1 μg)을 수행합니다. </li><li> cDNA를 템플릿의 2 μL, 2X PCR 마스터 믹스 10 μL, 프라이머 2 pmol의 2 μl를 포함하는 PCR 혼합물을 준비합니다(앞으로 1 μL 및 역방향 : 1 μl를) 및 DW 6 μL. 표 1에 기재된 프라이머로 RT-PCR을 수행하여 유전자 발현을 검출한다. </li><li> 증폭 반응의 효율을위한 제어로 GAPDH 유전자와 RT-PCR을 수행합니다. 시각화하고 1 % 아가로 오스 겔 전기 영동에 의해 PCR 생성물을 분석한다. </li></ol></li></ol>

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저자가 공개하는 게 없다.

hiPSCs에 인간의 체세포를 재 프로그램하는 것은 기본 생물학, 개인 의학 및 이식 12 전례없는 약속을 보유하고 있습니다. 이전에, 인간의 IPSC 세대는 의약품 개발과 이식 치료 13으로 추가 임상 응용 프로그램을 제한하는 바람직하지 않은 유전자 변이를 만들 수 있습니다 호스트 게놈으로 통합 위험이 DNA 바이러스를 요구했다. 이러한 이유로, 많은 연구는 여러 다른 방법으로 벡터 ?...

인간 배아 줄기 세포 (hESCs)는 약물 검사에 대한 질병 모델링을위한 잠재적으로 유용 할 수 있고, 질병 및 조직 손상을 치료하는 세포 기반 치료법을 개발하기 위해 자기 갱신 시험 관내 및가 능성에 대한 능력을 보유하고 있습니다. 그러나 인간 배아 줄기 세포 때문에, 면역 학적 종양학과 윤리적 장벽의 세포 대체 요법에 대한 제한이 있고, 질병 관련 유전자를 연구, 질병 특정 인간 배아 줄기 세포는 착상 전 유전자 진단 (PGD) 방식을 통해 분리 할 수​​ 있지만, 여전히 기술적으로 도전 및 배아 기증은 매우 드물다. 이러한 문제는 유도 만능 줄기 세포 (hiPSCs)의 개발을 주도하고있다 줄기 세포 생물학의 진행과 관련이 있습니다. 인간 유도 만능은 유 전적으로 인간의 성인 체세포에서 프로그램이 다시 그들에게 예 : D 재생 의학을위한 유용한 소스를 만드는 인간 배아 줄기 세포와 유사한 만능 줄기 세포와 같은 기능을 품고있다양탄자 발견, 질병 모델링 및 환자 특정 방식으로 1,2의 세포 치료. 지금까지 바이러스 매개 (레트로 바이러스 및 아데노 바이러스)를 포함하여 인간 유도 만능를 생성하는 여러 가지 방법, 3, 비 바이러스 매개 (BAC 시스템과 벡터의 형질) 4 유전자 transductions 및 단백질 전달 시스템 5-7이 있습니다. 바이러스 매개 유전자 전달 효율의 특정 수준을 보장 할 수 있지만 그들이 통제 할 수없는 방식으로 프로그램을 다시 만들 유전자를 표현하는 호스트의 염색체에 통합하기 때문에, 바이러스 성 벡터는 유전자 발자국을 떠날 수 있습니다. 전사 인자의 바이러스 통합을 활성화하거나 호스트 8 유전자를 불 활성화 할 수 있더라도, 이는 예기치 유전 수차 및 종양의 5,9의 위험을 일으킬 수있다. 한편, 체성 세포로 단백질 또는 RNA의 직접적인 도입은보고 있지만, 이러한 노동 집약적 반복 TRANSF 등의 몇 가지 단점을 가지고 있었다ection 및 7,10 재 프로그램의 낮은 수준입니다. 심지어 에피 솜 및 비 통합 아데노 바이러스, 아데노 - 관련 바이러스, 플라스미드 벡터는 여전히 11 비교적 덜 효율적이다. 이러한 이유로, 그것은 IPSC 세대의 높은 효능과 적은 유전자 이상과 비 통합 프로그램을 다시 만들 방법을 선택하는 그럴듯​​. 이 연구에서, 우리는 센다이 바이러스 기반 프로그래밍을 사용합니다. 이 방법은 호스트 게놈에 비 통합과 지속적으로 유전자 통합하지 않고 인간 유도 만능를 생산하는 것으로 알려져있다.

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			<td height="21" style="height:21px;width:543px;">SSEA-4</td>
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			<td height="21" style="height:21px;">DPBS (Dulbecco&#39;s Phosphate-Buffered Saline), 1X without calcium &#38; magnesium</td>
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			<td height="21" style="height:21px;width:543px;">QuantiTect Reverse Transcription Kit</td>
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			<td height="21" style="height:21px;">Trizol</td>
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			<td height="21" style="height:21px;">picking hood</td>
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			<td height="21" style="height:21px;width:543px;">dissecting scope&#160;</td>
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efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus

몇 년 전, 인간 유도 만능 줄기 세포 (유도 만능)의 설립은 생물 의학의 새로운 시대를 열었다. 인간 유도 만능의 잠재적 인 사용은 인간의 유전 질환의 모델링 발병 유전자 보정 후자가 세포 치료, 환자 별 증상과 관련된 세포의 소스를 제공함으로써 맞춤 약물 검사를 포함한다. 그러나, 이러한 인간 유도 만능 생산 후 남은 프로그래밍 인자 유전자의 발현을 제거하는 등의 극복하는 여러 장애물이있다. 더 중요한 것은, 미분화 된 인간 유도 만능 잔류 유전자 발현 적절한 분화를 방해하고 질병 관련 체외 표현형에서의 해석을 잘못지도 할 수 있습니다. 이러한 이유와 함께, 통합 - 무료 및 / 또는 유전자가없는 인간 유도 만능는 아데노 바이러스, 피기 백 (PiggyBac) 시스템, minicircle 벡터, 에피 솜 벡터, 직접 단백질 전달과 같은 몇 가지 방법을 사용하여 개발 및 mRNA의 합성되었다. 그러나 reprogra의 효율성통합없는 방법을 사용하여 mming하는 것은 대부분의 경우 매우 낮습니다. 여기서, 우리는 센다이 바이러스 (RNA 바이러스) 기반 프로그래밍 시스템을 사용하여 인간 유도 만능을 분리하는 방법을 제시한다. 이 프로그래밍 방법은 일관된 결과 및 비용 효율적인 방식으로 고성능을 나타낸다.

보통 감염 섬유 아 세포는 센다이 바이러스 전달 후 몇 일에 한 어떤 형태의 변화를 보여주지 않지만, 5 일 후, 그들은 다른 모양 (그림 1)를 가지고 시작합니다. 도 1의 오른쪽 패널에서 설명한 바와 같이, 세포는 전형적인 섬유 아세포의 형태를 가지고 있지 않는다. 그들은 세포질보다 둥근 모양과 더 큰 핵이있다. 전달이 80 %의 세포 포화 상태에서 수행되는 경우에도 그들...

Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus at JoVE.com

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

여기, 우리는 일관된 결과 및 향상된 효율성을 보여줍니다 센다이 바이러스와 유전자가없는 인간 유도 만능에 인간의 체세포를 재 프로그램하는 우리의 설립 방법을 제시한다.

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 ...

efficient-generation-human-induced-pluripotent-stem-cells-from-human

Research

JoVE Journal

Biology

22.2K 조회수.  Johns Hopkins University School of Medicine.  여기, 우리는 일관된 결과 및 향상된 효율성을 보여줍니다 센다이 바이러스와 유전자가없는 인간 유도 만능에 인간의 체세포를 재 프로그램하는 우리의 설립 방법을 제시한다. 

비디오: Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

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

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Stemgent 인간 TF의 Lentivirus와 인간의 섬유아 세포를 프로그래밍하여 유도 Pluripotent 줄기 세포의 생성 세트

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

연구

생물학

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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

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

인간 유도 만능의 줄기 세포를 Transfecting 및 Nucleofecting

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

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

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

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

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

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

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, ...

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 ...

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

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

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