Name: derivation and characterization of a transgene-free human induced pluripotent stem cell line and conversion into defined clinical-grade conditions
Uploaded: 2014-11-26T15:00:00.000+00:00
Duration: 10 min 48 s
Description: Watch this Scientific Journal Video about Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions at JoVE.com

We would like to thank Patrick C. Lee, Cyril Ramathal, and Saravanan Karumbayaram (SK) for their assistance in performing the iPSC derivation and characterization experiments; Aaron Cooper for performing the iPSC analysis experiments; Vittorio Sebastiano and Renee A. Reijo Pera for directing the initial reprogramming efforts; SK, William E. Lowry, Jerome A. Zack, and Donald B. Kohn for directing the establishment of the UCLA GMP facilities permitting the conversion and characterization of clinical-grade iPSCs; Gustavo Mostoslavsky for providing us with the STEMCCA polycistronic reprogramming vector. This work is based on a research collaboration with Fibrocell Science and the Clinical Investigations for Dermal Mesenchymally Obtained Derivatives (CIDMOD) Initiative to generate safe personalized cellular therapeutics. This work was supported by funding from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, The Phelps Family Foundation, Fibrocell Science, Inc., and the UCLA CTSI Scholar&#8217;s Award to JAB.

주 :.이 방법은 경외 등 (10)에보고 된 연구에 사용 하였다. STEMCCA 1. 재 프로그래밍 성인 인간 피부 섬유 아세포 <ol><li> 해동 성인 피부 인간 섬유 아세포 (한국 외대), 2 분 동안 37 ° C의 물을 욕조에 통로 (4) 이하, 물을 유리 병의 상단 잠수함 않도록주의. </li><li> 15 ML 원뿔 유리 병에 인간 섬유 아세포의 1 ml의 슬러리를 놓고 천천히 드롭 현명한 방식으로, 37 ° C로 미리 예열 표준 HUF 미디어, 4 ml의 배치, 디메틸 설폭 사이드 (DMSO)를 밖으로 희석하기 다시 중단 세포를. </li><li> 실온에서 10 분 동안 200 XG에서 바이알을 원심 분리기. 뜨는을 대기음 / ㎖ 100,000 세포 현탁액을 만들 매체의 적절한 볼륨에서 셀을 다시 중단하고 여섯 잘 플레이트 잘 하나에 2를 가하여, 0.2 % 젤라틴으로 20 분 동안 미리 코팅. 참고 : 라인과 조건에 따라 세포의 동일한 수의 잘 자매 fo를 접종해야R 셀 카운팅 / 감염 (MOI) 계산의 다양성. </li><li> 세포를 균일하게 분배하는 전후 좌우하고 판측 락. 가습 인큐베이터에서 37 ° C, 5 % CO 2에서 밤새 인큐베이션. </li><li> 전달의 날, 세포 자매의 수 (들) 잘 초기 HUF 도금 후 (들) 약 24 시간을 구하십시오. </li><li> 2 × 10 8 TU 또는 이와 동등한, 렌티 바이러스 농축 (STEMCCA)을 밖으로 해동 HUF 미디어 1 × 10 8 TU / ㎖로 희석. </li><li> 8 ㎍ / ㎖의 트랜 스펙 션 제로 보충 HUF 배지에서 10 MOI 비율로 세포를 형질 도입. 균등 형질 우물을 혼합하고 가습 배양기에서 37 ° C, 5 % CO 2에서 밤새 품어. </li><li> 렌티 바이러스 전달 후 약 24 시간은 표준 인간 만능 줄기 세포 (PSC) 매체에 HUF 매체를 전환합니다. </li><li> 일 2 ~ 6 일, 인간의 PSC 매체 매일 미디어를 변경합니다. </li><li> 하루 6 일, 두 0.2 % 젤라틴 코팅 10 cm 판을 판HUF 미디어 12 CF1 마우스에서 파생 된 1.5 10 × 1.75 6 MEFs에. </li><li> 7 일에, 조심 사이에 균등하게 세포를 도금, 1시 16분 비율로 6 웰 플레이트 및 통로의 한 우물에서 7 일 다시 프로그램 섬유 아세포를 들어 올릴 때는 100 개 이상의 XG, 사용 실온 트립신을 원심 분리하지 전날 MEFs에 도금 된 신선한 인간의 PSC 미디어, 두 10cm 플레이트. 37 ° C에서 시계 방향으로 나선형 운동과 장소에 세포 집합체를 배포, 5 % CO 2 하룻밤 인큐베이터를 가습. </li><li> 약 48 시간 이후 MEFs에 매일에 재 프로그램 세포를 파종 한 후, 신선한 매체와 소비 인간의 PSC 미디어를 변경합니다. </li><li> 3~4주 후, 21 게이지 바늘 형태처럼 전형적인 ESC 개별 콜로니를 선택하고 0.2 % 젤라틴 - 코팅 된 개별 웰에 특정 콜로니 약 8-10 개별적인 조각을 배치하여 24 웰 플레이트 아웃 서브 클로닝 24 웰 플레이트 전 씨인간의 PSC 매체 CF1 마우스에서 파생 된 MEFs에와 에드. 주 : 촬상 콜로니 전개 후, 콜로니를 단백질 수준에서 배아 체 형성 및 다 능성 마커의 발현 분석에 의하여 다 능성 특징으로 할 수있다. </li></ol> 2. 벡터 통합 사이트 분석 및 STEMCCA 절제술 <ol><li> 10 바와 같은 비 제한적인 선형 PCR에 의해 증폭 STEMCCA 통합 사이트를 분석한다. </li><li> 오래된 인간의 ESC 매체를 흡입하여 게놈, 소비세 STEMCCA의 원하는 영역으로 하나의 통합을 보장하기 위해 시금 후. 그 후, 24 시간 동안 표준 PSC 미디어 3 ㎖ 8 ㎍ / ㎖의 트랜 스펙 션 제로 농축 아데노 - 치운다 PuroR 바이러스의 45 μl를 결합한다. </li><li> 24 시간의 잠복기 후, 혼합 바이러스 뜨는을 대기음과 인간의 PSC 미디어로 두 번 세포를 씻으십시오. 5 일 동안 2 ㎍ / ㎖의 퓨로 마이신과 함께 신선한 인간의 PSC 매체를 놓고 미디어 매일 재치 변경시간 신선한 매체와 항생제. </li><li> 오일 후 서브 클론 12- 웰 플레이트에서 0.2 % 젤라틴 - 코팅 된 웰에 21 게이지 바늘을 가진 콜로니로부터 유도 된 잔류 CF1mice MEFs에 사전에 도포하고, 위에서 설명한 바와 같이 확장. 참고 : 충분한 확장은 냉동 보관을 얻을 후, 공급이없는 상태로 변환이 필요합니다. 대부분의 세포가 성공적으로 형질 도입되지 않고 배양 오일 걸쳐 항생제에 굴복하는 바와 같이 95 % 이상의 줄기 세포 콜로니 사멸 예상된다. 아데노 Cre 호텔 - PuroR는 요인이없는 식민지의 샘플을 reexposing, 감염된 세포의 호스트 염색체에 0.001 %의 효율로 통합 있지만 더 통합 13 발생하지 확인합니다 100 % 세포 사멸을 보장하기 위해 퓨로 마이신합니다. </li><li> 매트릭스가 액체가 될 때까지 약 2 시간 (제조자의 권고)에 걸쳐, 얼음에 사전 분취 기저막 매트릭스의 한 바이알을 해동하고 차가운 기저 매체 1:30 최종 농도로 희석 아웃. 공동물론 당 1 mL를 여섯 웰 플레이트에 웰 원하는 번호로. 실온에서 1 시간 동안 앉아 보자. </li><li> 물론 이전에 MEFs에 코팅 30 식민지 적어도 20 (18 게이지 바늘, 또는 유리 또는 플라스틱 &quot;팁&quot;을 사용하여) 크로스 해치. MEF는 고양 줄이고 판에서 전송할주의하십시오. <ol><li> 멸균 플라스틱 피펫 팁의 간단한 사용법으로, 화염에, 더 복잡한 가열에서, 다른 접근 방법을 통해 유리 파스퇴르 피펫의 당김, 및 마무리를 해칭 장치를 생성하거나, 우리의 경우에서와 같이, 21- 및 / 또는 18 게이지 바늘. </li></ol></li><li> 코팅 잘 후 배양에서 행렬을 기음. </li><li> 200 μL 피펫으로 이전 판에서 부드러운 스크랩하여 식민지의 조각을 제거하고 조심스럽게 매트릭스 코팅 접시에 신선한 결합 된 미디어 (1) 3 ㎖에 배치합니다. 37 ° C, 5 % CO 2 가습 배양기에서 하룻밤 품어. 미디어 변경, 48 시간 계대 후. </li><li> 상업 DNA 추출 키트를 사용하여 80 % 이상의 포화 상태에, 절제 포스트 및 피더없는 변환 된 hiPSCs에서 게놈 DNA를 추출합니다. </li><li> STEMCCA의 렌티 바이러스의 외생 적 통합을 위해 특정 프라이머와 적절한 절제에 대한 분석 : gDNA를-헨도-MycS 감기, 5&#39;-acgagcacaagctcacctct-3 &#39;; gDNA를-hWPRE-역, 5&#39;-tcagcaaacacagtgcacacc-3 &#39;. 10 μM 원액을 PCR 등급 물을 동결 건조 된 프라이머를 복원합니다. </li><li> 다음과 같은 5 단계 프로토콜 PCR을 실행하여 초기 변성을 3 분 동안 95 ° C에서; 15 초 동안 72 ° C에서 15 초, 및 확장을위한 62 ° C에서 20 초, 프라이머 어닐링 98 ° C에서 변성, 다음의 각 35주기 3 분 72 ° C에서 단일주기 최종 확장 하였다. </li><li> 아가 로스를 1.5 g 칭량하고 50 ㎖ 1X 트리스 - 아세테이트 - EDTA 버퍼에 배치하여 3 % 아가 로즈 젤을 확인. 아가로 오스 때까지 전자 레인지 용해 DNA 젤 얼룩의 장소는 p로퍼 희석, 혼합하고, 실온에서 1 시간 동안 고화 겔 카세트에 적절한 양을 분배. </li><li> PCR 산물의 부하 12 μl의 3 % 아가 로즈 겔에 로딩 염료 3 μL와 혼합 하였다. V. 80에서 30 분 동안 젤을 실행 </li><li> 적절한 절제를 나타내는 밴드의 부재에 대한 자외선 시각화. </li></ol> 사전 및 사후 적출 hiPSCs에서 정량 PCR을 이용한 유전자 발현의 차이 3. 정량 <ol><li> 상업 RNA 추출 키트를 사용하여 절제 포스트 및 피더없는 변환 된 hiPSCs에서 총 RNA를 추출합니다. </li><li> - 역 전사 제조업체의 지침에 따라 상업 키트를 사용하여 총 RNA 1 μg의 최대. 고정 - 올리고 (DT) (18) 임의 헥사 프라이머를 사용합니다. 참고 : PCR 프로토콜은 4 KB보다보다 더 많은 이하의 유전자 성적 증명서에 맞게 조정되어 있는지 확인합니다. 특정 프라이머 중 유전자 STEMCCA 원래 통합 전을 위해 만든해야합니다NTO. </li><li> 20 μM 원액을 PCR 등급 물을 동결 건조 된 프라이머를 복원합니다. </li><li> 10 μM의 UPL 프로브로 구성되어 반응 20 μL, 2 배의 LightCycler 480 프로브 마스터 당 샘플의 5 NG를 사용하고, 앞으로 20 μM 및 역방향 프라이머. </li></ol> GMP 수준의 조건으로 4. 변환 <ol><li> GMP 수준의 조건을 통해 후 절제 hiPSCs를 전환의 첫 날, 결합 된 인간의 PSC로 구성된 미디어의 혼합을 미디어 1 (결합 된 미디어 1) 임상 성적 우수 제조 기준 (GMP) 호환 미디어 (결합 된 미디어 2 ) 80 : 20의 비율로. </li><li> 대기음 오래 결합 매체 (1)과 80:20의 비율로 조합 된 새로운 매체 1 및 2, 예열 3 ㎖를 배치 오프. 이 특정 비율로 3 일 동안 매일 미디어를 변경합니다. 다시 37 ° C, 5 % CO 2 배양기에 장소 세포. </li><li> 하루에 4 일, 50 : 50의 비율로 결​​합 된 미디어 (1)과 (2)를 확인합니다. 기존 매체와 REPLAC 대기음50 대 50의 비율로 결​​합 된 미디어 1과 2와 미리 예열 미디어와 전자. 이 특정 비율로 3 일 동안 미디어를 변경합니다. 다시 37 ° C, 5 % CO 2 배양기에 장소 세포. </li><li> 7 일째, 20:80의 비율로 혼합 매체 1 및 2를 만든다. 대기음 기존 매체 오프와 20:80의 비율로 결​​합 된 미디어 1과 2, 예열 용지로 교체합니다. 이 특정 비율로 3 일 동안 미디어를 변경합니다. 다시 37 ° C, 5 % CO 2 배양기에 장소 세포. </li><li> 100 비 : 10 일에, 0에 결합 된 미디어 1과 2를합니다. 대기음 기존 매체 끄고 0 결합 된 미디어 1과 2, 미리 예열 용지로 교체 : 100 비율. 이 특정 비율로 매일 미디어를 변경합니다. 세포는 완전히 정의 이종 무료 매체에있다. 다시 37 ° C, 5 % CO 2 배양기에 장소 세포. 주 :이 전체 변환 프로세스 중, 18 게이지 바늘로는 적절한 루틴 계대 콜로니 크기 및 밀도를 유지하기 위해 필요하다. 페이지에 계획콜로니 크기, 포화 상태, 및 분화에 기초하여 모든 4~5일 세포 assaging. </li><li> 제조업체에서 권장 정의 이종 무료 기판과 여섯 잘 플레이트에 잘 코트 하나. </li><li> 기계적 통로 합류가 아니라, 이미 18 게이지 바늘 여섯 웰 플레이트에서, 이종없는 미디어 조건을 변환. 중요한 것은, 미디어를 조건을 사전에 계대 전에, 1 시간 동안 1 배 ROCK 억제제와 세포에 새로운 미디어 (결합 된 미디어 2)를 배치합니다. </li><li> 세포로 전달되고 있다는 새로운 플레이트 오프 합성 기질 용액 기음. 이전 판에서 줄기 세포 덩어리를 포함하는 모든 용지를 제거하고 새로운 판에 전송합니다. </li><li> (한 번 인큐베이터 내부, 직접 방식으로 접촉하지 않는 이상적 판) 다시 최소한의 물리적 방해 2 일 동안 37 ° C, 5 % CO 2 배양기에 장소 세포는 최대 첨부를 허용합니다. 참고 : 세포는 매일 미디어를 변경해야합니다. 지속적인 passaging 4 일마다이 중요 할 것이다. 21 게이지 바늘, 최적의 ESC와 같은 형태 (높은 nucleocytoplasmic 비율)와 식민지의 통로 부분 만 사용. 이것은 잘 성장하고 결국 동질적인 식민지를 얻을 것입니다 적절한 hiPSC 식민지에 대한 선택을 보장합니다. 식민지 같은 ESC의 유도에 전체 시간은 초기 결합 된 미디어를 변경 한 후 15~20일 사이에있다. </li><li> 18 게이지 바늘을 사용하여 첫 번째 크로스 해치에 의해 식민지를 모두 GMP 수준의 사후 적출 hiPSCs을 동결. 200 μL의 피펫을 사용하여 해제 모든 조각을 올리고 4 ℃에서 5 분 동안 200 XG에 15 ML 원뿔 유리 병 원심 분리기에 세포를 포함하는 모든 미디어를 전송합니다. </li><li> 세포를 원심 분리하는 동안 차가운 결합 매체 (2)의 같은 부피로 이루어진 7.5 %의 최종 DMSO 농도 매체 냉동 동결 매체를 만든다. </li><li> 세포 펠렛 후, 기존 매체를 대기음 및 냉동 매체와 펠렛을 다시 일시 중지 현명한 놓습니다. 즉시 TR동결 튜브에 ansfer와 C의 냉동고 °에서 -80했습니다. </li></ol> 5. 특성화 GMP 수준의 사후 적출 hiPSCs <ol><li> 3 단계 QPCR 방법론에서 언급 한 다음 키 능성 마커 (SOX2, 인 Oct4, 및 NANOG) 같은 단계를 표 1에 프라이머를 사용하여의 발현을 정량화하는 QPCR를 사용 능성 마커 변환 후 적절한 표현을 보장하기 위해 3 단계에 나열된 같은 단계를 반복합니다. </li><li> 사용 이전에 (10)이 발표 한 키트에 나열된 표준 약관에 따라 인간이 아닌 시알 산 Neu5Gc (N -glycolylneuraminic 산)을 감지하는 유동 세포 계측법. </li><li> 부드럽게 1X 인산 완충 식염수 (PBS)를 사용하여 세포를 세포 배양 플레이트를 세척한다. </li><li> 실온에서 5 분 동안 1X 해리 시약을 사용하여 콜로니를 떼어 놓다. </li><li> 단계 5.4의 5 분의 배양 시간 동안에 설치된 차단 용액 (제조얼음 - 냉각 PBS로 차단제 0.5 %함으로써 키트). </li><li> 유동 세포 계측법 분석을위한 튜브의 다음 세트 레이블 : 흠을; 4 &#39;, 6- Diamidino -2- 페닐 인돌 (DAPI) DAPI; 대조군 항체 1 : 200; 차 항체 1 : 200; 이차 항체 1 : 200 당나귀 안티 치킨의 IgG (H + L); 샘플 MEFs에; 매트릭스에 샘플 후 절제 세포; 및 샘플 합성 기판에 세포를 포스트 - 절제. </li><li> 조심스럽게 15 ML 원뿔 튜브에 솔루션 및 전송 내용을 차단 2 ㎖를 추가하여 단​​계 5.4에서 세포 집합체을 제거. 4 ℃에서 5 분 80 XG에 원심 분리기. </li><li> 단계 5.7에 명시된 솔루션과 원심 분리기를 차단로 두 번 세포를 씻으십시오. </li><li> 부드럽게 각 해당 항체의 희석 부피 차단 용액 100 ㎕에 약 1 × 106 세포를 다시 일시. 부드러운 1 시간 동안 4 ° C에서 락을 함께 품어. </li><li> 단계 5.8에서와 같이 세척을 세 번 반복합니다. </li><li> 희석제 니어 400 μL의 세포 펠렛을 다시 일시ffer은 1 (키트에서) : 튜브 2, 6, 7 DAPI 100, 8 단계 5.6 아래에 나열된. </li><li> 40 μm의 여과기를 통해 변형 세포와 사이토 흐름을 통해 실행합니다. </li></ol>

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James A. Byrne (JAB) and Agustin Vega-Crespo receive research funding from Fibrocell Science, Inc. JAB is a scientific consultant for Fibrocell Science, Inc. No other authors have any competing interests to disclose.

우리는 인자없는 hiPSCs를 유도하고 미래의 인간 치료제 하류 세포 분화에 대한 GMP 수준의 조건으로 이들 세포를 변환하여이를 임상 적으로 만드는 방법을 설명합니다. 이 프로토콜은 다양한 종류의 세포에 광범위하게 적용 할 수 있지만, 우리는 인해 환자에서 추출의 용이성과 개인화 된 인간의 치료제에의 적용에 인간 피부 섬유 아 세포를 재 프로그래밍하기로 결정했습니다. 제한이 임상 적으?...

Adult human cells have been shown to be capable of undergoing epigenetic remodeling and reprogramming, as a result of lentiviral-based expression of four key transcription factors4,5. An important advancement in the reprogramming field was the use of a single excisable lentiviral stem cell cassette (STEMCCA), which housed all four reprogramming transcription factors that allowed a precise stoichiometric ratio of protein expression6. Additionally, when transduced in specific multiplicity of infection ranges, STEMCCA can lead to predominantly single genomic integration events during the reprogramming process7. The introduction of an excisable version of STEMCCA, which utilizes Cre/loxP technology followed by excision of the reprogramming vector after derivation of the stem cell line, enabled factor-free human induced pluripotent stem cell (hiPSC) lines to be derived8. Additionally, in order to enhance therapeutic applications of hiPSCs, a novel, quick, and readily applicable methodology for good manufacturing practice (GMP)-grade cell line conversion, from xeno-containing to xeno-free conditions, needed to be implemented. Here, we discuss a relevant methodology that more precisely assesses integrated gene expression differences, specifically when integrated into an intron, and clinical-grade cell conversion into putative GMP conditions. 
Previous research has used only relatively insensitive microarray transcriptional analysis to analyze gene expression differences in integrated genes after STEMCCA transduction3,9. Here, we introduce the methodology of sensitive quantitative polymerase chain reaction (PCR) analysis, to further examine integrated gene expression differences. Importantly, current safe-harbor criteria discard hiPSCs that have genes with viral integrations, thus limiting the applicability of these cells for downstream human cellular therapeutics9. We propose that the status quo may change with the use of fully characterized and transgene-free intronically reprogrammed hiPSCs. Additionally, we introduce a robust GMP-grade cell conversion protocol that can be readily applied to a variety of different cell types, which were originally derived under xeno-containing conditions10. This provides significant opportunities for the development of future cell reprogramming experiments, which require clinical-grade conditions to maintain human therapeutic relevance. 
These methodologies provide a foundation upon which current safe-harbor criteria may be expanded to include characterized STEMCCA reprogrammed hiPSC lines that maintain a normal gene expression profile after STEMCCA excision from the integrated intron. Also, full conversion into clinical-grade conditions, free from non-human animal antigens, will help to incorporate many more cell types, which have previously been reprogrammed and characterized only in xeno-containing conditions. These methodologies combined, are persuasive grounds for the US Food and Drug Administration (FDA) to consider expanding their limited approval from human embryonic stem cell (ESC)-based therapeutics to hiPSC-based therapeutics11. 
We recently detailed the derivation of a factor-free hiPSC line that was fully characterized and converted into putative clinical-grade conditions10. Here, we detail the protocol for hiPSC derivation by utilizing the STEMCCA lentivirus. These stem cells then undergo an excision process followed by gene expression characterization. Finally, the hiPSCs are converted over into GMP-grade conditions by a slow conversion methodology.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Media and reagents&lt;/strong&gt;</td></tr><tr><td>DMEM/F12 (basal media)</td><td>Invitrogen (Carlsbad, CA, USA)</td><td>11330057</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Invitrogen</td><td>16000044</td><td></td></tr><tr><td>Minimum essential medium (MEM) non-essential amino acids (NEAA), 100x</td><td>Invitrogen</td><td>11140050</td><td></td></tr><tr><td>Glutamax, 100x</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>PenStrep, penicillin-streptomycin, 100x</td><td>Invitrogen</td><td>15140-122</td><td>Thaw at 4 &amp;deg;C, aliquot and store at &amp;minus;20 &amp;deg;C</td></tr><tr><td>Knockout serum replacement</td><td>Invitrogen</td><td>10828028</td><td>Thaw at 4 &amp;deg;C, aliquot and store at &amp;minus;20 &amp;deg;C</td></tr><tr><td>Trypsin/EDTA, 0.5%</td><td>Invitrogen</td><td>15400-054</td><td>Dilute stock out to 0.05% in 1x PBS</td></tr><tr><td>Basic fibroblast growth factor</td><td>GlobalStem (Rockville, MD, USA)</td><td>GSR-2001</td><td>Reconstitute to 10 &amp;micro;g/ml stock in 0.1% bovine serum albumin dissolved in 1x PBS and store at &amp;minus;80 &amp;deg;C</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Millipore (Billerica, MA, USA)</td><td>ES-007-E</td><td></td></tr><tr><td>Matrigel (basement membrane matrix)</td><td>BD Biosciences (San Jose, CA, USA)</td><td>356231</td><td>Dilute stock Matrigel vial with 10 ml of DMEM/F12 while on ice for a 1:2 dilution. Aliquot and store at &amp;minus;20 &amp;deg;C.</td></tr><tr><td>CELLstart (Synthetic Substrate)</td><td>Invitrogen</td><td>A1014201</td><td></td></tr><tr><td>Stemmolecule Y27632</td><td>Stemgent (Cambridge, MA, USA)</td><td>04-0012-02</td><td></td></tr><tr><td>Puromycin</td><td>Invitrogen</td><td>A1113802</td><td></td></tr><tr><td>LightCycler 480 Probes Master</td><td>Roche (Basel, Switzerland)</td><td>4707494001</td><td></td></tr><tr><td>ProFreeze-CDM Medium/freezing medium</td><td>Lonza (Basel, Switzerland)</td><td>12-769E</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich (St. Louis, MO, USA)</td><td>D8418</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14190-250</td><td></td></tr><tr><td>100 BP DNA Ladder</td><td>Invitrogen</td><td>15628019</td><td></td></tr><tr><td>SYBR Safe DNA Gel Stain 10,000x</td><td>Invitrogen</td><td>S33102</td><td></td></tr><tr><td>Agarose</td><td>Bio-Rad Laboratories, Inc. (Hercules, CA, USA)</td><td>161-3101</td><td></td></tr><tr><td>Gelatin, from porcine skin</td><td>Sigma-Aldrich</td><td>G1890-100G</td><td>Make stock at 0.2% in PBS, autoclave and store at room temperature.</td></tr><tr><td>mTeSR1</td><td>StemCell Technologies (Vancouver, BC, Canada)</td><td>5850</td><td>Combine Supplement 5x with the basic medium, aliquot and store at 4 &amp;deg;C for up to 2 weeks.</td></tr><tr><td>Stemedia NutriStem XF/FF Culture Medium</td><td>Stemgent</td><td>05-100-1A</td><td>Thaw at 4 &amp;deg;C O/N, aliquot and store at 4 &amp;deg;C for up to 2 weeks.</td></tr><tr><td>Primocin</td><td>InvivoGen (San Diego, CA, USA)</td><td>ant-pm-1</td><td></td></tr><tr><td>Accutase (Dissociation Reagent)</td><td>Invitrogen</td><td>A1110501</td><td></td></tr><tr><td>Donkey anti-Chicken IgG AlexaFluor 488</td><td>Jackson ImmunoResearch (West Grove, PA, USA)</td><td>703-546-155</td><td></td></tr><tr><td>Polybrene/transfection agent</td><td>Millipore</td><td>TR-1003-G</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Plasticware&lt;/strong&gt;</td></tr><tr><td>12-well plates</td><td>VWR (West Chester, PA, USA)</td><td>29442-038</td><td></td></tr><tr><td>6-well plates</td><td>VWR</td><td>29442-042</td><td></td></tr><tr><td>10-cm plates</td><td>Sigma-Aldrich</td><td>Z688819</td><td></td></tr><tr><td>18-gauge needle</td><td>Fisher Scientific (Pittsburgh, PA, USA)</td><td>148265D</td><td></td></tr><tr><td>21-gauge needle</td><td>Fisher Scientific</td><td>14-829-10D</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>BD LSRII Flow Cytometer</td><td>KSystem by Nikon (Tokyo, Japan)</td><td></td><td></td></tr><tr><td>BD FACSDiva Version 6.1.3 Software</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Kits&lt;/strong&gt;</td></tr><tr><td>PureLink Genomic DNA Mini Kit</td><td>Invitrogen</td><td>K182000</td><td></td></tr><tr><td>KAPA HiFi Hotstart ReadyMix PCR Kit</td><td>KAPA Biosystems (Wilmington, MA, USA)</td><td>KK2601</td><td></td></tr><tr><td>High Pure RNA Isolation Kit</td><td>Roche</td><td>11828665001</td><td></td></tr><tr><td>Transcriptor First Strand cDNA Synthesis Kit</td><td>Roche</td><td>4379012001</td><td></td></tr><tr><td>Sialix anti-Neu5Gc Basic Pack Kit</td><td>Sialix (Newton, MA, USA)</td><td>Basic Pack</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Media&lt;/strong&gt;</td></tr><tr><td>Combined media 1</td><td>StemCell Technologies and Stemgent</td><td></td><td>Consists of equal parts mTeSR1 and Nutristem</td></tr><tr><td>Combined media 2</td><td>StemCell Technologies and Stemgent</td><td></td><td>Consists of equal parts TeSR2 and Nutristem</td></tr><tr><td>HUF Media</td><td></td><td></td><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium/F12 [DMEM/F12] supplemented with 10% fetal bovine serum, 1x non-essential amino acids, 1x Glutamax, and 1x Primocin</td></tr><tr><td>Human Pluripotent Stem Cell Media</td><td></td><td></td><td>DMEM/F12 supplemented with 20% knockout serum replacement, 1x Glutamax, 1x non-essential amino acids, 1x Primocin, 1x &amp;beta;-mercaptoethanol, and 10 ng/ml basic fibroblast growth factor.</td></tr><tr><td colspan="4">DMEM/F12, Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium/F12; PBS, phosphate-buffered saline.</td></tr></tbody></table>

derivation and characterization of a transgene-free human induced pluripotent stem cell line and conversion into defined clinical-grade conditions

유도 인간 다 능성 줄기 세포 (hiPSCs)는 렌티 기반 재 프로그래밍 방법론으로 생성 될 수있다. 그러나, 게놈의 적극적 전사 영역에 남아있는 잠재적 발암 유전자의 흔적은 인간의 치료 응용 프로그램 (1)에 사용하기 위해 자신의 잠재력을 제한 할 수 있습니다. 또한, 치료 학적으로 중요한 유도체 줄기 세포 분화 또는 재 프로그래밍 유래의 비 - 인간 항원 인간 임상이 문맥에서 사용되는 이러한 hiPSCs는 배제. 이 비디오에서는, 우리는 재 프로그래밍 및 외래 유전자의 무료 요인이없는 hiPSCs를 분석하는 절차를 제시한다. 이러한 hiPSCs이어서 렌티 바이러스를 포함하는 특정 인트론 유전자 발현의 이상에 대해 분석 할 수있다. 이러한 분석은 이전에 유전자 발현의 차이 (3)를 검출하는 데 덜 민감한 기술 위에 이점을 갖는다 민감한 정량적 폴리머 라제 연쇄 반응 (PCR)을 이용하여 수행 될 수있다. 로 전체 변환임상 등급의 우수 제조 기준 (GMP) 조건, 인간의 임상 적 관련성을 할 수 있습니다. 우리의 프로토콜은 제공하는 또 다른 방법이 제공하는 현재의 안전 항구 기준이 확장 인간의 치료 응용 프로그램-을 위해 만기가 인간이 아닌 항원에 어떤 면역 원성의 위험을 제거해야한다 GMP 수준의 hiPSCs를 유도하는 요인이없는 특성화 된 hiPSC 기반의 파생 상품을 포함 할 것이다. 이 프로토콜은 모든 유형의 세포로 재 프로그램 렌티 폭넓게 적용되며, GMP 수준의 조건으로 세포를 재 프로그램을 변환하기위한 재생 가능한 방법을 제공한다.

우리 STEMCCA의 렌티 기반 리 프로그래밍 방법을 사용하여 임상 등급 인자없는 hiPSCs를 유도 프로토콜을 제안한다.도 1a는 MEFs에의 층에 STEMCCA 방식으로 재 프로그래밍 후, 세 가지 사전 절개 hiPSC 라인의 대표 사진을 보여준다. STEMCCA 재 프로그래밍 접근 방법의 주요 장점은 다른 연구 그룹 및 위치에, 여러 과학자들에 의해 달성 일관된 재 프로그램의 성공에있다. 그림 1B는 ?...

Watch this Scientific Journal Video about Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions at JoVE.com

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

의 유도 및 특성 형질 전환 유전자가없는 인간 유도 다 능성 정의 임상 수준의 조건으로 세포주 및 변환 줄기

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

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7.8K 조회수.  University of California, Los Angeles (UCLA). We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions. 

비디오: Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

피더가없는 조건에 센다이 바이러스를 사용하여 인간 말초 T 세포로부터 유도 된 다 능성 줄기 세포의 생성

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

연구

발생학

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

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

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

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

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

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

기본 양수 및 Pluripotency 이종 무료 조건에서 막 셀 프로그래밍

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Laminin 521 매트릭스를 이용한 인간 유래 다 능성 줄기 세포의 통합 된 자유 유도

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

피더 및 제노 프리 정의 상태에서 인간 다능성 줄기 세포에서 혈생 내피 분화

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

성인 인간 섬유 아세포의 유도 및 확장 신경 전구 세포에 자신의 직접 변환

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

신경과학

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 ...

생물학

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 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, ...