Name: establishment of proliferative tetraploid cells from nontransformed human fibroblasts
Uploaded: 2017-01-08T14:00:00.000+00:00
Duration: 10 min 4 s
Description: Watch this Scientific Journal Video about Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts at JoVE.com

We thank Mrs. Matsumoto for the technical assistance.

1. 세포 배양 <ol><li> tetraploidy을 유도하는 세포를 얻습니다. 최신으로이 기술은 인간 섬유 아세포 세포주 TIG-1, BJ, IMR-90 및 텔로 머라 - 불멸화 TIG-1 (TIG-HT)에 적용 할 수있는 것이 확인되었다. </li><li> α 변형 혹은 10 % (v / v)의 5 %에서 배양함으로써 열 - 불 활성화 소 태아 혈청 (FBS) (V / 보충 공부 될 세포 유형에 적합한 임의의 다른 세포 배양액 최소 필수 배지에서 세포 성장 V) CO 37 ° C에서 2 분위기. 통로 세포는 subconfluent 밀도를 초과마다 3 사일 없습니다. 주 : 인구 배증 레벨 (PDL)는 세포 성장 및 세포 연령 평가에서 계대마다 계산되어야한다. PDL은 접시 (N1) 및 다음 식 (X 초기 PDL이다)를 사용하여 다음 계대 (N2)에서 수확 한 세포 수로 접종 세포 수로부터 계산 될 수있다. PDL은 = X + (N1 로그 - N2 로그) / 2 로그 </li></ol> 2. 유도 및 사령관이배체 섬유 아세포에서 배체 세포의 blishment <ol><li> 흔들림 오프없이 tetraploidy 유도한다. 참고 : 일부 세포주 (예 : TIG-1)은 다음과 같이 데메 콜신 (DC)로 거의 완전히 배체 연속 처리에 의한 될 수 있습니다. <ol><li> 치료 전날에 하나 또는 두 개의 100mm 접시에 통로 세포는 tetraploidy 유도합니다. 일반적으로 배체 세포의 유도를 위해 접시 당 5 × 10 5 6 10 × 1 세포를 준비합니다. </li><li> 4 일 동안 0.1 μg의 / ㎖의 DC를 포함하는 매체가 기하 급수적으로 증가 세포 치료. </li><li> 마약없는 매체와 매체를 DC가 함유 교체하고 37 ° C에서 (v / v)의 CO 2 분위기 5 %에서 세포를 배양한다. 세포는 통상 1 주일 이내에 증식을 다시 시작합니다. </li></ol></li><li> 흔들림 오프로 tetraploidy 유도한다. 주 : 대부분의 세포는 DC와 간단한 처리의 결과로서 이배체 및 사배체 집단의 혼합물이 상기 (2.1)에 설명된다. 는 P의 따라서 일부 수정rocedure (그림 1)을 다음과 같이 이배체 인구가 필요합니다 제거합니다. <ol><li> 여러 T75에 통로 세포는 tetraploidy을 유도하는 치료 전날 플라스크. 일반적 배체 세포를 유도하기위한 적어도 5 × 106 세포 (플라스크 당 1 × 106 세포)를 준비한다. </li><li> 유사 분열에서 셀을 체포 18 시간 - 16 0.1 μg의 / ㎖의 DC를 포함하는 매체가 기하 급수적으로 증가 세포 치료. 유사 분열에서 세포를 저지하기 위해 필요한 시간은 표적 세포에 의존 할 수 있고, 가능한 한 많은 유사 분열 세포를 얻기 위해 예비 실험에 의해 결정되어야한다. 예를 들어, 느린 성장 세포는 빠르게 성장하는 세포보다 긴 시간 동안 DC로 처리해야합니다. 주 : 셀이 너무 길면,이 단계에서의 치료를하는 경우, 그들은 유사 분열 슬립을 겪을 수 있으며 불가능 다음 단계에서 흔들림 차단하여 세포 분열을 수집하게 식기 표면에 부착. </li><li> 어에서 흔들림 오프 법에 의해 DC-체포 유사 분열 세포를 수집ICH 느슨하게 부착 유사 분열 세포는 원심 분리 플라스크 중간 세척 부드러운 진탕 (300 XG, 5 분)에 의해 수집된다. 이 단계에서 적절한 방법으로 세포 수를 계산합니다. </li><li> 60mm 배양 접시에 수집 유사 분열 세포를 다시 시드 및 추가 3 일간 0.1 μg의 / ㎖의 DC와 세포를 치료. 세포의 10 % 미만이 처리를 견딜 수 있으므로, 접시 당 이상 1 × 10 6 세포 유사 분열을 시드. </li><li> 마약없는 매체와 매체를 포함-DC를 교체하고 세포는 통상 1 주일 이내에 발생 확산을 재개 할 때까지 기다립니다. </li></ol></li><li> 마찬가지로 원래 이배체 세포 통로 세포. </li></ol> DNA의 배수성 3. 시험 참고 :이 잘 설립 절차 및 기술 설명서는 자세한 절차 및 기술 도움말을 참조해야합니다. <ol><li> 수확 세포 (5 × 10 5 - 1 × 6) 솔루션 콘과 배양 세포에 의한37 ° C에서 10 분 동안 0.05 % 트립신 및 0.02 % EDTA를 이닝, 및 10 % FBS를 함유하는 배지에서 트립신을 중화. </li><li> 매체에 원심 분리기 세포와 원심 분리 (300 XG, 5 분) 다음 인산염 완충 생리 식염수 5 ㎖ (PBS)에 재현 탁하여 그들을 씻는다. </li><li> 다음 두 가지 방법 중 하나에 의해 DNA 분석을 위해 세포를 준비 <ol><li> 고정되지 않은 셀 (10)을 분석하기위한 방법 주 :이 방법은 세포주기 분석을 위해 상업적으로 이용 가능한 키트를 사용하여 수행 될 수있다. 세포가 절차에 고정 핵이 시간이 지남에 따라 손상되어 염색되지 않기 때문에이 방법으로 제조 된 샘플은 즉시 분석되어야한다. <ol><li> 250 MM의 자당을 함유하는 40 mM의 시트 레이트 버퍼와 세포를 씻으십시오. 이어서 실온에서 10 분 동안 250 mM의 자당을 함유하는 200 μL의 40 mM의 시트르산 완충액, 0.1 % (V / V)의 Nonidet P40, 30 μg의 / ㎖ 트립신 250 μL로 셀을 처리한다. </li><li> 0.5 μg의 / ㎖의 트립신 INH의 200 μL를 추가실온에서 10 분 동안 ibitor 250 mM의 수크로오스를 포함하는 40 mM의 시트르산 완충액 0.1 μg의 / ㎖의 RNase A, 및 세포 배양한다. </li><li> 0.4 mg을 200 μL를 추가 / 250 mM의 자당을 함유하는 40mm의 시트르산 완충액 ㎖의 프로피 디움 요오다 이드 (PI), 및 세포 핵을 염색을 실온에서 10 분 동안 세포를 배양한다. </li></ol></li><li> 고정 된 세포를 분석하기위한 방법 <ol><li> 세포 현탁액을 혼합하고 실온에서 30 분 동안을 남기고 100 % EtOH로 적가 4㎖를 첨가, PBS 1 ㎖로 세포를 현탁하여 80 % 에탄올로 세포를 고정한다. 이 정착액의 셀은 여러 주 동안 -20 ℃에서 저장 될 수있다. </li><li> PBS 5ml에 재현 탁하여이를 세포 씻어 원심 분리 (300 × g으로 5 분) 하였다. 0.5 ㎎ / ㎖의 RNase A의 세포를 취급를 50㎍ / ㎖의 프로피 디움 요오다 이드 (PI) (0.5 ㎎ / ㎖의 RNase (A)의 475 μL 1 ㎎을 25 μL / ㎖ PI)을 실온에서 30 분 동안로 얼룩. </li></ol></li></ol></li><li> t의 DNA 함량을 분석그는 488 nm의 적색 레이저 발광 채널 필터로 유동 세포 계측기를 사용하여 세포 (긴&gt; 내지 610 단계). 표적 세포의 배수성을 평가하는 동시에 정품 이배체 세포로부터 제조 된 기준 샘플을 분석한다. <ol><li> 대안 적으로, 이배체 세포 대 비드의 형광 강도의 비율을 평가하기 위해 표준 형광 비드를 추가한다. 사이토 단일 배체 세포와 이배체 세포 (11)의 덩어리를 구별하는 흐름의 옵션 &#39;펄스 폭 대 펄스 높이&#39;를 사용합니다. </li></ol></li></ol> 염색체 수와 핵형 분석 4. 시험 참고 :이 모든 잘 확립 된 절차이며, 기술 매뉴얼 또는 제조사의 프로토콜 세부 절차 및 기술 도움말을 참조해야합니다. <ol><li> 중기 세포를 체포하기 위해 4 시간 동안 0.1 μg의 / ㎖의 DC와 함께 기하 급수적으로 증가 세포 치료. </li><li> 염색체 슬라이드를 준비합니다. <ol><l난&gt; 트립신 처리하여 수확 세포 (적어도 5 × 10 5 세포)와 FBS를 포함하는 배지에서 일시 중지합니다. </li><li> 배지에서 세포 원심 분리 (300 × g으로 5 분), 10 분 동안 37 ° C에서 저장성 용액 (0.075 M KCl을 5 mL)로 처리한다. </li><li> 수정하고 Carnoy의 정착액 세포를 정지 (메탄올 : 아세트산 = 3 : 1). </li><li> 세포 현탁액의 - (35 μL 25) 작은 볼륨을 드롭하여 유리 슬라이드에 세포를 확산. 이러한 뜨거운 증기에 노출되는 등의 추가 단계가 확산 염색체를 개선해야 할 수도 있습니다. </li></ol></li><li> 현장 하이브리드의 염색체 개수 또는 핵형 분석 또는 여러 가지 빛깔의 형광에 대한 얼룩 세포 (mFISH). <ol><li> 염색체 수가. <ol><li> 20 분 - 5 % 김사 솔루션 (또는 형광 현미경 0.5 ㎎ / ㎖의 RNase A를 포함하는 5 μg의 / ML의 PI) 15와 얼룩 세포. </li><li> 디지털 적어도 50 중기 세포를 사진. </li><li> 이미지 분석을 이용 softwa 셀당 염색체 수를 계산터치 및 사진 편집 소프트웨어를 사용하여 염색체 중복 설명서를 분리 한 후 다시. 염색체 번호를 수동으로 득점 할 수 있지만, 컴퓨터 채점하는 것이 좋습니다. </li></ol></li><li> 핵형 분석 참고 :이 분석은 숙련 된 기술자 또는 전문 회사가 수행해야합니다. <ol><li> 표준 G 밴딩 기술 (12)에 따라 얼룩 세포. </li><li> 표준 karyograms (12)를 만들기 위해 염색체를 분석합니다. </li></ol></li><li> mFISH 참고 :이 단계는 선택 사항이며 필요한 경우 수행해야합니다. <ol><li> 제조 업체의 프로토콜 (13)에 따라 인간의 염색체에 대한 여러 가지 빛깔의 물고기 프로브를 사용하여 얼룩 세포. </li><li> 적절한 필터를 사용하여 형광 현미경 분석 mFISH (13)를위한 소프트웨어를 사용하여 염색체 분석. </li></ol></li></ol></li></ol>

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Oncol. 3, 198 (2013).</li><li>Ohshima, S., Seyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+proliferative+tetraploid+cells+from+telomerase-immortalized+normal+human+fibroblasts.">Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts.</a> Genes, Chromosome Cancer. 55 (6), 522-530 (2016).</li><li>Di Leonardo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+rereplication+in+the+presence+of+mitotic+spindle+inhibitors+in+human+and+mouse+fibroblasts+lacking+either+p53+or+pRb+function.">DNA rereplication in the presence of mitotic spindle inhibitors in human and mouse fibroblasts lacking either p53 or pRb function.</a> Cancer Res. 57, 1013-1019 (1997).</li><li>Andreassen, P., Lohez, O., Lacroix, F., Margolis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraploid+state+induces+p53-dependent+arrest+of+nontransformed+mammalian+cells+in+G1.">Tetraploid state induces p53-dependent arrest of nontransformed mammalian cells in G1.</a> Mol. Biol. Cell. 12, 1315-1328 (2001).</li><li>Vogel, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+of+the+mitotic+spindle+assembly+checkpoint+with+p53+to+prevent+polyploidy.">Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy.</a> Oncogene. 23, 6845-6853 (2004).</li><li>Aylon, Y., Oren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53:+Guardian+of+ploidy.">p53: Guardian of ploidy.</a> Mol. Oncol. 5 (4), 315-323 (2011).</li><li>Uetake, Y., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+progression+after+cleavage+failure+:+mammalian+somatic+cells+do+not+possess+a+&amp;#34;tetraploidy+checkpoint&amp;#34.">Cell cycle progression after cleavage failure : mammalian somatic cells do not possess a &amp;#34;tetraploidy checkpoint&amp;#34.</a> J. Cell Biol. 165, 609-615 (2004).</li><li>Ganem, N., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+the+proliferation+of+polyploid+cells.">Limiting the proliferation of polyploid cells.</a> Cell. 131, 437-440 (2007).</li><li>Ho, C., Hau, P., Marxer, M., Poon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+of+p53+for+maintaining+chromosomal+stability+during+tetraploidization.">The requirement of p53 for maintaining chromosomal stability during tetraploidization.</a> Oncotarget. 1 (7), 583-595 (2010).</li></ol>

저자는 더 경쟁 재정적 관심이 없음을 선언합니다.

화학 약품에 의한 이배체 세포에서 tetraploidy의 유도에 큰 문제 중 하나 세포질 분열 억제제 또는 스핀들 억제제에 의해 세포가 종종 배체 및 배체 집단의 혼합물이 될 것입니다, 그리고 배체 세포는 이배체 세포로부터 분리해야합니다. 이배체 세포의 무료 배체 인구의 절연을위한 가장 일반적인 방법은 희석을 제한하여 FACS 또는 복제를 사용합니다. 그러나 이러한 과정은 힘들고 수행하기 쉽지 않...

배수성은 포유 동물 종의 전문 조직에서뿐만 아니라, 암과 같은 퇴행성 질환의 병리 적 상태의 다양한뿐만 관찰되었다. 배수체 (주로 배체) 세포는 종종 경부 3,4의 바렛 식도 2 또는 편평 상피내 병변 인간 조직의 발암 병변 관찰하고, 그 조직을 5 이수성 악성 세포의 근원으로 간주되어왔다 6. 이 이수성 배체 세포로의 전환이 종양 형성의 초기 단계에서 중요한 이벤트가 될 수 있다고 제시되어 있지만,이 과정에 관여하는 메커니즘은 완전히 이해되지 않는다. 이 비 형질 배수체 인간의 세포를 전파 할 수있는 더 체외 모델을 사용할 수 없었다 부분적으로 있기 때문이다. 일부 연구자들은 INH로 이핵 세포의 생성을 통해 비 형질 인간의 상피 세포에서 tetraploidy을 유도 한세포질 분열 7-9 ibiting. 그러나이 방법 불필요한 이배체 세포 -7,8- 형광 - 활성화 세포 분류 (FACS)에 의해 제거해야 또는 희석 9를 제한하여 클로닝. 이러한 절차를 수행하기 쉬운 힘들고 없기 때문에 간단한 방법은 비 형질 배체 세포가이 분야의 연구를 위해 요구되는 설정합니다. 본 보고서에서, 우리는 비교적 간단한 절차에 의해 정상적인 인간 섬유 아세포 또는 텔로 머라 아제 - 불후의 인간 섬유 아세포의 증식 배체 세포를 확립하기위한 프로토콜을 설명합니다. 절차는 또한 DC로 처리 떨고에 의해 수집 된 유사 분열에서 이배체 세포 및 유사 분열 세포를 체포 스핀들 독 데메 콜신 (DC)를 사용합니다. 오랜 시간 동안 DC 처리 이배체 유사 분열 세포는 G1 배체 세포로 변환하고, 이들 세포는 약물의 제거 다음의 며칠 동안 성장 정지 후 같은 배체 세포 증식. 이 프로토콜은 제공염색체 불안정성 배수체 인간 종양 세포의 전위 사이의 관계를 연구하는 유용한 모델을 생성하는 효과적인 방법.

<table><tbody><tr><td>MEM-&amp;alpha;</td><td>Sigma-Aldrich</td><td>M8042-500ML</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>172012-500ML</td><td></td></tr><tr><td>Demecolcine solution (10 &amp;mu;g/mL in HBSS)</td><td>Sigma-Aldrich</td><td>D1925-10ML</td><td></td></tr><tr><td>BD CycleTES Plus DNA Reagent Kits</td><td>BD Biosciences</td><td>#340242</td><td>For examination of DNA ploidy by flow cytometry</td></tr><tr><td>Human chromosome multicolor FISH probe 24XCyte</td><td>MetaSystems</td><td>#D-0125-060-DI</td><td>Specialized filter set and software for mFISH analysis are necessary</td></tr><tr><td>Isis imaging system with mFISH&amp;nbsp; software&amp;nbsp;</td><td>MetaSystems</td><td></td><td>Specialized probe kit is necessary</td></tr></tbody></table>

establishment of proliferative tetraploid cells from nontransformed human fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

우리의 경험에 의하면, TIG-1 세포 4 일 0.1 μg의 / mL의 DC (그림 2A)와 간단한 연속 처리에 의해 거의 완전히 배체 할 수있다. 대조적으로, 예 BJ 또는 IMR-90 및 TIG-HT 세포와 같은 다른 섬유 아세포 균주, 흔들림 오프 법에 의해 이배체 동일한 처리 다음 배체 집단, 및 유사 분열 세포의 분리 혼합물되었다이 과정에서 필요한 (- 치료 시작 후 18 시간 일반적으로 16) <stron...

Watch this Scientific Journal Video about Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts at JoVE.com

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

비 형질 인간 섬유 아세포의 증식 배체 세포의 수립

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

establishment-proliferative-tetraploid-cells-from-nontransformed

Universidad de Cartagena UDEC

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Developmental Biology

7.2K 조회수.  Saitama Medical University. Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

비디오: Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

인간 Mammary 섬유아 세포에서 암종 - 관련 섬유아 세포의 실험 세대 (CAFs)

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

연구

의학

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) ...

발생학

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

인간의 기본 섬유 아 세포의 유도 만능 줄기 세포로 재프로그래밍 RNA 기반

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

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 ...

생물학

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

전사 요인의 강제 표현에 의한 인간 섬유아세포의 혈생 재프로그래밍

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

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

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 ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

비통합 방법을 사용하여 복부 피부에서 성인 인간 진피 섬유 아세포의 분리 및 유도 만능 줄기 세포의 생성

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

생체공학

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

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

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-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 ...