Name: generation of integration-free human induced pluripotent stem cells using hair-derived keratinocytes
Uploaded: 2015-08-20T13:00:00.000+00:00
Duration: 8 min 36 s
Description: Watch this Scientific Journal Video about Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes at JoVE.com

The authors wish to thank Harene Ranjithakumaran and Stacey Jackson for technical support. This work was supported in part by grants from the National Health and Medical Research Council (R.C.B. Wong, A. P&#233;bay), the University of Melbourne (R.C.B. Wong), Retina Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay) and the Ophthalmic Research Institute of Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay); Australian Research Council Future Fellowship (A. P&#233;bay, FT140100047), Cranbourne Foundation Fellowship (R.C.B. Wong); intramural funding from the National Institutes for Health (R.C.B. Wong, S.S.C. Hung) and operational infrastructure support from the Victorian Government.

개인의 인간의 머리카락 샘플의 수집은 호스트 기관에서 인간의 연구 윤리위원회 윤리의 승인을 필요로하며, 제도적 지침에 따라 수행해야합니다. 뽑아 머리에서 각질 1. 분리 <ol><li> 얼음 O / N에 (즉, 마트 리겔) 해동 세포 외 기질 (ECM) 솔루션입니다. </li><li> 사전 냉장 피펫 팁을 사용하여, 냉장 DMEM / F12 배지 12 ml를 200 μL ECM 솔루션을 추가합니다. 코트의 각 웰에 희석 된 ECM 용액 1 ㎖와 함께 12 웰 플레이트. 37 ° C에서 코팅 된 플레이트 O / N을 품어. 사용하기 전에 미리 예열 DMEM / F12 배지로 코팅 된 판을 씻으십시오. </li><li> 머리카락의 뿌리 부근에 손가락을 배치 한 빠르고 부드러운 동작으로 머리를 따 버릴에 의해 개인의 머리에서 차 인간의 머리카락을 수집합니다. 각 수집 머리를 그대로 모낭이 포함되어 있는지 확인하고 헤어 유엔의 성장 단계를 결정하기 위해 확인해부 현미경 (그림 1) 데르. <ol><li> 각질 세포를 추출하는 각 개인으로부터 5 ~ 10 성장기의 머리카락을 수집합니다. 프로세스는 각질 세포의 성공적인 유도를 보장하기 위해 가능한 한 빨리 털을 뽑아. 참고도 1은 성장주기의 다른 단계에서의 모발 형태를 도시한다. 성장기 모발 ORS 알려진 모발의 길이를 둘러싸는 상피의 가시 외층과, 성장 단계에있다. 한편, 휴지기 모발 휴식 단계에서 머리를 나타내고 가시 ORS이 없지만 휴지기 융기 알려진 모발의 뿌리를 덮는 셀 명확한 공을 갖는다. </li></ol></li><li> 실온에서 5 분간 씻어 항생제 믹스 10 ㎖를 포함하는 페트리 접시에 머리 샘플을 놓습니다. </li><li> 머리 샤프트​​의 1cm - 멸균 가위와 집게를 사용하여, 그대로 모낭 주위에 0.5으로 머리 조각을 떠나, 과도한 헤어 샤프트를 잘라. </li><li> 조심스럽게 TRA겸자를 이용하여 ECM 코팅 플레이트의 각 웰에 1 nsfer 머리 단편. </li><li> 모발 샘플 - (200 μL ~ 100) 1 mL를 피펫을 사용하여, 녹아웃 세럼 교체 (KSR) 매체의 약 3 방울을 추가합니다. 주 : 부착 및 후속 각질 생장을 허용하도록 플레이트 표면에 밀착 머리 단편 유지. 이 단계에서 과도한 미디어를 추가 할 수있다 머리 조각이 떠있는 원인과 성공적인 헤어 첨부 파일의 기회를 감소시킨다. </li><li> 5 % CO 2와 37 ° C 배양기에서 머리카락 샘플을 품어과 모낭은 / N을 O를 첨부 할 수 있습니다. 촉촉한 모발 샘플을 유지하기 위해 - (200 μL ~ 100) 다음 날, 부드럽게 KSR 매체의 또 다른 3 방울을 추가합니다. </li><li> 모낭이 성공적으로 연결되었는지 확인하기 위해 매일 머리 샘플을 관찰한다. 일반적으로 모낭의 부착 1-3 일 이내에 알 수있다. 잔털이 높은 가지고 두꺼운 머리카락, 잔털에 비해 부착 일반적으로 쉽게경향은 첨부 파일 프로세스를 방해하는 떠. <ol><li> 머리가 코팅 된 플레이트에 부착 한 후, 웰에 KSR 매체의 500 μl를 추가합니다. 머리 샘플이 단단히 연결되지 않았을 수 있습니다으로 매체를 추가 할 때 좀 더주의를 기울여. 이 시점에서, 2 일마다 미디어를 변경합니다. 참고 : 케 라티노 사이트의 outgrowths는 3-7일 (그림 2A-B) 후 머리 조각의 ORS 지역에서 볼 수 있어야합니다. 각질은 다음 섹션에 설명 된대로 세포를 계대 14 일 전으로 성장하도록 허용합니다. </li></ol></li></ol> 2. 유지 관리 및 각질과 Passaging <ol><li> 사전 코트 코팅 매트릭스 키트를 사용하여 6 웰 플레이트. 코팅 매트릭스 솔루션의 6.8 μL 뒤에 아니라 각 키트에서 희석 매체의 680 μl를 추가합니다. 플레이트에 도포 매트릭스 용액의 균일 한 분포를 보장하기 위해 플레이트를 흔들어. </li><li> 코팅 된 플레이트는 실온에서 30 분 동안 품어하도록 허용합니다. 제거각질 세포의 도금하기 전에 코팅 매트릭스 솔루션입니다. </li><li> 37 ℃에서 2-5 분 동안 웰 당 0.25 % 트립신 500 μL와 단일 세포 현탁액 내로 해리 각질. 매체를 포함하는 혈청 (예, 우 태아 혈청 (FBS) 배지) 500 μL와 트립신을 비활성화하고 멸균 15 ㎖의 튜브에 수집한다. 멸균 집게를 사용하여 머리 조각을 제거합니다. </li><li> 혈구를 사용하여 수확 세포의 수를 결정합니다. </li><li> 200 X g에서 3 분 동안 세포를 원심 분리기. PBS를 반복 원심 분리와 세포를 씻으십시오. PBS를 대기음 신선한 각화 세포 배지에서 세포 펠렛을 재현 탁. </li><li> 각질 세포 배지의 존재 ECM 코팅 플레이트의 한 웰 플레이트에 4 × 10 아래 4 각화 세포. 5 % CO 2 (그림 2C)와 37 ° C 배양기에서 각질 세포를 유지합니다. 문화가있는 경우 ~ 용지를 80 % 합류를 매일 통과 세포를 변경, WHI채널은 각질 세포의 통과 번호와 확산 속도에 따라 약 2-7일 걸릴 수 있습니다. 유래 각질 세포는 표준 느린 냉동 냉동 보존 방법 (16)를 사용하여 충분한 냉동 주식을 생성하기 위해 확장되어야한다. 참고 : 설명 배양 조건은 전형적인 조약돌 형태 (그림 2C)와 각질 형성 세포의 균일 한 인구의 확장을 지원합니다. 때때로, 몇 가지 차별화 된 대형 평면 형태와 각질 세포 및 복제 성 잠재력을 잃었다는 문화에서 관찰 할 수있다. 각화 세포의 특성화 cytokeratin 14 대해 항체를 이용한 면역 염색에 의해 수행 될 수는 앞서 ​​설명한 17. 설립 각질 세포주는 또한 시판 키트를 사용하여 1-3 통로 이후 마이코 플라즈마에 대해 시험한다. </li></ol> 에피 솜 벡터를 사용하여 각질 형성 세포에서 hiPSCs의 3 세대 <ol><li> 6 웰 플레이트와 사전 코트0.1 % 젤라틴 (2 ㎖ / 웰). RT에서 적어도 20 분 동안 코트에 젤라틴을 허용. </li><li> 리 프로그래밍 실험, 사용 각질 세포는 더 이상 통과 6.보다 해리는 37 ° C에서 2-5 분 동안 잘 당 0.25 % 트립신 1 ml의 단일 세포 현탁액에 각화 없습니다. 혈청 함유 매체 (예를 들어, FBS 배지)의 1 ml의 트립신 불활 및 15 ㎖의 튜브에 수집한다. </li><li> 각질 세포 배지에서 200 XG와 재현 탁 세포에서 3 분 동안 원심 분리기 세포. 젤라틴 화 판 O / N 잘 플레이트 당 1 × 10 5 -1.5 × 10 5 각질. </li><li> 다음 날 (0 일)에서 세포 50-60 %의 합류 있는지 확인하기 위해 판을 확인합니다. 참고 : 각질 세포가 ~ 50 %의 합류가 아니라 형질 전환 후 성장하지 않을 수보다 작은 경우. </li><li> 0 일에, 2 μg의 총 플라스미드 DNA에 8 ㎕를 형질 전환 시약의 비율을 이용하여 각질 세포에서 에피 좀 벡터의 재 프로그래밍 형질 전환을 수행한다.<ol><li> 4 플라스미드 (pCXLE-eGFP는, pCXLE-hOct3 / 4 shP53F, pCXLE - HSK, pCXLE - 훌)의 각각 0.5 μg의를 타고 감소 혈청 배지 (예를 들어, OPTI-MEM) 100 ㎕에 희석. 주 : 플라스미드 pCXLE-eGFP는 각질 세포는 형질에서 효율을 모니터링하기 위해 사용되며, 성공적 프로그래밍에 필요한 것은 아니다. </li><li> 희석 된 DNA와 형질 전환 시약의 8 μl를 추가합니다. 튜브를 쓸어 넘겨 섞는다. DNA 반응 혼합물을 실온에서 15 분 동안 휴식을 흐트러 허용. </li><li> 신선한 매체와 각질 세포 매체를 교체합니다. </li><li> 부드럽게 4 시간 동안 5 % CO 2, 37 ℃ 배양기에서 배양 각질 DNA 반응 혼합물을 추가 배양한다. 참고 : 그것은 그들이 낮은 생존 후 형질을 나타내는으로 각질 세포에 장기간 형질 전환 O / N을 수행하지 않는 것이 좋습니다. </li><li> 4 시간 포스트 형질 전환 한 후, 신선한 매체와 각질 세포 매체를 교체합니다. </li></ol></li><li> 1 일에 (1 일 이후 트랜스fection), 각질 세포 배지를 변경하고, 형광 현미경 하에서 eGFP는 양성 세포의 비율을 추정하여 형질 감염 효율을 확인한다. 전형적&gt; 60~70% 형질 효율이 프로토콜을 이용하여 일차 각질 세포에 대해 관찰된다. </li><li> 단계 3.5의 설명에 따라 2 일에서 형질를 반복합니다. 두 개의 연속 된 형질 감염은 일반적으로&gt; 90 %의 형질 전환 효율을 달성하는 것이다. 각질 세포 문화는 일반적으로 두 번째 형질의 말에 합류에 도달한다. <ol><li> 각 프로그래밍 반응에 마우스 배아 섬유 아세포 (MEF) 피더 10cm 접시를 준비합니다. 이전 18 설명 된대로 mitotically 불 활성화 MEF 피더를 준비합니다. </li><li> 프리 코트를 적어도 20 분 동안 0.1 % 젤라틴을 5 ml와 10cm 접시. FBS 배지에서 10cm 접시 당 플레이트 4 × 10 6 mitotically 비활성화 MEF. 세포가 정착 5 % CO 2와 37 ° C 배양기에서 O / N을 첨부 할 수 있습니다. </li></ol></li><li> 3 일에, HAR단계 3.2 및 3.3에 설명 된대로 0.25 % 트립신과 각질을 가득. KSR 배지에서 각질 세포를 재현 탁하고 KSR 배지 MEF 10cm 접시에 피더 6 웰 플레이트의 한 웰에서 각질 세포의 90 %를 접시. </li><li> 이후 4 일부터 매일 KSR 매체를 변경합니다. <ol><li> 또한 18 일 후, 모든 둘째 날 문화 매체를 교체합니다. 참고 : 인간 배아 줄기 세포 (인간 배아 줄기 세포)를 닮은 정의 된 경계와 hiPSC 식민지가 날 14에서 등장 반면 비 재 각질 세포가 증식 중단됩니다 - (21). </li></ol></li><li> 이후의 날 (21)로부터 수동 절개로 hiPSC 식민지를 분리합니다. 긴밀 클러스터 만 명확하게 다른 사람에서 분리 hiPSC 식민지를 선택하고 있습니다 hiPSC 식민지를 피하십시오. 참고 : hiPSC 식민지 이전 포착 할 수 있지만 정의 된 경계와 식민지 작은 식민지 크기 주어진 이전 시점에서 명확하지 않을 수 있습니다. 재 프로그래밍 효율은 DIFF에 따라 다를 수 있습니다erent 환자의 각질 세포 및 또한 이러한 통로 번호 또는 각질 세포의 증식 속도와 같은 다른 요인들에 의해 영향을 받는다. <ol><li> hiPSC 식민지의 수동 해부를 들어, 해부 현미경 hiPSC 식민지를 잘라 26G 바늘을 사용합니다. 길이 600 μm의 - (300)의 주위에 작은 조각으로 hiPSC 식민지를 잘라. hiPSCs의 클론 라인을 설정하는 새로운 MEF 공급 판 (2.5 × 10 4 MEF / ㎠)로 한 hiPSC 식민지에서 조각을 전송합니다. 처음에, 문화는 12 웰 플레이트 또는 기관 배양 요리 잘 하나에 각 hiPSC 클론 라인은 다음 6 웰 플레이트 형식으로 확장합니다. </li><li> MEF 피더에 KSR 매체에 hiPSCs의 설립 클론 라인을 유지한다. 문화 일단 70에 도달 - hiPSC 식민지와 80 %의 포화 상태 ~ 직경 1.5 mm, 제조업체의 지시에 따라 표준 효소 계대 방법 (Accutase, 스파 아제 (Dispase) 또는 콜라게나 IV)를 사용하여 통과 hiPSCs. </li></ol></li><li> 설립 hiPSC 라인을 특성화S는 만능과 시험 관내 및 / 또는 생체 내 13, 19, 21, 23의 3 배엽 세포의 대표로 분화 할 수있는 능력을 확인합니다. 또한, 시판 키트를 사용하여 마이코 플라스마 정기적으로 설립으로 테스트합니다 hiPSC 라인은 무균 있는지 확인합니다. 정기적으로 핵형 또는 어레이 기반 복제 수 변이 (CNV) 분석에 의한 hiPSC 라인의 게놈 안정성을 모니터링 할 수 있습니다. </li></ol>

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The authors have no conflict of interest.

환자 별 hiPSCs의 생성은 시험관 내에서 질환 세포 유형의 발병 기전 연구를위한 독특한 방법을 제공하고, 또한 질환의 표현형을 구출 할 신규 한 분자를 식별하는 약물 스크리닝에 대한 플랫폼을 제공한다. hiPSCs를 이용하여 질병이 모델링 접근법은 긴 QT 증후군, 헌팅 톤병, 파킨슨 질환 및 근 위축성 측삭 경화증 (22)을 포함하여 다양한 질환에 대한 유망한 결과를 수득했다. 여러 이?...

유도 된 인간 다 능성 줄기 세포 (hiPSCs)의 발견은 환자 맞춤형 줄기 세포의 생성을위한 1-3 가능한 방법을 제공하는 재생 의료 분야 혁명을 일으켰다. hiPSCs 성공적 4,5- 섬유 아세포, 조혈 세포 6,7, 소변 8에서 신장 상피 세포와 각화 9,10 포함하는 다양한 종류의 체세포에서 생성되었다. 현재까지, 피부 섬유 아세포 및 조혈 세포는 환자 별 iPSCs를 생성하기위한 가장 일반적으로 사용되는 셀의 소스를 나타낸다. 틀림없이, 이것은 피부 생검과 채혈 일상 의료 절차 및 여러 나라에서 확립 된 환자의 혈액 샘플의 피부 또는 대형 바이오 뱅크 있다는 사실에 기인한다. 혈액 세포 및 침윤성 추출 방법을 필요로 피부 섬유 아세포는 달리, 케 라티노 사이트는 hiPSC 생성에 쉽게 접근 세포 유형을 나타낸다. KeratinocytES는 피부의 외관 상피 장벽을 형성하고 또한 손톱 및 모발 (11)에서 발견되는 케라틴 풍부한 상피 세포이다. 특히 각질 세포가 모낭 내부 근초 (IRS) 셀 (12,도 1)와 함께 모발 덮여 셀룰러 외부 층의 외측 근초 (ORS)에서 찾을 수있다. 헤어 컬렉션 의료인의 도움을 필요로하지 않는 간단한 절차이므로 환자 수집 크게 hiPSC 생성을위한 환자 샘플의 수집을 용이하게 할 실험실에 자신의 머리 샘플을 보낼 수 있도록, 그 기회를 제공한다. 표피 각화 세포도 생성 hiPSC 9, 13의 시작으로 각질 형성 세포를 사용하는 장점에 추가하여 섬유 아세포에 비해 높은 효율 및 프로그래밍 빠른 프로그래밍 속도론있다. 또한, hiPSCs 또한 모낭 내의 다른 세포 집단을 사용하여 생성 될 수 있고,모낭 14,15 바닥에있는 모유두 세포를 포함. 헤어 유래 세포를 사용하여 IPSC 세대의 이전 보고서들은 레트로 바이러스 또는 렌티 바이러스 기반의 프로그래밍 방법 9,14,15을 사용합니다. 그러나 이러한 바이러스 성 방법은 재 프로그래밍시 외국 유전자의 바람직하지 않은 게놈 통합을 소개합니다. 비교하여, 에피 솜 벡터의 사용은 통합 프리 iPSCs 4를 생성 가능한, 비 바이러스 프로그래밍 방법을 나타낸다. 우리는 이전에 효과적으로 에피 솜 벡터 (13)를 사용 hiPSCs으로 각질 세포를 재 프로그램하는 간단하고 비용 효율적인 비 바이러스 성 방법을 개발 하였다. 여기에 우리가 뽑아 머리, 확장 및 각질 세포의 유지 보수 및 hiPSCs를 생성 이후의 재 프로그래밍에서 각질 세포의 유도를 포함하여 각질 세포에서 파생 된 hiPSCs,의 생성에 대한 자세한 프로토콜을 제공합니다.

<table><tbody><tr><td>Antibiotic Mix:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>250 ng/ml Antimycotic amphotericin B</td><td>Sigma</td><td>A2942-20ml</td><td>Antibiotic mix is made up in PBS.&amp;nbsp;</td></tr><tr><td>1X Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>PBS (-)</td><td>Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>Knockout Serum Replacement (KSR) medium:&amp;nbsp;</td><td></td><td></td><td>KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use.</td></tr><tr><td>20% knockout serum replacement (KSR)</td><td>Invitrogen</td><td>10828-028</td><td></td></tr><tr><td>DMEM/F12 with glutamax</td><td>Invitrogen</td><td>10565-042</td><td></td></tr><tr><td>1&amp;times; MEM non-essential amino acid</td><td>Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>&amp;nbsp;0.5&amp;times; Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>&amp;nbsp;0.1 mM &amp;beta;-mercaptoethanol</td><td>Invitrogen</td><td>21985</td><td></td></tr><tr><td>&amp;nbsp;bFGF (10 ng/ml, added fresh)</td><td>Millipore</td><td>GF003</td><td></td></tr><tr><td>Keratinocyte medium:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;EpiLife with 60&amp;nbsp;&amp;micro;M Calcium</td><td>Invitrogen</td><td>M-EPI-500-CA</td><td></td></tr><tr><td>1&amp;times; Human keratinocyte growth supplement (HKGS)</td><td>Invitrogen</td><td>S-001-5</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) medium:&amp;nbsp;</td><td></td><td></td><td>FBS medium is filtered using Stericup (Millipore, #SCGPU05RE) before use.</td></tr><tr><td>10% fetal bovine serum (FBS)</td><td>Invitrogen</td><td>26140079</td><td></td></tr><tr><td>DMEM&amp;nbsp;</td><td>Invitrogen</td><td>11995-073</td><td></td></tr><tr><td>0.5x Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>2 mM L-glutamine</td><td>Invitrogen</td><td>25030</td><td></td></tr><tr><td>0.25% trypsin-EDTA</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>Extracellular Matrix (ECM):</td><td></td><td></td><td></td></tr><tr><td>Matrigel</td><td>Corning&amp;nbsp;</td><td>354234</td><td>Aliquot Matrigel stock and store in -80&amp;deg;C following manufacturer&amp;rsquo;s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200&amp;micro;l matrigel for coating a 12-well plate (~150&amp;micro;g/well).&amp;nbsp;</td></tr><tr><td>Coating Matrix Kit&amp;nbsp;</td><td>Invitrogen</td><td>R-011-K</td><td></td></tr><tr><td>Plasmids:&amp;nbsp;</td><td></td><td></td><td>Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming.</td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-eGFP</td><td>Addgene</td><td>27082</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hOct3/4-shP53F</td><td>Addgene</td><td>27077</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hSK</td><td>Addgene</td><td>27078</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hUL</td><td>Addgene</td><td>27080</td><td></td></tr><tr><td>Transfection reagent Fugene HD</td><td>Promega</td><td>E231B</td><td></td></tr><tr><td>Gelatin (from porcine skin)</td><td>Sigma</td><td>G1890</td><td>Make up 0.1% gelatin in distilled water. Autoclave before use.&amp;nbsp;</td></tr><tr><td>Reduced Serum medium: OPTI-MEM</td><td>Invitrogen</td><td>31985062</td><td></td></tr><tr><td>Accutase</td><td>Sigma</td><td>A6964-100ml</td><td></td></tr><tr><td>Mouse embryonic fibroblast (MEF) feeder</td><td></td><td></td><td>MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16.</td></tr><tr><td>26G needle</td><td>Terumo</td><td>NN2613R</td><td></td></tr><tr><td>6-well plate (tissue culture treated)</td><td>BD Biosciences</td><td>353046</td><td></td></tr><tr><td>12-well plate (tissue culture treated)</td><td>BD Biosciences</td><td>353043</td><td></td></tr><tr><td>10 cm dish (tissue culture treated)</td><td>BD Biosciences</td><td>353003</td><td></td></tr><tr><td>Dispase</td><td>Invitrogen</td><td>17105-041</td><td>Use at 10 mg/ml</td></tr><tr><td>Collagenase IV</td><td>Invitrogen</td><td>17104-019</td><td>Use at 1 mg/ml</td></tr><tr><td>TRA-160 antibody</td><td>Millipore</td><td>MAB4360</td><td>Use at 5 &amp;micro;g/ml</td></tr><tr><td>OCT4 antibody</td><td>Santa Cruz</td><td>SC-5279</td><td>Use at 5 &amp;micro;g/ml</td></tr><tr><td>NANOG antibody</td><td>R&amp;amp;D Systems</td><td>AF1997</td><td>Use at 10 &amp;micro;g/ml</td></tr><tr><td>MycoAlert Detection kit</td><td>Lonza</td><td>LT07-418</td><td></td></tr></tbody></table>

generation of integration-free human induced pluripotent stem cells using hair-derived keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

성장기 (성장 단계), 퇴행기 (회귀 상) 및 휴지기 (나머지 상) (20, 21) : 머리는 3 가지 성장주기의 단계를 통해 간다. 성장기 모낭은 상피 세포의 여러 계층을 포함, 이들 층은 ORS, IRS 및 모간 (도 1)를 포함한다. 성장기의 머리는 결국 ORS 헤어 샤프트 차별화 종료의 사멸로 표시됩니다 퇴행기 단계로 전환 겪는다. 마지막으로, 세포 사멸의 멈추고 휴지기 모낭이 휴지기 특성과 정지...

Watch this Scientific Journal Video about Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes at JoVE.com

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

헤어 유래 각질 세포를 사용하여 통합없는 인간 유도 만능 줄기 세포의 생성

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

generation-integration-free-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

11.5K 조회수.  University of Melbourne. This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors. 

비디오: Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

비 통합 에피 솜 플라스미드를 사용하여 냉동 버피 코트에서 유도 만능 줄기 세포의 생성

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

연구

발생학

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

에피 솜 벡터를 이용하여 인간 말초 혈액 단핵 세포로부터의 통합없이 유도 다 능성 줄기 세포의 생성

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

인간의 모공에서 신경 크레스트 줄기 세포의 분리와 문화

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

신경과학

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) ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

생성 및 기본 인간 Keratinocytes 성인 피부에서의 경작

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

코드 혈액 파생에서 3D 피부 Organoid의 생성 유도 만능 줄기 세포

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

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 ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

완료 녹아웃 세럼 교체 피더 - 무료 매체를 사용하여 인간의 IPS 세포 피더 - 자유 적응력, 문화와 Passaging

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

생물학

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Cryopreserving와 인간 IPS 세포 복구 완료 마네에게 세럼 교체 피더 - 무료 매체를 사용하여

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 ...