A.F.-P. was supported by the National Science Foundation Graduate Research Fellowship under Grant No.2015165336. N.V.M was supported by grants from the NIH (HL094699), Burroughs Wellcome Fund (1009838), and the March of Dimes (#5-FY14-203). We acknowledge Young-Jae Nam, Christina Lubczyk, and Minoti Bhakta for their important contributions to protocol development and data analysis. We also thank John Shelton for valuable technical input and members of the Munshi lab for scientific discussion.

동물 관행과 관련된 모든 실험 절차는 UT 사우스 웨스턴 의료 센터의 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. Hcn4-GFP E12.5 마우스 배아 섬유 아세포 1. 분리 (MEFs에) <ol><li> 동형 접합 Hcn4-GFP의 남성과 CD-1 여성 사이의 시간 제한 교배를 설정합니다. </li><li> 이산화탄소 안락사 이후 자궁 전위에 의해 E12.5에서 임신 여성을 희생. <ol><li> 이전 23 22 설명 된대로 해부 집게와 자궁 뿔을 제거하고 칼슘 또는 마그네슘없이 1X 인산 완충 식염수 (PBS)와 얼음에 페트리 접시 2+에 배치합니다. </li></ol></li><li> 멸균 기법을 사용하여 조직 배양 후드에서 모든 후속 단계를 수행한다. <ol><li> 가위를 사용하여 집게를 해부 골목 자궁과 양수에서 배아를 제거합니다. 더 나은 처리를 위해 첨부 된 태반을 유지합니다. 레그나NT CD-1 여성은 일반적으로 10 ~ 14 새끼를 낳을. </li><li> 집게를 해부 사용하여 격리 된 배아을 빠르게 70 % (v / v)의 EtOH로 두 번을 씻어. 주 : 세척은 세포 사멸을 최소화하기 위해 빠른해야한다. </li><li> 격리 된 배아에서 심장을 포함, 머리, 사지, 꼬리와 내장을 제거합니다. </li><li> 약 1mm 3 크기로 멸균 면도날을 사용하여 1X PBS와 잘게 다진 1 mL를 10 cm 접시에 남아있는 조직을 놓습니다. </li><li> PBS로 50 ML 원뿔 튜브에 다진 조직을 전송합니다. </li><li> 3 분 동안 300 XG에 스핀. 조심스럽게 과량의 PBS를 대기음. </li><li> 배아 당 멸균 0.25 % 트립신 - EDTA의 1 ML을 추가합니다. 15 분 동안 37 ° C의 수조에서 세포를 부화. 부드럽게 튜브마다 4 분을 섞는다. 오버 소화 조직의 크게 수율을 낮출 것이다. </li><li> 4 초 동안 최대 속도 (3200 RPM)에서 소용돌이 세포 혼합물. </li><li> 배아 당 섬유 아세포 미디어의 2 mL를 넣고 섞는다. 필터 t스트레이너를 통해 세포를 돕기 위해 피펫을 사용하여 100 μm의 셀 스트레이너 hrough. 이후의 모든 매체의 배합은 표 1을 참조하십시오. </li><li> 4 분 동안 300 XG에 스핀. 조심스럽게 뜨는을 대기음. </li><li> 3 배아 당 신선한 섬유 아세포 미디어의 10 ㎖를 추가하고 6 ~ 10 번 씹다. </li><li> 준비 매 3 배아 1 15 cm 조직 배양 접시에 세포를 플레이트. 37 ° C, 5 % CO 2 배양기에서 문화 하룻밤. </li><li> 밤새 배양 한 후, 접시 당 미디어의 신선한 30 mL로 미디어를 교체합니다. 밤새 다시 인큐베이터로 세포를 놓습니다. 참고 : 형광 현미경 Hcn4-GFP + 세포의 오염을 확인합니다. 문화는 GFP해야한다 - 만 프로그래밍에 GFP +를하게된다. </li><li> 다음날, 신선한 미리 예열 0.25 % 트립신 EDTA 3 mL를 수확 세포. 카운트 세포를 동결. 일반적으로 용액 당 3 × 10 6 세포에서 세포를 동결. 예상 yieLD는 배아 당 3 × 10 6 세포해야한다. </li></ol></li></ol> 2. 레트로 바이러스 생산 및 재 프로그래밍 주의 : 다음 프로토콜은 감염성 레트로 바이러스의 생산 및 처리가 필요합니다. BSL-2 가이드 라인 및 무균 기술 하에서 바이오 안전성 레벨 2 장에서 다음 단계를 수행합니다. 레트로 바이러스에 노출 된 모든 자료를 폐기하는 10 % 표백제를 사용합니다. <ol><li> 레트로 바이러스의 생산 및 MEFs에 준비 참고 : 다음과 같은 프로토콜을 24 웰 플레이트에서 레트로 바이러스 6 웰 플레이트에서 생산 및 MEF 감염입니다. 다른 형식은 표 2를 참조하십시오. 타이밍 (섹션 2.3 및 그림 2 참조) 각 실험에 대해 적절하게 조정해야합니다 있도록 MEFs에이 일 -1에 도금되어있다. <ol><li> 플랫-E (PE) 제조업체의 권장 사항에 따라 세포를 유지한다. 간단히, DMEM에서 배양 PE 세포는 10 % FBS, 1 μg의 / ㎖로 보충 된 puromyCIN, 10 μg의 / ㎖의 블라스트, 페니실린 및 스트렙토 마이신. 통로 세포 1 : 문화 70~90%의 포화 상태에 도달 4 이틀. </li><li> 일 -2 : 형질 전날, 형질 미디어에서 6 웰 플레이트에 / 잘 1 × 10 6 세포에서 플랫 - E 세포를 시드. 세포를 형질 감염시 70~80% 합류해야한다. </li></ol></li><li> 상업적 형질 전환 제를 이용하여 형질 전환. 참고 : 상용 시약은 형질 전환 전에 실온 (RT)에 있어야합니다. DNA를 형질 들어 GHMT 칵테일 구를 형성하여 레트로 바이러스 플라스미드 DNA 각각 (G, H, M, 및 T)를 추가한다. <ol><li> 일 -1 : 15 ML 원뿔 폴리스티렌 튜브에서, 6 웰 플레이트 형식 반응 당 형질 감염 시약 6 μL의 혈청 감소 배지 중 60 μL를 혼합한다. 실온에서 5 분 동안 혼합물을 인큐베이션. 주 : 여기에서 사용 된 트랜 스펙 션 시약 플라스틱 결합 때문에,직접 환원 혈청 배지에 형질 전환 효율의 임의의 감소를 피하기 위해 DD. </li><li> 반응 당 GHMT 칵테일 2 μg의 총을 추가하고 부드럽게 섞어 누릅니다. 소용돌이하지 마십시오. RT에서 15 분 동안 반응물을 인큐베이션. </li><li> 드롭 현명한 방식으로 PE 세포 단계 2.2.3에서 혼합물을 추가합니다. </li><li> 37 ° C, 5 % CO 2 배양기에서 하룻밤 형질 플랫 - E 세포를 품어. 형질 전환의 시간을 기록한다. </li></ol></li><li> Hcn4-GFP 마우스 배아 섬유 아 세포의 시드. <ol><li> 1 시간 MEFs에 도금하기 전에, 면역 세포 화학에 대한 24 웰 플레이트를 준비합니다. <ol><li> 물론 당 12mm의 피브로넥틴의 커버 슬립을 추가합니다. </li><li> (예 SureCoat) 소 콜라겐 용액 300 μL 및 코트 웰을 1 시간 동안 37 ° C의 배양기에서 배양한다. </li><li> 바로 MEF 도금 전에 대기음 코팅 용액. </li></ol></li><li> Hcn4-GFP MEFs에의 냉동 병을 녹여 및 사전 예열 fibrobl에 1 개를 세척5 분 500 XG에 AST 미디어. </li><li> 트리 판 블루 배제 또는 유사한 염료를 사용하여 세포 생존을 결정합니다. 다음 식을 사용하여 배양 용액 당 생존 세포의 수를 계산한다 : % 가능한 세포가 = [1.00 - (총 세포의 파란색 셀 수 / 수)] × 100 <ol><li> 다음 식을 이용하여 생존 세포의 수를 계산한다 : 세포 현탁액의 생균 = % 생존 세포의 X 희석 인자 X 10,000 X 전량 </li></ol></li><li> 이전에 제조 된 소 콜라겐 솔루션 피브로넥틴 coverslip에있는 24 웰 플레이트 상에 잘 당 종자 3 × 104 세포. </li></ol></li><li> 형질 도입 및 MEFs에의 재 프로그래밍 참고 : 제조업체의 메모에 따르면, 제대로 플랫 - E 세포를 1 × 10 7 감염 단위 / mL의 평균 역가를 생산 유지했다. 역가가 직접 각 실험 측정되지 않지만하는 GFP 제어는 항상 감염 효율에 대한 대용으로 포함된다.높은 GFP 발현 강도 (GFP +&gt; 95 %)는 일반적 iCLMs 성공적 GHMT 매개 세대 연관. <ol><li> 0 일 24 시간 후 형질 감염은, 0.45 ㎛의 세공 크기의 계면 활성제가없는 셀룰로오스 아세테이트 필터를 통해 PE 레트로 매체 필터링하고 15ml의 원뿔형 튜브에 옮긴다. 8 μg / ML의 최종 농도로 폴리 브렌을 추가한다. 조심스럽게 새로운 형질 감염 배지 2 ㎖의 세포를 보충. 주 : 매체가 너무 빠르게 변경되면 세포는 쉽게 판에서 분리합니다. </li><li> 배양 MEFs에의 매체를 기음과 갓 수집 된 레트로 바이러스 매체를 추가; 그것은 6 웰 플레이트의 웰 당 미디어 1.7 mL로 산출한다. 24 웰 플레이트의 웰 당 ~ 800 μL를 추가합니다. 인큐베이터에 MEF 플레이트를 반환하고 밤새 품어. </li><li> 1 일 : 반복 2.4.1와 2.4.2 단계를 반복합니다. 2 차 바이러스 수집 한 후 세포를 폐기하십시오. 인큐베이터에 감염된 MEFs에를 반환하고 하룻밤 휴식을 할 수 있습니다. </li><li> 주 2 : 48 시간 후 유도, 1X PBS로 세포 조절 미디어 및 세척 1 개를 대기음. 24 웰 플레이트의 웰 당 500 μL 사전 예열 iCLM 미디어를 추가합니다. </li><li> 삼일 - 매 2 iCLM 미디어를 교체합니다. 십사일 심장 프로그래밍의 면역 세포 (ICC) 분석을 위해 바이러스 유도 후 처리 플레이트. </li></ol></li></ol> 재 프로그램 MEFs에 3. 면역 염색 <ol><li> 14 일 후 유도, 신중하게 미디어를 대기음. </li><li> 얼음처럼 차가운 1X PBS 300 μL 잘 각을 씻어. 초과 솔루션을 대기음. </li><li> 24 웰 플레이트의 웰 당 4 % 파라 포름 알데히드 (PFA) 용액 250 μL와 세포를 수정. 실온에서 15 분을 품어. 참고 : - 염색 전에 이주 고정 세포를 1 4 ° C에서 PBS에 저장할 수 있습니다. </li><li> 300 μL 0.1 % PBS - 트리톤 100 (PBST)로 세척 웰 X3에 의해 Permeabilize 하시려면 세포. 세척 사이 RT에서 5 분을 품어. 마지막 세척 후 과잉 솔루션을 대기음. </li><li> 10 블록300 μL / 웰의 1 배 범용 블록 버퍼와 RT에서 분. </li><li> (ICC) 염색 버퍼를 준비합니다 버퍼를 차단 1X PBS 및 1X 유니버설의 1 : 1을 추가합니다. ICC 염색 버퍼에 차 항체를 희석하고 4 ℃에서 밤새 항체를 품어. 권장 희석 용 재료 섹션을 참조하십시오. <ol><li> Nppa IPM 및 IAM 식별을위한 마우스 α-티닌, 닭 αGFP, 토끼와 슬라이드의 한 쌍의 얼룩. </li><li> IPM 및 IVM 식별을위한 마우스 α-티닌, 닭 αGFP, 토끼 Myl2으로 슬라이드 한 쌍의 얼룩. </li></ol></li><li> 다음 날, 300 μL 0.1 % PBST로 웰 X3 씻는다. 세척 사이 RT에서 5 분을 품어. 마지막 세척 후 과잉 솔루션을 대기음. </li><li> ICC 염색 버퍼에 차 항체 희석을 준비합니다. 권장 희석 용 재료 섹션을 참조하십시오. 이차 항체를 빛으로부터 보호 실온에서 1 시간을 품어. <ol><li> 다음 보조 antibodie 모든 슬라이드를 얼룩S : 마우스 알렉사-555, 닭 알렉사-488을, 토끼 알렉사-647. </li></ol></li><li> 300 μL 0.1 % PBST로 웰 X3을 씻으십시오. 세척 사이 RT에서 5 분을 품어. 빛으로부터 보호합니다. </li><li> 유리 현미경 슬라이드에 4 &#39;, 6-diamidino-2-페닐 인돌 (DAPI)의 1.5 μg의 / mL로 포함하는 설치 매체의 2.4 μL를 추가합니다. 조심스럽게, 24 웰 플레이트의 우물에서 커버 슬립을 제거 여분의 용액을 제거하고 설치 매체를 사용하여 유리 슬라이드에 전송할 수 있습니다. 부드럽게 초과 볼륨과 공기를 제거하기 위해 커버 슬립을 누릅니다. </li><li> 선호 매니큐어 또는 플라스틱 밀봉 제와 인감 슬라이드. 상점은 빛으로부터 보호 4 ° C에서 슬라이드를 장착. </li></ol> 공 초점 현미경을 사용하여 심장 서브 타입의 4 확인 주 : 촬상 들어, 공 초점 레이저 주사 현미경은 적어도 2 형광체 (405)에 스펙트럼 감지 가능한 감지기, 488, 555, 및 639 nm의 파장을 구비 IPMS, IAMS 및이 iVMS를 식별하기 위해 필요하다. IMAG플랜 - 고차 색지움 20X / 0.75 목적 또는 더 나은 사용하여 전자 셀. 제조업체의 이미지 분석 소프트웨어를 사용하여 40X 오일 침지 품질의 이미지를 얻을 수 확대 이미지를 스캔. <ol><li> 이미지 라이브러리 : DAPI, 알렉사-488, 알렉사-555, 알렉사-647 채널 8 비트 이미지를 가지고 (CH.). 픽셀을 2 6의 1024 프레임 크기, 라인 단계의 체류 시간, 및 (2)의 평균은 높은 해상도 이미지에 충분하다. </li><li> 각 슬라이드를 들어, 하나의 가장자리에서 시작하고 최대 스캔 시작 α-티닌 + 근절 + 세포의 적색 형광 채널 (CH.)에서 아래 (예 3을 참조하십시오 그림 5A). 근절 줄무늬는 555 nm 파장에서 시각적으로 쉽게 식별 할 수 있습니다. <ol><li> α-티닌 + 근절 + 세포가 확인되면, 녹색 CH 전환. 이 긍정적 인 경우 (IPM)을 메모를 유지합니다. 원적외선 647 채널을 평가하기 위해 컴퓨터로 전환합니다. (IAM 또는 IVM). 참고 : 있습니다 셀α-티닌 + / 근절 + / Hnc4-GFP + / Nppa - / Myl2은 - IPMS로 지정되어 있습니다. GFP 발현 세포 (도 4a)에 걸쳐 알 수있다. </li><li> α-티닌 (마우스 Alexa555), Hcn4-GFP (닭 Alexa488)와 얼룩 슬라이드, Nppa (토끼 Alexa647) 및 DAPI. α-티닌 + / 근절 + / Hnc4-GFP있는 세포 - / Nppa +는 IAM 있습니다. Nppa 염색은 핵 주변 및 점상 (그림 4B)를 나타납니다. </li><li> α-티닌 (마우스 Alexa555)와 얼룩 슬라이드, Hcn4-GFP (닭 Alexa488), Myl2 (토끼 Alexa647). α-티닌에 대한 긍정적 인 세포 + / 근절 + / Hnc4-GFP는 - / Myl2 +이 iVMS 있습니다. Myl2 염색은 근절 필라멘트를 따라 가로 무늬 양식을 전시 할 예정이다. 때문에 염색 및 Z 평면의 품질 변화에 줄무늬가 항상 (그림 4C) 쉽게 표시되지 않을 수 있습니다. </li></ol></li></ol> 주 : 잠재적으로 재 프로그램 된 MEFs의 실제 수를 평가하기 위해, 24 웰 플레이트의 웰이 실험 웰에 병렬로 시딩하고, 도금 후 하루 수확. 도금 셀의 총 개수는 두 개의 웰을 평균함으로써 결정된다. 이 도금 실제 총 세포 (aTotal)을하게된다. <ol><li> 근절 + <ol><li> 시각적으로 적절한 α-티닌 + / 근절 + (오른쪽 패널 그림 3)과 기록 (그림 5B-1)에 대한 커버 슬립 각 셀을 검사합니다. </li><li> 도금 실제 총 세포 (aTotal)에 의해 각각의 커버 슬립과 분할에 α-티닌 + / 근절 +의 총 수를 집계 (그림 5B-III). aTotal = 12,500 세포 및 100 세포가 α-티닌 된 경우, 예를 들어 + / + 근절 후, 도금 MEFs에 0.8 %가 다시 프로그램되었다. 평균reprograming 실험은 1 % α-티닌은 + / 근절 + 세포 (그림 5C)를 얻을 것입니다. </li></ol></li><li> 하위 유형 + 참고 : 다음 단계는 대표 iCLM 정량 워크 플로우 5B / C 그림을 참조하십시오. 이 중 하위 유형 (그림 5B-1)에 대한 고유 한 경우 간단히, 각 근절 + 셀, 집계. 평균 근절 + X 100 세포 (도 5b-1)를 통해 부속 + 세포의 수를 나누어 % 하위 유형 (도 5b-III)을 계산한다. 절대 %의 하위 효율 (그림 5B-IV)를 계산하려면, 그림에서 하위 유형 + 세포 수를 분할 5B-난 × 100 (그림 5B-II) 시드 세포의 총 수에 의해. <ol><li> 그들은 GFP +, Nppa +, 또는 Myl2있는 경우, α-티닌의 각 + / 근절 + 세포의 경우, 평가 <s&gt; +입니다. </li><li> / Myl2 - -, 티닌 + / 근절 + / Hnc4-GFP - / Nppa +, 및 α-티닌 + / 근절 + / Hnc4-GFP α-티닌 + / 근절 + / Hnc4-GFP + / Nppa의 총 수를 집계 - / Myl2 + 세포 (도 5b-1). </li><li> 각 하위 유형의 비율을 계산하려면, 잘한다는 점에서 + / 근절 + 세포 α-티닌의 총 수에 의해 서브 타입 + 세포의 수를 분할하고 100 GHMT 곱 IPMS, IAMS, 그리고이 iVMS 대략 동일한 비율을 생성한다 (그림 5B-III). </li><li> 아형 + 세포의 절대 수를 계산하기 aTotal하여 실험 조건 아형 + 세포의 총 수를 100 분할하고 (도 5b-II)로 곱한다. 평균 IPMS는 전체 감염 세포 인구의 0.3 %, IAMS 0.3 %를 나타낼과이 iVMS 0.25 % (그림 5B-IV). </li></ol></li></ol>

<ol>
	<li>Sissman, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+landmarks+in+cardiac+morphogenesis:+comparative+chronology.">Developmental landmarks in cardiac morphogenesis: comparative chronology.</a> Am J Cardiol. 25 (2), 141-148 (1970).</li><li>Buckingham, M., Meilhac, S., Zaffran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+mammalian+heart+from+two+sources+of+myocardial+cells.">Building the mammalian heart from two sources of myocardial cells.</a> Nat Rev Genet. 6 (11), 826-835 (2005).</li><li>Ali, S. R., 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=Existing+cardiomyocytes+generate+cardiomyocytes+at+a+low+rate+after+birth+in+mice.">Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice.</a> Proc Natl Acad Sci U S A. 111 (24), 8850-8855 (2014).</li><li>Bergmann, O., 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=Evidence+for+cardiomyocyte+renewal+in+humans.">Evidence for cardiomyocyte renewal in humans.</a> Science. 324 (5923), 98-102 (2009).</li><li>Senyo, S. E., 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=Mammalian+heart+renewal+by+pre-existing+cardiomyocytes.">Mammalian heart renewal by pre-existing cardiomyocytes.</a> Nature. 493 (7432), 433-436 (2013).</li><li>Lin, Z., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+cardiac+regeneration+and+repair.">Strategies for cardiac regeneration and repair.</a> Sci Transl Med. 6 (239), 239rv231 (2014).</li><li>Writing Group, M., 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=Heart+Disease+and+Stroke+Statistics-2016+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association.</a> Circulation. 133 (4), e38-e360 (2016).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Song, K., 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=Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors.">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485 (7400), 599-604 (2012).</li><li>Ieda, M., 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=Direct+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142 (3), 375-386 (2010).</li><li>Qian, L., 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=In+vivo+reprogramming+of+murine+cardiac+fibroblasts+into+induced+cardiomyocytes.">In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.</a> Nature. 485 (7400), 593-598 (2012).</li><li>Fu, J. D., 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=Direct+reprogramming+of+human+fibroblasts+toward+a+cardiomyocyte-like+state.">Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state.</a> Stem Cell Reports. 1 (3), 235-247 (2013).</li><li>Zhou, H., Dickson, M. E., Kim, M. S., Bassel-Duby, R., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt1/protein+kinase+B+enhances+transcriptional+reprogramming+of+fibroblasts+to+functional+cardiomyocytes.">Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes.</a> Proc Natl Acad Sci U S A. 112 (38), 11864-11869 (2015).</li><li>Zhou, Y., 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=Bmi1+Is+a+Key+Epigenetic+Barrier+to+Direct+Cardiac+Reprogramming.">Bmi1 Is a Key Epigenetic Barrier to Direct Cardiac Reprogramming.</a> Cell Stem Cell. 18 (3), 382-395 (2016).</li><li>Zhao, Y., 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=High-efficiency+reprogramming+of+fibroblasts+into+cardiomyocytes+requires+suppression+of+pro-fibrotic+signalling.">High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.</a> Nat Commun. 6, 8243 (2015).</li><li>Miki, K., Yoshida, Y., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+steady+progress+on+direct+cardiac+reprogramming+toward+clinical+application.">Making steady progress on direct cardiac reprogramming toward clinical application.</a> Circ Res. 113 (1), 13-15 (2013).</li><li>Atkinson, 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=Anatomical+and+molecular+mapping+of+the+left+and+right+ventricular+His-Purkinje+conduction+networks.">Anatomical and molecular mapping of the left and right ventricular His-Purkinje conduction networks.</a> J Mol Cell Cardiol. 51 (5), 689-701 (2011).</li><li>Bootman, M. D., Smyrnias, I., Thul, R., Coombes, S., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+cardiomyocyte+calcium+signalling.">Atrial cardiomyocyte calcium signalling.</a> Biochim Biophys Acta. 1813 (5), 922-934 (2011).</li><li>Miquerol, L., Beyer, S., Kelly, R. G. <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+the+mouse+ventricular+conduction+system.">Establishment of the mouse ventricular conduction system.</a> Cardiovasc Res. 91 (2), 232-242 (2011).</li><li>Sp&#228;ter, D., Hansson, E. M., Zangi, L., Chien, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+cardiomyocyte.">How to make a cardiomyocyte.</a> Development. 141 (23), 4418-4431 (2014).</li><li>Nam, Y. J., 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=Induction+of+diverse+cardiac+cell+types+by+reprogramming+fibroblasts+with+cardiac+transcription+factors.">Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors.</a> Development. 141 (22), 4267-4278 (2014).</li><li>Conner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Molecular Biology. , (2001).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells.">Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.</a> J Vis Exp. (64), (2012).</li><li>Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., Yee, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+G+glycoprotein+pseudotyped+retroviral+vectors:+concentration+to+very+high+titer+and+efficient+gene+transfer+into+mammalian+and+nonmammalian+cells.">Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.</a> Proc Natl Acad Sci U S A. 90 (17), 8033-8037 (1993).</li><li>Ichim, C. V., Wells, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+high-titer+viral+preparations+by+concentration+using+successive+rounds+of+ultracentrifugation.">Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation.</a> J Transl Med. 9, 137 (2011).</li><li>Qian, L., Berry, E. C., Fu, J. D., Ieda, M., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+mouse+fibroblasts+into+cardiomyocyte-like+cells+in+vitro.">Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.</a> Nat Protoc. 8 (6), 1204-1215 (2013).</li><li>Yang, R., 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=Direct+conversion+of+mouse+and+human+fibroblasts+to+functional+melanocytes+by+defined+factors.">Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors.</a> Nat Commun. 5, 5807 (2014).</li><li>Muraoka, N., Ieda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+of+fibroblasts+into+myocytes+to+reverse+fibrosis.">Direct reprogramming of fibroblasts into myocytes to reverse fibrosis.</a> Annu Rev Physiol. 76, 21-37 (2014).</li><li>Coffin, J. M., Hughes, S. H., Varmus, H. E., Coffin, J. M., Hughes, S. H., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retroviruses. , (1997).</li><li>Bass, G. T., 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=Automated+image+analysis+identifies+signaling+pathways+regulating+distinct+signatures+of+cardiac+myocyte+hypertrophy.">Automated image analysis identifies signaling pathways regulating distinct signatures of cardiac myocyte hypertrophy.</a> J Mol Cell Cardiol. 52 (5), 923-930 (2012).</li></ol>

The authors have nothing to disclose.

본 연구는 심장 전사의 레트로 바이러스 - 매개 발현을 통해 심장 서브 타입의 다양한 세트로 MEFs에의 변환을위한 직접 프로그래밍 전략을 제공하는 Gata4, Mef2c, Tbx5 및 Hand2 (GHMT) 요인. PM 특정 리포터 마우스와 함께 면역 다중화 방식을 사용하여, 우리는 단일 세포 해상도에서 IAM,이 iVMS 및 IPMS를 식별 할 수있다. 이러한 분석은 하위 유형의 다양성과 근절 개발에 대한 개별 전사 인자의 기여를 분리 ?...

핵심은 배아 (1), (2)에서 개발 한 최초의 기능 기관이다. 순환계와 함께에서는 산소, 영양분, 개발시의 폐기물 처리 장치에 공급한다. 3 주 후, 수정, 인간 심장 처음 박동 및 적절한 조절은 심근 (CMS)에 의해 유지된다. 이들 특화된 세포의 비가역적인 손실 따라서 진보적 심부전의 기초가되는 기본적인 문제이다. 이러한 제브라 피쉬 제노와 같은 일부 유기체는 심장 재생 잠재력을 가지고 있지만, 성인 포유류 심장 3, 5, 6 개의 제한된다. 따라서, 심장의 중요한 기능을 제공, 심장 질환 혼자 7 미국에서 60 만 명이 사망를 차지, 세계에서 사망의 주요 원인이다 놀라운되지 않습니다. 그만큼효율적으로 복구하거나 손상된 심근을 대체 할 refore, 세포 기반 치료는 큰 임상 적 관심이다. 야마나카 동료 8 정액 연구는 네 개의 전사 인자 강제 발현이 다 능성 줄기 세포로 완전히 분화 된 섬유 아세포를 변환하기에 충분한 것으로 나타났다. 그러나, 모든 다 능성 줄기 세포의 종양 전략 용량은 치료 목적의 사용에 중요한 문제였다. 이것은 능성 단계를 피하면서 다른 방법은 세포를 transdifferentiate하는 검색 할 과학 분야 동기를 부여. 최근, 여러 그룹이 전사의 자궁외 식으로 유도 된 심근 유사 세포 (iCLMs)에 마우스 섬유 아세포의 직접 변환을 표시하여이 전략의 가능성을 보여 주었다 Gata4, Mef2c, Tbx5, 나중에, Hand2 (GMT 및 GHMT 요인 각각) 9,10. 작고에hermore 동일한 전략은 생체 내에서 인간 유래 조직 9, 11, 12에서 수행 될 수있다. 최근 연구에 추가적인 요소 또는 심부전 리 프로그래밍 효율을 13, 14, 15을 향상시키는 변조 될 수있는 신호 전달 경로를 강조하고있다. 함께 찍은, 이러한 연구는 재생 요법에 대한 감독 분화의 가능성을 보여줍니다. 그러나, 방법 론적 차이 16 CM 리 프로그래밍, 미지의 분자 메커니즘 일관성 재현성의 저 효율성 및 iCLMs의 이질적인 성질은 어드레싱 남아있다. 직접 iCLM 얼룩을 평가하기 위해, 우리는 근절 개발 및 심장 계통 specificatio의 식별 이산 견고한 단일 세포 분석 설계N-이 기능 심근의 필요한 특성. 심방 (AM), 심실 (VM)와 조정기 (PM) 17, 18, 19, 20의 위치와 고유의 전기적 특성에 의해 정의되는 심장 CM의 적어도 세 가지의 유형이있다. 조율 된 조합, 그들은 혈액의 적절한 펌핑을 할 수 있습니다. 심장 손상 중에, 하나 또는 모든 아형의 영향을 야기 할 수 있으며 세포 치료의 유형은 경우에 따라 해결해야 할 것이다. 작은 일이 부속 명세서를 조절하는 분자 메커니즘을 연구하기 위해 수행되는 동안 현재 대부분의 전략은 심근 세포의 전체적인 생성에 초점을 맞춘다. 다음 연구는 제대로 잘 조직 된 근섬유 분절을 정량화 및 심근 서브 타입의 다양한 세트를 식별하는 방법을 자세히 설명합니다. 페이스 메이커 (PM) - 특정 기자 마우스를 사용하여, 우리는 난을 적용 할 수 있습니다mmunocytochemical 접근 방식은 유도 심방 같은 근육 세포 (IAM), 유도 심실 같은 근육 세포 (IVM)과 유도 PM-같은 근육 세포 (IPMS) (21)를 구별한다. 우리의 관찰에 기초하여, 전용 근절 조직을 나타내는 세포는 자율 박동 할 수있다. 이 독특한 프로그래밍 플랫폼 역할을 평가하기 위해 허용 단일 셀 해상도 근절 조직, 하위 사양 및 CM의 재 프로그래밍의 효율성의 특정 매개 변수를 설정합니다.

<table><tbody><tr><td>DMEM</td><td>Sigma</td><td>D5796</td><td>Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td></tr><tr><td>Medium 199</td><td>Thermo Fisher Scientific</td><td>11150059</td><td>Component of iCLM media</td></tr><tr><td>Fetal bovine serrum (FBS)</td><td>Sigma</td><td>F2442</td><td>Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td></tr><tr><td>Insulin-Transferrin-Selenium G</td><td>Thermo Fisher Scientific</td><td>41400-045</td><td>Component of iCLM media</td></tr><tr><td>MEM vitamin solution</td><td>Thermo Fisher Scientific</td><td>11120-052</td><td>Component of iCLM media</td></tr><tr><td>MEM amino acids</td><td>Thermo Fisher Scientific</td><td>1601149</td><td>Component of iCLM media</td></tr><tr><td>Non-Essential amino acids</td><td>Thermo Fisher Scientific</td><td>11140-050</td><td>Component of iCLM media</td></tr><tr><td>Antibiotic-Antimycotics</td><td>Thermo Fisher Scientific</td><td>15240062</td><td>Component of iCLM media</td></tr><tr><td>B-27 supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td>Component of iCLM media</td></tr><tr><td>Heat-Inactivated Horse Serum</td><td>Thermo Fisher Scientific</td><td>26050-088</td><td>Component of iCLM media</td></tr><tr><td>NaPyruvate</td><td>Thermo Fisher Scientific</td><td>11360-70</td><td>Component of iCLM media</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>1514022</td><td>Component of Plat-E media and fibroblast media</td></tr><tr><td>Puromycin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A11139-03</td><td>Component of Plat-E media</td></tr><tr><td>Blasticidin&amp;nbsp;&amp;nbsp;</td><td>Gemini Bio-Products</td><td>400-128P</td><td>Component of Plat-E media</td></tr><tr><td>Glutamax</td><td>Thermo Fisher Scientific</td><td>35050-061</td><td>Component of Fibroblast media</td></tr><tr><td>Confocal laser scanning LSM700</td><td>Zeiss</td><td></td><td>For confocal analysis</td></tr><tr><td>FuGENE 6 transfection Reagent</td><td>Promega</td><td>E2692</td><td>Transfection reagent&amp;nbsp;</td></tr><tr><td>Opti-MEM Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985-070</td><td>Transfection reagent&amp;nbsp;</td></tr><tr><td>Polybrene</td><td>Millipore</td><td>TR-1003-G</td><td>Induction reagent. Use at a final concentration of 8 &amp;mu;M/mL</td></tr><tr><td>Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic</td><td>CellBiolabs</td><td>RV-101</td><td>Retroviral pacaking cell line</td></tr><tr><td>Trypsin 0.25% EDTA</td><td>Thermo Fisher Scientific</td><td></td><td>For MEFs and Plat-E dissociation</td></tr><tr><td>Mouse anti &amp;alpha;-Actinin (Clone EA-53)</td><td>Sigma</td><td>A7811</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Chicken anti-GFP IgY</td><td>Thermo Fisher Scientific</td><td>A10262</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Rabbit Pab anti-NPPA&amp;nbsp;</td><td>Abgent</td><td>AP8534A</td><td>Antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Rabbit Pab anti Myl2 IgG&amp;nbsp;</td><td>ProteinTech</td><td>10906-1-AP</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Vectashield solution with DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Vector Labs</td><td>H-1500</td><td>Dye for confocal analysis</td></tr><tr><td>Superfrost Plus Microscope slides</td><td>Thermo Fisher Scientific</td><td>12-550-15</td><td>25 x 75 x 1.0 mm</td></tr><tr><td>BioCoat Fibronectin 12 mm coverslips</td><td>NeuVitro Corp</td><td>GG-12-1.5</td><td>Coverslips for confocal analysis</td></tr><tr><td>100 &amp;mu;m cell strainer</td><td>Thermo Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>0.45 &amp;mu;m Syringes filters SFCA 25MM</td><td>Thermo Fisher Scientific</td><td>09-740-106</td><td>For virus filtration</td></tr><tr><td>6 mL Syringes</td><td>Covidien</td><td>8881516937</td><td>For virus filtration</td></tr><tr><td>Goat anti-Chicken IgY (H&amp;amp;L) A488</td><td>Abcam</td><td>AB150169</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Donkey anti-rabbit A647 IgG(H+L)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A31573</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Goat anti-mouse IgG(H+L) A555</td><td>Thermo Fisher Scientific</td><td>A21422</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>93443-100ml</td><td>For cell permeabilization&amp;nbsp;</td></tr><tr><td>Dulbecco&#039;s PBS without CaCl&lt;sub&gt;2&lt;/sub&gt; and MgCl&lt;sub&gt;2&lt;/sub&gt; (D-PBS)</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Power Block 10x Universal Blocking reagent</td><td>Thermo Fisher Scientific</td><td>NC9495720</td><td>Dilute to 1x in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>16% Paraformaldehyde aqueous solution (PFA)</td><td>Electro Microscopy Sciences&amp;nbsp;</td><td>15710</td><td>Use at 4% diluted in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>6 cm&amp;nbsp; plates</td><td>Olympus</td><td>25-260</td><td></td></tr><tr><td>6-well plates</td><td>Genesee Scientific</td><td>25-105</td><td></td></tr><tr><td>24-well plates</td><td>Genesee Scientific</td><td>25-107</td><td></td></tr><tr><td>10 cm Tissue culture dishes</td><td>Corning</td><td>4239</td><td></td></tr><tr><td>15 cm&amp;nbsp; Tissue culture dishes</td><td>Thermo Fisher Scientific</td><td>5442</td><td></td></tr><tr><td>15 mL Conical tubes</td><td>Corning</td><td>4308</td><td></td></tr><tr><td>50 mL Conical tubes</td><td>Corning</td><td>4249</td><td></td></tr><tr><td>0.4% Trypan blue solution</td><td>Sigma</td><td>T8154</td><td>For viability</td></tr><tr><td>Ethyl Alcohol 200 proof</td><td>Thermo Fisher Scientific</td><td>7005</td><td></td></tr><tr><td>Bleach</td><td>Thermo Fisher Scientific</td><td>6009</td><td></td></tr></tbody></table>

assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

PM을 특정 리포터 마우스 활용, 우리는도 1에 도시 된 바와 같은 다양한 내인성 근세포를 식별하기 위해 멀티 플렉스 면역 전략을 개발했다. 도 2에 도시 된 프로그래밍 단계에 따라, 특정 아형의 CM 유도이라도 낮은 속도 빠르면 4 일째 21로 감지 할 수있다. 14 일으로 실험을 중지 할 수 있습니다 및 근절 조직 (그림 3)과</strong...

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Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

마우스 배아 섬유 아 세포의 전사 인자 매개 재 프로그래밍에 따라 심근 서브 타입을 평가

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

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JoVE Journal

Developmental Biology

7.4K 조회수.  UT Southwestern Medical Center. This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5. 

비디오: Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. &#8220;Biological pacemakers&#8221; generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines &#8220;forward programming&#8221; of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of &#62;80% physiologically functional pacemaker cells. These &#8220;induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These &#8220;Induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

Myh6 발기인 - 선택과 함께 ES-세포 프로그래밍을 바탕으로 설치류 심장 페이스 메이커 세포 집합체의 생성

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

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Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Gata4, Mef2c 및 Tbx5의 최적 비율을 표현하는 폴리 시스 트론 구조를 사용하여 유도 된 심근의 향상 발전

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

전사 인자와 마우스 배아 섬유 아 세포를 재 프로그램하는 것은 Hemogenic 프로그램을 유도하는

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

생체 외에서 기능 Cardiomyocyte-같은 세포로 인간 중간 엽 줄기 세포의 분화

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

프로 거리 신호 억제 유도 Cardiomyocytes에 마우스 미 발달 섬유 아 세포의 프로그래밍 요소 중재 Potentiates

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Assessing Cardiac Reprogramming using High Content Imaging Analysis

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a reprogramming technique. Cardiac reprogramming provides a strategy to generate new cardiomyocytes. By introducing core cardiogenic transcription factors into fibroblasts; fibroblasts can be converted to iCMs without transition through the pluripotent stem cell state. However, the conversion rate of fibroblasts to iCMs still remains low. Accordingly, there have been numerous additional approaches to enhance cardiac reprogramming efficiency. Most of these studies assessed cardiac reprogramming efficiency using flow cytometry, while at the same time performed immunocytochemistry to visualize iCMs. Thus, at least two separate sets of reprogramming experiments are required to demonstrate the success of iCM reprogramming. In contrast, automated high content imaging analysis will provide both quantification and qualification of iCM reprogramming with a relatively small number of cells. With this method, it is possible to directly assess the quantity and quality of iCMs with a single reprogramming experiment. This approach will be able to facilitate future cardiac reprogramming studies that require large-scale reprogramming experiments such as screening genetic or pharmacological factors for enhancing reprogramming efficiency. In addition, the application of high content imaging analysis protocol is not limited to cardiac reprogramming. It can be applied to reprogramming of other cell lineages as well as any immunostaining experiments which need both quantification and visualization of immunostained cells.

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a ...

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

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

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

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

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

생물학

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

성인 마우스 Cardiomyocytes의 단세포 Transcriptional 프로파일

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

유동 세포 계측법에 의한 고효율 심근에 인간 다 능성 줄기 세포의 분화 및 특성

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

GCaMP3 리포터와 심장 특정 칼슘 플럭스를 측정하여 심장 재프로그래밍의 체외 평가

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...