We thank Dr. Leonid Gnatovskiy for his carefully and critical reading of the paper. This work was supported in part by grants from the National Institutes of Health (HL109054) and the Samuel and Jean Frankel Cardiovascular Center, University of Michigan (Inaugural Fund) to WZ and from the Leon H Charney Division of Cardiology, New York University School of Medicine to BL.

마우스는 ESC에서 마우스 배아 클릭당 비용 (CPC) 1. 유도 <ol><li> 마우스 배아 섬유 아 세포 (MEFs) 세포층을 준비합니다. <ol><li> 따뜻한 MEF 매체 37 ° C에 (DMEM에 10 % FBS). </li><li> 젤라틴 코팅 된 플레이트를 준비합니다. <ol><li> 10cm 접시에 6 웰 플레이트의 우물 또는 5 ㎖에 물에 0.1 % 젤라틴의 1 ML을 추가합니다. </li><li> 적어도 30 분 동안 판 또는 요리 37 ° C에서 또는 실내 온도를 남겨주세요. 사용하기 전에 젤라틴을 열망. </li></ol></li><li> 해동은 부드러운 흔들림으로, 37 ° C의 물을 욕조에 신속하게 MEFs에를 조사. </li><li> 15 ml의 50 ML 원뿔 튜브에 부드럽게 세포를 전송합니다. 예열 MEF 매체의 5 ML을 추가합니다. 천천히 세포를 소용돌이 및 5 분 동안 200 XG에 원심 분리기. </li><li> MEF 매체에서 세포를 재현 탁하고 가능한 세포를 계산합니다. 10cm 접시에서 세포를 2 ml를 6 웰 플레이트에서 웰 당 세포 및 10 ml의 다음 MEF 매체의 볼륨을 사용합니다. </li><li> 6 웰 플레이트에 플레이트 세포 또는 1-2에서 10cm 요리플레이트 / 접시 당 × 10 (6). </li><li> 37 ° C에서 MEF 플레이트 / 접시를 품어, 5 % CO 2 최소 24 시간 이전에 사용할 수 있습니다. 주 : 주보다 오래된 MEF 판 / 요리를 폐기하십시오. </li></ol></li><li> 마우스는 ESC를 준비 <ol><li> 문화 Isl1-CRE; Rosa26YFP / Mef2c-CRE; 90 %의 합류까지 MEFs에에 Rosa26YFP 마우스는 ESC. 사용 ES 세포 배지 410 ml의 DMEM, 75 ㎖의 FBS, 0.1 mM의 MEM NEAA, 2 mM L- 글루타민, 0.1 mM의 피루브산 펜 100 U / 스트렙토 마이신, 및 0.1 밀리미터 β 머 캅토 에탄올로 구성. </li><li> 1.1.2에 설명 된대로 젤라틴 코팅 6 웰 플레이트를 준비합니다. </li><li> 접시에서 대기음 매체와는 DPBS로 세포를 씻어. <ol><li> 기음 DPBS 6 웰 플레이트 잘 하나에 0.4 ml의 0.25 % 트립신을 추가합니다. 5 분 동안 37 ℃로, 5 % CO 2에서 세포를 배양한다. 트립신 반응을 중단 1 ml의 예열 ES 매체를 추가합니다. </li></ol></li><li> 수확 세포와 원심 분리기 세포 200 XG와 대기음 매체에서. 재현 탁 된 1 ml의 ES 세포 배지에서 세포와 세포를 카운트. &lt;/ 리&gt; <li> 3 × 104 / ㎠의 밀도에 젤라틴 코팅 플레이트 플레이트는 ESC. 는 ESC는 약 90 % 합류에 도달했을 때 약 2 일 동안 37 ° C, 5 % CO 2에서 배양한다. 매체 일상을 변경합니다. </li><li> 는 ESC가 90 % 합류 경우, 반복 완전히 MEF 피더를 제거하는 1.2.2-1.2.5 단계. </li></ol></li><li> EB 형성에 의해 CPC 분화를 유도 <ol><li> 다음으로 분화 배지 준비 : 36 ml의 IMDM, 12 ml의 햄 F12, 2 mM L- 글루타민, 0.34 ml의 BSA (7.5 %), 0.25 ml의 N2, 0.5 ㎖의 B27, 50 ㎍ / ㎖의 AA, 및 0.45 mM의 1- 티오 글리세롤. </li><li> 기음 세포로부터 중간 DPBS 씻어. 기음 DPBS 6 웰 플레이트 잘 하나에 0.4 ml의 0.25 % 트립신을 추가합니다. 5 분 동안 37 ℃에서 배양한다. </li><li> 하나의 세포로 세포 클러스터를 분산 아래로 반응 및 피펫 업을 중단하고 1 ml의에게 차별화 매체를 추가합니다. </li><li> 원심 분리기는 ESC 5 분 200 XG에와 매체를 폐기합니다. <ol><li> 재현 탁 1 × 6 &lt;/ SUP&gt; 1 ml의 차별화 매체는 ESC. 제 EB 응집 37 ° C에서 분화 배지에서 1 × 105 세포 / ml의 낮은 농도에서 분리 배양 접시에서 세포를 세포 수 및 배양. </li></ol></li><li> 이틀 EB 형성 한 후, 50 ㎖ 튜브에 요리에서 사채를 전송할 수 있습니다. 1 분 동안 200 XG에 스핀. 매체를 제거하고 칼슘과 마그네슘 2+없이 10 ML의 DPBS로 사채를 씻어. </li><li> 기음 DPBS 1 ml의를 0.25 % 트립신을 추가합니다. 5 분 동안 37 ℃에서 배양한다. 반응을 정지 4.5 ml의 차별화 매체를 추가합니다. 피펫 위아래로 하나의 세포로 사채를 해리합니다. </li><li> 5 분 동안 200 XG에 세포를 원심 분리기. 1 ml의 분화 배지에서 대기음 매체에 resuspend 세포. </li><li> 세포 수 및 분화 배지에서 1 × 105 세포 / ㎖로 2 × 106 세포를 희석. 5 NG / ㎖, 0.8 ng를 / ㎖의 최종 농도 매체에 VEGF, BMP4과를 Activin이 추가, 5 NG / ㎖, 각각. Cultu37 ° C에서 두 번째 EB 형성을위한 배양 접시에 세포 재. </li><li> 두 번째 EB 형성 후 40 시간은 50 ㎖ 튜브에 사채를 전송할 수 있습니다. 5 분 동안 37 ° C에서 1 ml의 0.25 % 트립신과 EBS을 (를) 해리, DPBS로 한번 씻어. 피펫 위아래로 하나의 세포로 사채를 해리합니다. </li><li> 5 NG / ㎖ VEGF, 10 NG / ㎖의 bFGF 12.5 NG / ㎖로 FGF10 Stempro-34 배지에서 세포를 재현 탁. 젤라틴 - 코팅 된 플레이트에서 2 × 105 / cm 2에서 셀을 접시. CPC 분리하기 전에 약 32 시간 동안 37 ° C의 배양기에서 문화. </li></ol></li><li> mCPCs를 분리 <ol><li> 대기음 매체와 DPBS 씻어. 5 분 동안 37 ℃에서 배양 플레이트에 0.5 ml의 0.25 % 트립신을 추가합니다. 반응을 정지 4.5 ml의 차별화 매체를 추가합니다. 피펫 위아래로 하나의 세포로 사채를 해리합니다. FACS 정화 2 % FBS / PBS에서 원심 분리기에 resuspend 세포에 의한 세포를 수집합니다. </li><li> 음성 대조군으로 정렬에 미분화는 ESC를 실행합니다. 변경 전압은 앞으로 배설물을 조정합니다터 (FSC)와 측면 산란 (SSC)를 원하는 인구를 선택합니다. 파편과 이중를 제외 측면 분산과 폭 대 앞으로 분산 높이 대 흩어 앞으로 사용합니다. 선택한 하위 집단에서, ESC 컨트롤의 분포에 따라, 세포의자가 형광을 제거하기 히스토그램에 긍정적 인 YFP의 게이트를 그립니다. YFP + 게이팅에서 YFP + CPC를 수집합니다. </li><li> 차별화 매체 (1.3.1) 6 웰 플레이트에서 정제 된 클릭당 비용 (CPC) (YFP + 셀) 플레이트. 심근 및 SMCs에 클릭당 비용 (CPC)의 차별화를위한 37 ° C에서 문화 추가 3~5일. </li></ol></li></ol> 인간는 ESC에서 인간 배아 클릭당 비용 (CPC) 2. 유도 <ol><li> 공급기를 준비 <ol><li> 2 × 104 / ㎠의 밀도로 상기 한 바와 같이 마우스 배아 섬유 아세포 (MEFs에) 세포층을 준비한다. </li></ol></li><li> 인간는 ESC를 유지 <ol><li> CRE; M에 정기적으로 pCAG - 플록스-STOP-플록스-GFP 또는 RFP는 ESC : 인간 ISL1 유지정규 hESC의 매체 (녹아웃 DMEM / F-12 385 ㎖, 넉 아웃 SR 100 ㎖, 2 mM L- 글루타민, 0.1 mM의 NEAA, 0.1 밀리미터 β 머 캅토 에탄올, 10 NG / ㎖ 염기성 FGF)을 사용 EF. 심장 분화 이전에, 적어도 2 개의 통로의 피더 자유로운 상태는 ESC 인간 옮긴다. </li></ol></li><li> 피더 무료 조건에서 인간는 ESC를 준비 <ol><li> 인간는 ESC 문화 코팅 된 플레이트를 준비합니다. <ol><li> 4 ° C에서 하룻밤 리겔을 녹여. 얼음에 50 ML 튜브를 넣고 튜브에 30 ml의 차가운 DMEM / F12 배지를 추가합니다. </li><li> 차가운 매체에 1 ml의 마트 리겔을 넣고 잘 섞는다. 10cm 접시에 6 웰 플레이트의 우물 또는 5 ㎖에 1 ml의 차가운 리겔 - DMEM / 12 매체를 추가합니다. </li><li> 2 시간 동안 판 / 4 ° C에서 하룻밤 요리 또는 실내 온도를 남겨주세요. 대기음 매체를 사용하기 전에. </li></ol></li><li> MEF 플레이트에서 대기음 미디어와 DPBS 인간의는 ESC를 씻어 : 인간 플레이트 상에는 ESC를 분할합니다. 이어서 6- 웰 플레이트의 웰에 1 ㎖의 디스 파제 (1 mg / ㎖)을 추가한다. 37 ° C에서 세포를 품어인큐베이터는 식민지의 가장자리가 분리 시작할 때까지. </li><li> 디스 파제 솔루션을 제거합니다. 다음 2.5 ml에 추가 / 잘 mTeSR 매체 또는 MEF-조정 배지 당 판에 두 번 DPBS 씻어. </li><li> 작은 덩어​​리로 인간의 ESC 식민지 아래 휴식을 셀 스크레이퍼를 사용하여 세포를 긁고 조심스럽게 피펫합니다. </li><li> ESC 덩어리를 전송하고 1을 희석 : 3 코팅 된 판에. </li><li> 2 ㎖ / 잘 mTeSR 매체 또는 MEF 에어컨 매체를 추가하고 매일 매체를 변경합니다. </li><li> mTeSR 매체 또는 적어도 두 구절 코팅 접시에 MEF 에어컨 매체 문화 인류는 ESC. </li></ol></li><li> 인간는 ESC에서 CPC의 분화를 유도 <ol><li> 세포가 ~ 90 % 컨 플루 언트 될 때, 배지를 흡인하고 DPBS로 헹군다. 그런 다음 20 분 동안 37 ℃에서 0.5 ㎎ / ㎖의 인간 배아 줄기 스파 아제 (Dispase)를 배양한다. </li><li> 셀 리프터와 접시에서 세포를 제거, 두 번 DPBS와 인간 배아 줄기 린스, 다음 DMEM / F12는 18 % FBS, 0.1 mM의 NEAA, 2 mM의를 포함하는 (분화 배지에서 세포 클램프를 재현 탁L- 글루타민, 0.1 MM의 β-머 캅토 에탄올 및 50 ㎍ / ml의 아스 코르 빈산 산). </li><li> EB 형성을위한 6 자 초저 부착 판에 세포를 전송합니다. </li><li> 다음날 차별화 매체를 변경합니다. </li><li> 3 일마다 중간 교체 및 현탁 배양에 사채을 유지한다. </li></ol></li><li> HCPCS를 분리 <ol><li> 더 이상 인간의 CPC 격리를위한 차별화의 날 (9)보다 사채를 수집하지 않습니다. </li><li> 대기음은 중간 두 번 DPBS로 사채를 씻어. 5 분 동안 37 ° C에서 6 웰 배양 플레이트의 각 웰에 0.5 ml의 0.25 % 트립신을 추가한다. </li><li> 반응을 정지 분화 배지의 동일한 볼륨. 피펫 위아래로 하나의 세포로 사채를 해리합니다. 2 % FBS / DPBS에 5 분에 resuspend 세포 200 XG에서 원심 분리하여 세포를 수집합니다. 40 μm의 셀 스트레이너 및 FACS 정제 RFP + CPC 세포와 필터 세포로 1.4.3에 설명. </li><li> 플레이트 geltin 코팅 된 플레이트 상에 HCPCS 정렬. 7 분화 배지에서 배양 HCPCS-9 일 심근 세포 및 평활근 세포로 분화한다. 칠일 도금 후, 문화 DMEM / F12 배지에서 5 % 녹아웃 SR과 세포를 분화. </li></ol></li></ol>

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S., Barbas, T. A. L. 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=ZFN,+TALEN,+CRISPR/Cas-based+methods+for+genome+engineering.">ZFN, TALEN, CRISPR/Cas-based methods for genome engineering.</a> Trends in Biotechnology. 31 (7), 397-405 (2013).</li><li>Joung, J. K., Sander, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALENs:+a+widely+applicable+technology+for+targeted+genome+editing.">TALENs: a widely applicable technology for targeted genome editing.</a> Nature Reviews. Molecular Cell Biology. 14 (1), 49-55 (2013).</li><li>Yang, H., 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=One-step+generation+of+mice+carrying+reporter+and+conditional+alleles+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.</a> Cell. 154 (6), 1370-1379 (2013).</li><li>Mali, P., 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li><li>Laflamme, M. 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=Cardiomyocytes+derived+from+human+embryonic+stem+cells+in+pro-survival+factors+enhance+function+of+infarcted+rat+hearts.">Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.</a> Nature. biotechnology. 25 (9), 1015-1024 (2007).</li><li>Burridge, P. W., 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=A+universal+system+for+highly+efficient+cardiac+differentiation+of+human+induced+pluripotent+stem+cells+that+eliminates+interline+variability.">A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.</a> PloS One. 6 (4), e18293 (2011).</li><li>Kouskoff, V., Lacaud, G., Schwantz, S., Fehling, H. 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The authors declare no competing financial interests.

This protocol combines a method using growth factors to guide mESCs differentiation and spontaneous differentiation of human ESCs into CPCs. CPC lineage marked with fluorescent reporter is used to efficiently identify and isolate CPCs by FACS. The FACS-purified CPCs retain the capacity to differentiate into cardiomyocytes, smooth muscle, and endothelial cells and have a comparable expression profile to the in vivo cells. Thus, these CPCs can serve as a great resource for cell based heart therapy because of their ability to form both cardiac muscle and vessel structures. 
One critical step in this protocol is to identify the correct timing associated with high purity and quantity of CPCs during in vitro differentiation. There are many conditions (e.g., serum lot number, cell line, etc.) that might affect differentiation of pluripotent stem cells into CPCs. Pilot experiments for optimization of differentiation and determination of the right time window for isolation of CPCs are strongly recommended.
The purification of CPCs in this protocol depends on the fluorescent reporter which is activated by tissue specific cre recombinase. The reporter specificity relies on the fidelity of the promoter of cardiovascular progenitor markers and cre recombinase. The recent development of genome editing tools such as CRISPR/Cas9 system allows efficient generation of a fluorescent reporter in ESCs16-21. Therefore, these technologies may be used to engineer more stringent and better CPC-reporter cell lines in future. Subsequently, the use of FACS-purification of CPCs may be widely applied to these superior lines.
Another important consideration is the combination of CPC reporters with growth factor treatment. In this protocol, human ESCs are not treated with growth factors to enhance the differentiation efficiency. Many approaches have been described for directed and efficient cardiomyocyte differentiation of human ESCs/iPSCs with growth factors. For example, highly efficient cardiomyocyte generation can be achieved by monolayer culture of hESCs/iPSCs with high levels of Activin A and BMP422, and the formation of human EBs with growth factors13,23-25. Recently, a sequential application of Glycogen synthase kinase 3 (GSK-3) inhibitor CHIR99021 and Wnt/&#946;-catenin pathway inhibitor IWP2/4 has also been reported to drive robust cardiomyocyte differentiation of human pluripotent stem cells26. Even though these methods are focused on cardiomyocyte differentiation but not on CPCs, combining these methods with the CPCs reporter system should enable a higher percentage of CPCs differentiation. Furthermore, by combining the genetic modifications with these cardiomyocyte differentiation protocols, it will be possible to isolate and then differentiate CPCs into distinct cell lineages as desire, such as ventricular and atrial myocytes27. 
It is worth noting, that the efficacy of ESCs EB/monolayer based differentiation and the effects of growth factors vary considerably among different lines. Therefore, selection of optimal ESC lines for CPC differentiation should be considered carefully.

A correction to the author list was made to: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/52047/derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells">Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells</a>.
The following author is now listed as a co-corresponding author:
Lei Bu

A correction to the author list was made to: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells.

Erratum: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

심장 질환은 세계에서 가장 주요 원인 오늘날 남아 사망률은 지난 20 년 (미국 심장 협회)에서 이뤄지지 않고있다. 효과적으로 방지 또는 심부전를 취소하는 신규 한 치료 전략의 개발에 대한 중요한 필요가있다. 한가지 유망한 전략은 줄기 세포 생물학 (1)의 급속한 발전 다음 세포 기반 치료이다. 이와 관련, 다 능성 클릭당 비용 (CPC)으로 인해 증식하지만 심장 혈통 차별화하기 위해 최선을 다하고 자신의 능력으로 치료를위한 우수한 셀 소스 수 있습니다. 따라서, 효율적이고 강력한 방법은 생성과 심장 세포 치료제 연구를위한 매우 중요하다 클릭당 비용 (CPC)을 분리합니다. 이 프로토콜은 초기 배 발생 및 방법는 ESC에서 자신의 세대에서 확인 된 배아 클릭당 비용 (CPC)에 초점을 맞추고있다. 다양한 클릭당 비용 (CPC)에도 뼈 된 marrows 2에서, 배아와 성인 마음에서 고립되었다. 배아 개발하는 동안, 뼈 morphoge마그네틱 단백질 (BMP 형식)은, 날개 형 MMTV 통합 사이트 가족 (Wnts)와 마디 신호는 Mesp1 + 능성 중배엽 3의 헌신을 유도한다. Mesp1 + 세포는 배아의 CPC 사로 분화. 이 클릭당 비용 (CPC)은 일반적으로 NK2의 호 메오 박스 (5) (Nkx2-5가), ISL LIM의 호 메오 박스 (1) (Isl1가), T-상자 5 (Tbx5) 및 심근 증강 인자 2C (Mef2c가), 차 및 제 마음 필드를 형성, HCN4으로 표시하고 있습니다 cardiogenesis ~ 10시 심장의 주요 부분에 기여하고 있습니다. Nkx2-5 + 및 Isl1 + / Mef2c + 두 클릭당 비용 (CPC)가 심근 세포로 분화 할 수있는, 평활근 세포 (SMCs) 및 내피 세포 5-8. 따라서 이들의 CPC 심장 혈관계뿐만 아니라 심장 조직을 야기하고 심장 세포 기반 치료를위한 이상적인 셀 소스있는 것이다. 결과적으로, 시험 관내에서 CPC를 생성하는 심혈관 연구에서 주요 연구 초점이되어왔다. 는 ESC 무제한 확장 용량을 가지고 있기 때문에ND 포배기에서 ICM 세포를 나타내고, 천연 배아 배아 다음의 CPC는 ESC로의 분화는 CPC를 획득하기 위해 논리적이고 효과적인 방법으로 간주된다. 는 ESC에서 클릭당 비용 (CPC)을 얻을 수있는 한 넓게 적용 방법은 사채 11로는 ESC를 집계하는 것입니다. 분화 효율을 향상시키기 위해, 심장 개발 기술에 기초하여 정의 화학적 및 성장 인자 12-14 사용되고있다. 그러나, 현장에서 널리 허용되는 명확한 CPC 마커, 특히 아니 세포 표면 마커는 없다. 이 문제를 해결하기는 ESC는 Isl1 + 또는 Mef2c + 클릭당 비용 (CPC) 및 Cre 호텔 /에 loxP 시스템을 사용하여 형광 기자들과 이들의 유도체를 표시하도록 설계되었습니다. CRE 재조합 효소는 Isl1 / Mef2c 프로모터 / 인핸서의 제어에 노크한다. 구성 적 프로모터에 의해 구동되는 형광 단백질의 변성 또는 RFP YFP 유전자 CRE 재조합 효소와 플록스 정지 코돈의 절단에 의해 활성화 될 수있다(ISL1 : CRE;-pCAG - 플록스-STOP-플록스 GFP 또는 RFP / Isl1-CRE; Rosa26YFP / Mef2c-CRE; Rosa26YFP) 5,6. 는 ESC가 두 번째 심장 필드 클릭당 비용 (CPC)로 분화되면, Isl1 / Mef2c 프로모터 / 증강 구동 CRE는 형광 기자를 활성화하고 클릭당 비용 (CPC)은 FACS 정화에 의해 강화 될 수있다. 간략하게, EB 집계 방법은 ESC의 분화를 시작하는 데 사용된다. 분화 효율을 향상시키기 위해, 분화 세포는 아스코르브 산 (AA) 및 BMP4, 티빈 13,15 VEGF와 같은 성장 인자로 처리된다. 이 프로토콜은 마우스와 인간는 ESC를 모두 사용하여 강력하고 효율적인 CPC 차별화 할 수 있습니다.

<table><tbody><tr><td>FBS</td><td>Thermo scentific</td><td>SH30070.03E</td><td></td></tr><tr><td>Knockout SR</td><td>Life technology</td><td>10828028</td><td></td></tr><tr><td>Knockout DMEM</td><td>Life technology</td><td>10829018</td><td></td></tr><tr><td>DMEM</td><td>Life technology</td><td>11965118</td><td></td></tr><tr><td>NEAA</td><td>Life technology</td><td>11140050</td><td></td></tr><tr><td>GlutaMAX</td><td>Life technology</td><td>35050061</td><td></td></tr><tr><td>N2</td><td>Life technology</td><td>17502048</td><td></td></tr><tr><td>B27</td><td>Life technology</td><td>12587010</td><td></td></tr><tr><td>Ham&amp;rsquo;s F12</td><td>Life technology</td><td>11765062</td><td></td></tr><tr><td>IMDM</td><td>Life technology</td><td>12440061</td><td></td></tr><tr><td>Pen/Strep</td><td>Life technology</td><td>15140122</td><td></td></tr><tr><td>Pyruvate</td><td>Life technology</td><td>11360070</td><td></td></tr><tr><td>Dispase</td><td>Life technology</td><td>17105041</td><td></td></tr><tr><td>Stempro-34</td><td>Life technology</td><td>10639011</td><td></td></tr><tr><td>DMEM/F12</td><td>Life technology</td><td>11330032</td><td></td></tr><tr><td>BSA&amp;nbsp;</td><td>Life technology</td><td>15260037</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Life technology</td><td>25200056</td><td></td></tr><tr><td>Ascobic Acid&amp;nbsp;</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>1-Thioglycerol</td><td>Sigma</td><td>M1753</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>VEGF</td><td>R&amp;amp;D</td><td>293-VE</td><td></td></tr><tr><td>Bmp4</td><td>R&amp;amp;D</td><td>314-BP</td><td></td></tr><tr><td>ActivinA</td><td>R&amp;amp;D</td><td>338-AC</td><td></td></tr><tr><td>bFgf</td><td>R&amp;amp;D</td><td>233-FB</td><td></td></tr><tr><td>Fgf10</td><td>R&amp;amp;D</td><td>345-FG</td><td></td></tr><tr><td>mTeSR</td><td>Stemcell technologies</td><td>5850</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354277</td><td></td></tr></tbody></table>

derivation of cardiac progenitor cells from embryonic stem cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

이 프로토콜은 여러 ES 세포주에서 클릭당 비용 (CPC)의 유도를 보여줍니다. 는 ESC는 클릭당 비용 (CPC)로 분화하는 교환 사채를 형성하기 위해 집계됩니다. 는 ESC 정기적으로 MEF 피더 (그림 1A, F)가 유지 관리 및 공급 장치는 차별화 전에 제거됩니다. 분화 배지에서 EB를 (도 1b, G)는 ESC에 응집하면, EB를 형성 제는 마우스 세포주 (도 1C)에서 중배엽 분화를 향상 BMP4 티빈과 함께 처리된다. 형광 단백질 YFP / RFP (그림 1D)에 의해 식별는 ESC는 심장 계보로 분화된다. 클릭당 비용 (CPC) (그림 1H) FACS 정제 및 심근 세포 및 평활근 세포로 분화하는 것이 더 배양 하였다. cTnT의 MHC-SM과의 염색 심근 및 평활근 세포 (그림 1 E, I, J)의 CPC로의 분화 능력을 보여 주었다. 일반적으로 80-90 %의 클릭당 비용 (CPC) 마우스 ESC 차별화 제자에서 얻을 수 있습니다ocol와 0.5 % 클릭당 비용 (CPC)은 인간의 ESC 분화 프로토콜에서 얻을 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/52047/52047fig1highres.jpg" /> 그림 1 : 다 능성 클릭당 비용 (CPC)으로는 ESC의 차별화. (AE) 마우스 ESC 차별화. 피더 세포의 마우스 ESC의 (A) 형태학. (B) 마우스는 ESC 먼저 EB 형성. (C) 성장 인자 분화 배지에서 둘째 EB 형성. (D)의 CPC는 YFP을 표현한다. (E) 심근 (cTnT의 +) FACS - 정제 클릭당 비용 (CPC)에서 유래. 인간는 ESC의 (FJ)의 분화. 인간 ESC의 (F) 형태학 피더 세포에서 유지. 인간 ES 세포 (G) EB 형성. (H) FACS 정제 클릭당 비용 (CPC)의. (IJ) 클릭당 비용 (CPC) 심근 (cTnT의 +) 및 평활근 세포로 분화 (SM-MHC + </s)&gt;까지. 스케일 바 : AC, F, G, 100 ㎛. D, E, I, J, 5 μm의. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52047/52047fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells at JoVE.com

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

배아 줄기 세포에서 심장 전구 세포의 유도

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

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8.1K 조회수.  University of Michigan. In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

비디오: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murine 배아 줄기 세포로부터 조혈 줄기 세포의 유도

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

연구

생물학

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

BMP 길항제 Grem2를 사용하여 다 능성 줄기 세포에서 심방 심근의 차별화

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

발생학

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

심장 세포 치료를 위한 줄기 세포 DNA 무결성을 평가

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

유전학

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 ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

첫 번째 심장 필드 같은 심장 창시자와 같은 심 실 Cardiomyocytes 인간 만능 줄기 세포에서의 세대

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

낮은 혈 청 조건 하에서 심장 조상 세포에서 내 피 분화의 유도

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

마우스 심장 필드 특정 심장 전구 세포의 시험관 내 생성

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

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 ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

인간의 Cardiomyocytes의 생성 : 피더없는 인간 유도 만능 줄기 세포에서 분화 프로토콜

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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 ...