Name: generation of first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells
Uploaded: 2018-06-19T14:00:00
Description: Watch this Scientific Journal Video about Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells at JoVE.com

우리는 유용한 토론 Colas 실험실의 구성원 및 원고의 중요 한 리뷰 감사합니다. 이 연구는 NIH/NIEHS R44ES023521-02에 의해 지원 되었다 고 CIRM DISC2-10110 박사 Colas를 부여 합니다.

1. Id1 바이러스 준비 및 감염
<ol><li>공동 transfecting pCMVDR8.74, pMD2.G, 및 pCDH EF1 Id1 PGK PuroR Id1 overexpressing lentivirus 생성 (Addgene: 플라스 미드 #107735) HEK293T 세포로. 바이러스 성 입자를 수집 하 고, 여과 하 고, 상쾌한에서 정화 하 고 무라 외 에서-80 ° C에서 저장 (2003) 19. 또는 pCDH EF1 Id1 PGK PuroR lentivirus 생산 공급 업체에 전송 하 여 Id1 lentivirus를 상업적으로 생산. 주의: lentiviral ?...

<ol>
	<li>Chong, J. 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=Human+embryonic-stem-cell-derived+cardiomyocytes+regenerate+non-human+primate+hearts.">Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.</a> Nature. 510, 273-277 (2014).</li><li>Kodo, 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=iPSC-derived+cardiomyocytes+reveal+abnormal+TGF-beta+signalling+in+left+ventricular+non-compaction+cardiomyopathy.">iPSC-derived cardiomyocytes reveal abnormal TGF-beta signalling in left ventricular non-compaction cardiomyopathy.</a> Nature cell biology. 18, 1031-1042 (2016).</li><li>Kim, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+arrhythmogenic+right+ventricular+dysplasia+with+patient-specific+iPSCs.">Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs.</a> Nature. 494, 105-110 (2013).</li><li>Colas, A. 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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=Chemically+defined+generation+of+human+cardiomyocytes.">Chemically defined generation of human cardiomyocytes.</a> Nature methods. 11, 855-860 (2014).</li><li>Kattman, S. 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=Stage-specific+optimization+of+activin/nodal+and+BMP+signaling+promotes+cardiac+differentiation+of+mouse+and+human+pluripotent+stem+cell+lines.">Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.</a> Cell stem cell. 8, 228-240 (2011).</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, 1015-1024 (2007).</li><li>Lian, X., 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=Robust+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 1848-1857 (2012).</li><li>Willems, 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=Small+molecule-mediated+TGF-beta+type+II+receptor+degradation+promotes+cardiomyogenesis+in+embryonic+stem+cells.">Small molecule-mediated TGF-beta type II receptor degradation promotes cardiomyogenesis in embryonic stem cells.</a> Cell stem cell. 11, 242-252 (2012).</li><li>Yang, 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=Human+cardiovascular+progenitor+cells+develop+from+a+KDR++embryonic-stem-cell-derived+population.">Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.</a> Nature. 453, 524-528 (2008).</li><li>Palpant, N. 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성공적인 차별점에 대 한 밀접 하 게 지침에 따라 위에 나열 된 확인 합니다. 또한, 여기 우리가 강력 하 게 차별화 결과 영향을 주는 주요 매개 변수 강조 표시 합니다. 감 별 법을 시작 하기 전에 다음 세 개의 매개 변수 형태를 관찰 해야 합니다: hPSCsId1, 높은 세포 압축 및 높은 합류의 줄기 형태 (> 90%) 0에서 문화. 그런 측면에서 최적의 차별화 조건 단일 세포로 도금 해리 hPSCsi d 1 ...

인간 만능 줄기 세포 (hPSCs)의 대규모 생산-파생된 심장 창시자 및 cardiomyocytes는 줄기 세포 기반 요법1,2,3 와의 빠른 특성을 모델링 하는 질병에 대 한 필수 새로운 통로 규제 심장 차별화4,,56 과 생리학7,8. 비록 연구9,10,11,12,,1314의 번호,15 는 앞에서 설명한 고효율 심장 차별화 프로토콜 hPSCs에서, 아무도 중요 한 분자 차이 왼쪽의 식별도 결과 cardiomyocytes의 심장 필드 근원 해결 (첫 번째 심장 분야) 및 오른쪽 (두 번째 심장 분야) 심 실 cardiomyocytes16 과 심장 분야 특정 선 천 성 심장 질환;의 존재 즉, hypoplastic 좌 심 혼 증후군17 또는 arrhythmogenic 오른쪽 심 실 발육 이상18. 따라서, 심장 창시자의 생성 및 hPSCs에서 정의 된 심장 분야 원산지 cardiomyocytes 되고있다 치료로 그들의 관련성을 증가 하기 위하여 필요성 및 질병 모델링 도구.
이 프로토콜의 Id1, 최근 확인 된5 첫 번째 심장 필드 지정 큐 필요 하 고 충분 한 hPSCs에서 cardiogenesis를 시작 하는 Activin A와 함께에서 하는 제정 overexpression에 의존 합니다. 특히, 커닝 햄 외. (2017) 5 표시는 Id1 유도 창시자 구체적으로 표현 첫 심장 필드 (HCN4, TBX5) 하지만 하지 두 번째 심장 필드 마커 (SIX2, ISL1) 그들은 심장 차별화를 받 다. 또한, 저자는 또한 표시 유전자 변형 마우스 배아의 유전자 (i d 1-4), 전체 Id 가족 부족 첫 심장 필드 심장 창시자, 더 많은 중간과 뒷부분 심장 창시자 (동안 형성 없이 개발 두 번째 심장 필드) 아직도 그로 인하여 Id 단백질 필수적인 첫 번째 심장 필드 cardiogenesis에서 vivo에서시작 하는 제안, 형성할 수 있다. 편리 하 게, Id1 유도 창시자 cryopreserved 수 고 저절로 cardiomyocytes 표시 심 실 같은 특성을 포함 하 여 심 실 전용 마커 (IRX4, MYL2) 식으로 차별화 하 고 같은 심 실 활동 전위.
여기는 첫 번째 심장 필드 같은 (FHF-L) 심장 창시자와 같은 심 실 cardiomyocytes Id1 overexpressing hPSCs에서 생성 하는 간단 하 고 확장 가능한 방법에 설명 합니다. 이 프로토콜의 중요 한 특징은 편리한 cryopreservation 단계를 사용 하 여 후속 cardiomyocyte 생산에서 심장 조상 세대 연결을 푸십시오 가능성. 요약 하자면,이 프로토콜 (1) 생성 Id1 overexpressing hPSCs, (2) hPSCs에서 FHF L 심장 창시자를 생성, (3) cryopreserve 결과 창시자, 및 (4) 재개 FHF L 심장 조상 차별화에 필요한 단계를 자세히 설명 하 고 생성 매우 농축 (> 70-90%) 심 실 같은 cardiomyocytes 박동.

<table border="0" cellpadding="0" cellspacing="0" style="width:911px;" width="910">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:204px;">ACTC1 antibody</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:119px;">A7811</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activin A</td>
			<td>Stem Cell Technologies</td>
			<td>Hu Recom Activin A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic Antimycotic (Anti-Anti)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement w/o - insulin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement w/o - vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CDH5 antibody</td>
			<td style="width:144px;">R&#38;D Systems</td>
			<td>AF938</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoStor CS10</td>
			<td>Stem Cell Technologies</td>
			<td>7930</td>
			<td style="width:444px;">Cryopreservation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM high Glucose</td>
			<td>Mediatech</td>
			<td>10-013-CV&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS w/ Ca &#38; Mg</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>VWR</td>
			<td>89510-186</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FluoVolt membrane potential kit&#160;&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>F10488</td>
			<td>For optical action potential acquisition, please refer to McKeithan et al. 2017</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel, Growth Factor Reduced</td>
			<td>Corning</td>
			<td>356231</td>
			<td style="width:444px;">Coating reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 media kit</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS w/o Ca &#38; Mg</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Puromycin&#160;</td>
			<td>Acros</td>
			<td>227422500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td style="width:444px;">Enzyme-free dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TAGLN antibody</td>
			<td style="width:144px;">Abcam</td>
			<td>ab14106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thiazovivin</td>
			<td>Stem Cell Technologies</td>
			<td>72254</td>
			<td style="width:444px;">RHO/ROCK pathway inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE Express</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605 -010</td>
			<td style="width:444px;">1X enzyme-containing dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tyrodes solution mix packets&#160;&#160;</td>
			<td>Sigma</td>
			<td>T2145-10X1L</td>
			<td>(For optical action potential acquisition, please refer to McKeithan et al. 2017)</td>
		</tr>
	</tbody>
</table>

generation of first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells

많은 양의 기능 인간의 pluripotent 줄기 세포 파생 심장 창시자의 정의 심장 분야 원산지 cardiomyocytes 세대 셀 기반 심장 치료 및 질병 모델링에 대 한 필수입니다. 우리는 최근 Id 유전자는 필요 하 고 척추 개발 하는 동안 첫 번째 심장 필드 창시자 지정 하려면 충분 한 나타났습니다. 이 분화 프로토콜 이러한 연구 결과 활용 하 고 첫 번째 심장 필드 같은 (FHF-L) 창시자를 생산 하 강력한 지정 큐로 Activin A와 함께에서 Id1 overexpression를 사용 합니다. 중요 한 것은, 결과 창시자 효율적으로 심 실 같은 cardiomyocytes에 (~ 70-90%)를 구분합니다. 여기는 1) 생성 Id1 overexpressing hPSCs 2) 차별화 cryopreservable FHF L 창시자와 같은 심 실 cardiomyocytes의 확장 가능한 수량을 상세한 방법에 설명 합니다.

HPSCs의 생성 I d 1 라인 hPSCs 중재 Id1 overexpression (그림 1A) lentivirus 감염 됩니다. HPSCId1 생성 됩니다 일단 transgene 식 qRT-PCR (그림 1B)에 의해 정량입니다. 그 GAPDH 0.005 배 보다 높은 레벨에서 Id1 mRNA 표현으로 하는 hPSCId1 선만 차별?...

Watch this Scientific Journal Video about Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells at JoVE.com

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

여기 우리 Activin A와 overexpression-lentivirus 중재 Id1의 간단한 조합을 사용 하 여 첫 번째 심장 필드 같은 심장 창시자와 같은 심 실 cardiomyocytes 인간 만능 줄기 세포에서 생성 하는 확장 메서드를 설명 합니다.

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 ...

The main advantage of this simple technique is to generate cardiac progenitors and cardiomyocytes of defined heart field origin and subtypes. In turn, resulting cells can be used for a wide range of research application, including basic developmental biology, proof-of-concept cell-based therapy, and cardiac disease modeling studies. Demonstrating the procedure will be Sean Spiering and Michael Yu from my laboratory.To begin, coat enough wells of a 96-well plate with 50 microliters of coating reagent to accommodate subsequent cell seeding and viral infection steps, then add one milliliter of dissociation reagent to a well in the six-well plate to detach the pluripotent stem cells seeded. Then, use a sterile pipette to transfer the cell suspension in a 15-milliliter plastic centrifuge tube. In the tube, add five milliliters of PBS containing calcium and magnesium to neutralize the dissociation reagent.Then centrifuge the cells at 200 times g for three minutes. Post-centrifugation, discard the supernatant and then gently flick the tube for the pellet to be dislodged. Then dissolve the cell pellet in one milliliter of stem cell medium.Next, use an automated cell counter to count the number of cells. Once the counting is done, seed 20, 000 viable cells in each well of a 96-well plate containing 50 microliters of stem cell medium supplemented with two-micromolar RHO/ROCK pathway inhibitor. After 24 hours, monitor the cell attachment, then replace the medium with 100 microliters of stem cell medium one hour prior to the lentiviral infection.Next, thaw the Id1 lentivirus on ice, then add three microliters of purified lentivirus per well. After adding the lentivirus, transfer the plate back to cell culture incubator at 37 degrees Celsius with 5%carbon dioxide. 24 hours post-viral infection, add 100 microliters of fresh stem cell medium to the infected cells.After 48 hours, replace the virus-containing medium with 200 microliters of fresh stem cell medium supplemented with one microgram per milliliter of puromycin. Every day, keep changing the medium with fresh 200 microliters of stem cell medium supplemented with one microgram per milliliter of puromycin. Start the process of differentiation on Day 0 by replacing the stem cell medium with 1.5 milliliters of induction medium with Activin A in a 12-well plate.24 hours after differentiation has started, replace the medium with two milliliters of induction medium without Activin A.On the third day, replace with two milliliters of induction medium without Activin A.On the fifth day, aspirate the entire medium from the well containing the FHF-L CPs, then immediately add one milliliter of warm enzyme-containing dissociation reagent in the well, then incubate the culture plate at 37 degrees Celsius. After a minute, shake the plate inside the incubator and remove it from the incubator after two minutes, then add one milliliter of 10%fetal bovine serum-containing medium in the well. Detach the cells from the plate by gently pipetting the cells up and down.Next, transfer the cell suspension to a centrifuge tube and subject to centrifugation. After the centrifugation, dissolve the cell pellet in two milliliters of cryopreservation reagent, then count the cell number. Then transfer the cells in the cryopreservation vials and leave the vials in the cooling device.Then transfer the device at minus 80 degrees Celsius overnight. After 24 hours, transfer the vials in liquid nitrogen for long-term storage. Just before thawing the FHF-L CPs, coat the wells of the 12-well plate with two milliliters of coating reagent.Next transfer the five-days old FHF-L CPs from the liquid nitrogen in a dry ice container. From the dry ice, transfer the vial in a water bath at 37 degrees Celsius for two minutes to ensure complete thawing. After the cells are completely thawed, transfer the cells to a centrifuge tube, then pipette 10 drops of pre-warmed cardiogenic medium every 30 seconds to the cell solution.Keep adding the cardiogenic medium til the cryopreservation and cardiogenic medium ratio reaches one to three. Next subject the cells to centrifugation. Post-centrifugation remove the supernatant and carefully flick the tube, then dissolve the cell pellet in cardiogenic medium supplemented with two-micromolar RHO/ROCk pathway inhibitor.Further dilute the cell suspension with cardiogenic medium to achieve the desired cell concentration. Then seed the cells on the culture plate containing two milliliters of cardiogenic medium. Leave the plate in the tissue culture hood for 20 minutes before transferring to the incubator.Monitor the cell attachment on the sixth day of differentiation. On the seventh day, aspirate half of the cardiogenic medium and replace it with fresh medium. From here on out, replace 50%of the cardiogenic medium every other day, then, on the 15th day, quantify the cardiac differentiation.Here the expression level of Id1 mRNA is quantified in the hESC and hPSC cell lines. Only hPSC lines expressing Id1 mRNA at levels greater than 0.005-fold GAPDH are used for differentiation. Shown here are brightfield images post-differentiation of optimal, sub-confluent, and over-confluent hPSCs at Day 0.Upon further differentiation, the cell confluency reaches 75%after 24 hours. At Day 3, the differentiating cells form a cluster, and, by Day 5, the cells remain in tightly-connected clusters with homogenous morphology. At Day 6, the FHF-L CPs cover more than 90%of the surface area of the wells.In fact, by Day 12, the cardiomyocytes start to beat and display cardiac contractions. Further into the differentiation process, there are increased mRNA expression levels of MYL2 and IRX4 after Day 15. Both proteins represent ventricular-specific markers within the cardiomyocytes.Here the action potential trace represents the rapid upstroke and downstroke velocities as well as the ventricular plateau. These rates were characteristic of ventricular-like cardiomyocytes at Day 25 of differentiation. In summary, it takes about two weeks to generate Id1 over-expressing human pluripotent stem cells, then it takes about five days to generate first heart field-like progenitors and about 10 days to generate beating ventricular-like cardiomyocytes.

Research

JoVE Journal

Developmental Biology

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