이 연구는 NIH 4R00HL094708-03에 의해 지원되었다, MCW 리서치 업무위원회 새로운 교수 상, 위스콘신 의과 대학 (RLG)에서 컨 재단 (시작 자금); 홍콩 테마 기반의 연구 계획 T13-706 / 11 (KRB)의 연구 보조금위원회; U01 HL099776, CIRM TR3-05556, CIRM DR2A-05394 및 AHA는 탐정 수상 (JCW) 설립; AHA 박사후 원정대 12POST12050254 (PWB). 우리는 데이터 수집 및 원고의주의 깊은 검토에 대한 지원은 위스콘신의 혈액 연구소의 유동 세포 계측법 코어에 희망 캠벨 감사합니다.

저자가 공개하는 게 없다.

분화 프로토콜의 성공에 중요 분화의 개시 전에 적어도 다섯 통로위한 단일 세포 수준에서 계대되었다 hPSCs 고품질 배양의 사용이다. 그들은 세포주 독립적 분화 개시시 100 % 합류, 경우 유사 분화 효율 일상적 다양한 hPSC 라인 중에서 관찰된다. 분화의 개시시 세포의 합류 ≤95 % 또는&gt; 100 % 인 경우 차선 효율이 관찰된다. 따라서, 파종 밀도 및 계대의 타이밍은 분화의 시작에 100 % 컨 플루 언트 단...

hESC의와 hiPSC 포함 hPSCs,에서 심근의 발생은 기계 론적 연구, 그렇지 않으면 접근 할 수없는 단계에 대한 통찰력을 제공하고, 초기 인간의 심장 발달 과정의 체외 모델로 작동 할 수 있습니다. 이 모델 시스템은 심장 계통의 의지와 세포 운명 사양을 제어하는​​ 분자 경로를 연구하는 독특한 기회를 제공합니다. 최근, 효율적 hPSCs에서 cardiomyogenic 세포를 생성하는 능력이 크게 향상되었다 1-15. 그러나, 프로토콜들 세포는 챔버 관련 마커 (예를 들어, 심실과 심방)를 발현하는 세포와 cardiomyogenic 타이밍을 생성하는 효율에 대해 세포주 편차가있다. 이상적으로,이 모델 시스템의 미래 응용 프로그램, 기능적으로 정의 된 세포의보다 균일 한 집단이 요구된다. 이전 방법과 대조적으로, 심근 분화 프로토콜 CE 필요없는 여기서 설명한LL 집계 또는 견고 티빈 또는 뼈 형태 형성 단백질 4 (BMP4)과의 추가는 일에 의해 TNNI3, TNNT2, IRX4, MLC2v 및 MLC2a에 대한 문화가 매우 긍정적 인 일에 테스트 한 모든 hESC의와 hiPSC 라인에 걸쳐 10 세포를 생성합니다. 이 전략은 특히 입체 문화, 대중 문화, 또는 배아 체를 기반으로 전략 4-9에 비해 구현하기가 기술적으로 간단하고, 최근 hPSCs에 대한 선택적 독성을 가진 작은 분자를 설명하는 연구 (Boheler 등으로 정의 하였다. ) 65. 이 프로토콜의 특징은 작은 분자를 사용하여 완전하게 정의 미디어 (아무런 구별 1,2,7,13에서 유사) Wnt 신호를 변조 hESC의 자격을 갖춘 매트릭스 (마트 리겔)의 단일 층을 사용하여 단일 층 문화 hPSCs의 분화를 포함하고, 분화 효율 및 셀 아이덴티티 평가 염색 방법 최적화 유동 세포 계측법. 이전 보고서는 비용 effectiv을 포함에 요약하면,이 프로토콜의 장점을 비교eness, 재현성 및 hiPSC hESC의 라인을 포함하여 여러 hPSC 라인 중 심근를 생성하기위한 고효율. 유동 세포 계측법에 배양 세포의 품질을 평가하고 모집단의 균질성을 결정하기위한 강력한 분석 도구이며, 적절한 실험 설계로, 정량적 측정을 제공 할 수있다. 모든 항체 기반의 전략으로, 실험 결과의 정확한 해석은 하위 최적의 조건이 크게 항체의 효율성에 영향을 미치는로 항체 농도와 고정 및 permeabilization 조건 (세포 내 항원을 표적)을 포함하여 분석 디자인의 요소주의 깊게 각 항체 테스트해야 결과의 해석을 결합하고, 따라서. 정량 분석​​이 필요하다면 폴리 클로 날 항체가 여러 에피토프를 인식하고 뱃치 변화하는 경향이있다 중요한 것으로, 단일 클론 항체는 필수적이다. 현재 항체의 다양한 (포lyclonal 및 모노클로 날) 및 염색 프로토콜 어려운 프로토콜 1,2,9,11 중 cardiomyogenesis의 효율을 비교하기 위하여, 시험 관내 분화의 평가를 위해 설명되었다. 모든 유동 세포 계측법 분석을 위해 사용 가능한 경우 그 이유를 들어, 단일 클론 항체가 사용된다. 향후, 그것은 더 나은 차별화 전략 간의 비교를 허용해야, 특히 정량와 관련하여,이 염색 프로토콜의 표준화를 것으로 예상된다. 체외 cardiomyogenesis의 순도를 평가하기 위해 사용 된 마커의 선택 및 해당 항체는, 리포트 중에서 변한다. TNNT2는 cardiomyogenic 운명 커밋 세포의 지표로 간주되었으며 심장 일상적 분화 프로토콜의 효율을 평가하기 위해 사용된다. 그러나 TNNT2은 초기 병아리와 쥐 개발 (16, 17) 중 골격근에서 발현과 인간의 평활근 (18)에 존재한다 </sup&gt;. 따라서, TNNT2 반드시 ​​체외에서 인간의 cardiomyogenesis의 특정 마커 없습니다. MLC2v 및 MLC2a 일상적으로 각각 심실 및 심방 하위 유형의 대리 지표로 사용된다. 그러나, 체외 분화의 컨텍스트에서 심근 하위 유형을 판별 MLC2v 및 MLC2a에 의존하고있는 도전이 유전자 산물이 성인 통해 중심 튜브에서, 심장 개발 걸쳐 특정 챔버로 제한되지 않을 수 있다는 사실로부터 발생한다. 짐승의 마음을 루프에서 MLC2a의 mRNA는 심방 / 유입 관의 지역에서 우세하고 MLC2v의 mRNA는 심실 / 유출 요로 지역에서 지배적이다. 루프 중심부에 위치한 MLC2a 및 MLC2v의 mRNA의 공동 발현은 유입 경로, 방실 운하 및 유출 관 (19, 20)에서 관찰된다. 출생 후 삼일으로 MLC2v의 mRNA는 심실로 제한되고, 출생 후 십일에 의해, MLC2a는 신생아 쥐의 심장 (19)에 심방으로 제한됩니다. 따라서 해석cardiomyogenesis 효율성과 하위 정체성은 기준 마커 레벨의 존재와 양을 고려하지해야하지만, 분석 분화 timepoints이 해당하는 발달 단계 (들)을 고려해야한다에 관한 데이터. 이 hPSCs의 체외 분화에 의해 생성 cardiomyogenic 세포의 성숙 단계가 가장 밀접하게 배아 / 태아 발달 21-25들과 유사한 점을 고려 특히 중요합니다. 따라서, 출생 후 심장 마커의 공간 표현에 의존하는 것은 적어도 어떤 경우에는 hPSC 유래 세포의 평가를 위하여는 적절하지 않을 수 있습니다. 체외에서 심근 ID를 정의하는보다 구체적인 기준의 개발을 용이하게하기위한 노력으로, TNNI3는 그것이 병아리 및 지브라 피쉬 15,20에서 배아 걸쳐 심근로 제한으로서 체외에서 cardiomyogenesis을 평가하기위한 유용한 마커로 간주인간 태아의 골격 근육 (26)의 존재이다. TNNI1 인간 태아 심장에 존재하지만, TNNI3 정상 성인의 심장 (27, 28)의 유일한 TNNI 이소 존재입니다. 심근 하위 정체성과 관련하여, IRX4 29 ~ 31는 심실 운명 세포의 정보 마커입니다. 단백질 수준에서 IRX4 최근 마우스 (32)에 신생아 단계를 통해 선형 심장 관에서 심실로 제한하는 것으로 나타났다. 따라서, 유동 세포 계측법에 의해 TNNI3의 분석 및 최적화를위한 IRX4 염색 프로토콜을 설명한다. 우리가 알기로는,이 유동 세포 계측법에 의한 효율적인 항체 기반 염색 인간 심근에 IRX4 수준의 분석을위한 방법의 제 설명이다.

<table border="0" cellpadding="0" cellspacing="0" width="922">
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		<tr height="34">
			<td height="34" style="height:34px;width:431px;">Name of Reagent / Equipment</td>
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			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Cell Culture</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">BD Matrigel, hESC-qualified matrix</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:111px;">354277</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Accutase cell detachment solution</td>
			<td style="width:152px;">Stem Cell Technologies</td>
			<td style="width:111px;">7920</td>
			<td></td>
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		<tr height="34">
			<td height="34" style="height:34px;width:431px;">Dubelcco&#39;s phosphate buffered saline (DPBS), no calcium, no magnesium</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">14190-136</td>
			<td></td>
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			<td height="17" style="height:17px;width:431px;">Dimethyl sulfoxide (DMSO, Hybri-Max, sterile-filtered)</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">D2650</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Sodium bicarbonate</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">53817</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Citric acid</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">C2404-100G</td>
			<td></td>
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			<td height="17" style="height:17px;width:431px;">Y-27632 dihydrochloride selective p160ROCK inhibitor</td>
			<td style="width:152px;">R&#38;D Systems</td>
			<td style="width:111px;">1254</td>
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			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">A8960-5G</td>
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			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">S5261-10G</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Transferrin (Optiferrin &#8211; Defined, Animal-free, Recombinant Human)</td>
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			<td style="width:111px;">777TRF029</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Fibroblast growth factor 2 (FGF2)</td>
			<td style="width:152px;">R&#38;D Systems</td>
			<td style="width:111px;">4144-TC-01M</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Transforming growth factor beta 1 (TGF&#946;1)</td>
			<td style="width:152px;">Peprotech</td>
			<td style="width:111px;">100-21</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">DMEM/F12 with L-Glutamine and 15 mM HEPES</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">11330-032</td>
			<td></td>
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			<td height="17" style="height:17px;width:431px;">Insulin</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">I9278-5ML</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">CHIR-99021 HCl</td>
			<td style="width:152px;">Sellekchem</td>
			<td style="width:111px;">S2924</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">IWR-1</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">I0161</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">RPMI 1640 with L-Glutamine</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">B-27 Supplement Minus Insulin</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">A1895601</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">B-27 Supplement (with Insulin)</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">17504-044</td>
			<td></td>
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			<td height="17" style="height:17px;width:431px;">Fetal bovine serum</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">10437</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;"></td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
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			<td height="20" style="height:20px;width:431px;">Flow cytometry reagents</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Phosphate buffered saline (10X)</td>
			<td style="width:152px;">Quality Biological Inc.</td>
			<td style="width:111px;">119-069-151</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:431px;">Hank&#39;s balanced salt solution without Ca2+ and Mg2+</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">14175-095</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">BD Cytofix</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:111px;">554655</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">BD Phosflow perm buffer III</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:111px;">558050</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">16% Paraformaldehyde</td>
			<td style="width:152px;">Thermo Scientific</td>
			<td style="width:111px;">28906</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Methanol</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">179957-4L</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Acetone</td>
			<td style="width:152px;">Mallenckrodt Chemicals&#160;</td>
			<td style="width:111px;">2440-16</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Triton X-100</td>
			<td style="width:152px;">Amresco</td>
			<td style="width:111px;">M236-10ML</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Goat serum</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">16210-064</td>
			<td></td>
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			<td height="17" style="height:17px;width:431px;">Trypan blue solution, 0.4%</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">15250-061</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Materials</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">5 ml round bottom polystyrene tube (without cap)</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">352008</td>
			<td>for preparation of cells for flow cytometry</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">5 ml round bottom polystyrene tube (with 35 &#181;M cell strainer snap cap)</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">352235</td>
			<td>for preparation of cells for flow cytometry</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">2 ml rubber bulbs</td>
			<td style="width:152px;">Fischer Scientific</td>
			<td style="width:111px;">03-448-24</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">9&#34; borosilicate unplugged glass Pasteur pipets</td>
			<td style="width:152px;">BioExpress</td>
			<td style="width:111px;">P-2904-2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">0.65 mL microcentrifuge tubes, graduated, green</td>
			<td style="width:152px;">GeneMate</td>
			<td style="width:111px;">C-3259-G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">1.5 mL microcentrifuge tubes, graduated, red</td>
			<td style="width:152px;">GeneMate</td>
			<td style="width:111px;">C-3260-R</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">TC20 automated cell counter</td>
			<td style="width:152px;">BioRad</td>
			<td>145-0102</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Counting slides for TC20</td>
			<td style="width:152px;">BioRad</td>
			<td style="width:111px;">145-0011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">6 well flat bottom tissue-culture treated plate</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">353046</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">20 mL syringes</td>
			<td style="width:152px;">BD Plastipak</td>
			<td style="width:111px;">300613</td>
			<td>For filter sterilization of aliquots</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Sterile syringe filters, 0.2 &#181;m polyethersulfone</td>
			<td style="width:152px;">VWR</td>
			<td style="width:111px;">28145-501</td>
			<td>For filter sterilization of aliquots</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">250 mL filter system, 0.22 &#181;m polyethersulfone, sterilizing, low binding&#160;</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">431096</td>
			<td>For filter sterilization of bulk media&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">500 mL filter system, 0.22 &#181;m polyethersulfone, sterilizing, low binding&#160;</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">431097</td>
			<td>For filter sterilization of bulk media&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Antibodies and Isotype Controls</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Anti-TNNI3 (clone: 284 (19C7))</td>
			<td style="width:152px;">Abcam</td>
			<td>ab19615</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-TNNT2 (clone: 1C11)</td>
			<td>Thermo Scientific</td>
			<td>MA1-16687</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-MYL2 (MLC2v, clone: 330G5)</td>
			<td>Synaptic Systems</td>
			<td>310111</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-MYL7 (MLC2a, clone: 4E7)</td>
			<td>Abcam</td>
			<td>AB131661</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-IRX4&#160;&#160;</td>
			<td>Bioss</td>
			<td>BS-9464R</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Alexa Fluor 488 Goat anti-mouse IgG1&#160;</td>
			<td style="width:152px;">Life Technologies</td>
			<td>A21121</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Alexa Fluor 647 Goat anti-mouse IgG2a</td>
			<td style="width:152px;">Life Technologies</td>
			<td>A21241</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Alexa Fluor 488 Goat anti-mouse IgG2b</td>
			<td style="width:152px;">Life Technologies</td>
			<td>A21141</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Phycoerythrin(PE) Goat anti-rabbit IgG</td>
			<td style="width:152px;">R&#38;D Systems</td>
			<td style="width:111px;">F0110</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mouse IgG1</td>
			<td>BD Biosciences</td>
			<td>557273</td>
			<td>Isotype Control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mouse IgG2a</td>
			<td>eBiosciences</td>
			<td>16-4724-81</td>
			<td>Isotype Control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rabbit IgG-PE</td>
			<td>R&#38;D Systems</td>
			<td>IC105P</td>
			<td>Isotype Control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;"></td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">TaqMan Probesets for qRT-PCR</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ACTB</td>
			<td>Life Technologies</td>
			<td>Hs01060665</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Brachyury T</td>
			<td>Life Technologies</td>
			<td>Hs00610080</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CASQ2</td>
			<td>Life Technologies</td>
			<td>Hs00154286</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IRX4</td>
			<td>Life Technologies</td>
			<td>Hs01100809</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ISL1</td>
			<td>Life Technologies</td>
			<td>Hs00158126</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MESP1</td>
			<td>Life Technologies</td>
			<td>Hs01001283</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYH11 (SMMHC)</td>
			<td>Life Technologies</td>
			<td>Hs00224610</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">MYH6</td>
			<td>Life Technologies</td>
			<td>Hs01101425</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYL2 (MLC2v)</td>
			<td>Life Technologies</td>
			<td>Hs00166405</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYL7 (MLC2a)</td>
			<td>Life Technologies</td>
			<td>Hs01085598</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYOG</td>
			<td>Life Technologies</td>
			<td>Hs01072232</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NKX2.5</td>
			<td>Life Technologies</td>
			<td>Hs00231763</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NPPA</td>
			<td>Life Technologies</td>
			<td>Hs01081097</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">POU5F1</td>
			<td>Life Technologies</td>
			<td>Hs04260367</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SOX17</td>
			<td>Life Technologies</td>
			<td>HS00751752</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNI1</td>
			<td>Life Technologies</td>
			<td>Hs00913333</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNI2</td>
			<td>Life Technologies</td>
			<td>Hs00268536</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNI3</td>
			<td>Life Technologies</td>
			<td>HS00165957</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNT2</td>
			<td>Life Technologies</td>
			<td>Hs00165960</td>
			<td></td>
		</tr>
	</tbody>
</table>

high efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry

, 심장 손상을 복구하는 심장 독성이없는 새로운 치료제를 발견하고 정확하게 심장 질환을 모델링하기위한 전략을 향상시키기위한 방법을 개발하기위한 시급한 필요성이있다. 이러한 응용 프로그램은 &quot;접시에&quot;심장 근육을 생성하는 인간의 유도 만능 줄기 세포 (hiPSC) 기술을 악용 가능성이 높은 열정을 생성하고 있습니다. 최근, 효율적으로 인간 다 능성 줄기 세포 (hPSCs) 발 cardiomyogenic 세포를 생성하는 능력이 크게 다르게 접근 할 수없는 인간 심장의 초기 개발 단계를 모델링 우리에게 새로운 기회를 제공하는, 개선했다. 많은 이전의 방법과는 대조적으로, 심근 분화 프로토콜 세포 응집 또는 티빈 또는 BMP4의 추가를 요구하지 않는 여기서 설명하고 견고 심장 트로포 닌 고도로 양성 세포의 배양을 생성하여 I 및 T (TNNI3, TNNT2) iroquois- 클래스 homeodomain을 단백질IRX-4 (IRX4), 마이 오신 규제 경쇄 2, 심실 / 심장 근육의 이소 형 (MLC2v)과 미오신 규제 경쇄이 모든 인간 배아 줄기 세포 (hESC의)와 hiPSC 라인에서 일 10에 의해 심방 이소 형 (MLC2a는) 테스트 날짜입니다. 세포는 계대와 문화에 90 일 이상 유지 될 수있다. 전략은 기술적으로 구현하기에 간단하고 비용 효과적이다. 다 능성 세포로부터 유도 된 심근 세포의 특성은 종종의 mRNA 및 단백질 수준 모두에서, 기준 마커의 분석을 포함한다. 단백질 분석을 위해, 유동 세포 계측법에 배양 세포의 품질을 평가하고 모집단의 균질성을 결정하기위한 강력한 분석 도구이다. 그러나, 샘플 제조에서 기술 변화는 크게 유동 세포 계측법 데이터의 품질에 영향을 미칠 수있다. 따라서, 염색 프로토콜의 표준화는 다양한 차별화 전략 간의 비교를 용이하게한다. 따라서, IRX4, MLC2v, MLC2a, TNNI3 및 TNN의 분석을 위해 염색 프로토콜을 최적화흐름에 의해 T2 세포 계측법에 설명되어 있습니다.

0 일에, 세포는 콤팩트 형태 및 세포 파편 최소 100 % 융합 성이다. 1-2 일에, 그것은 중요한 세포 죽음 (40~50%)을 관찰하는 것이 일반적이지만, 부착 세포는 콤팩트 형태 (도 1a)을 유지한다. 이 시간 동안 미디어는 오렌지와 혼탁입니다. 핑크 미디어 과도한 세포 사멸을 나타내며,이 경우, 트리 판 블루로 확인하고 세포 사멸이 70 %를 초과하는 경우 중단. 세포는 3 ~ 4 일 동안 복구하고 밀도?...

Watch this Scientific Journal Video about High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry at JoVE.com

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

이 기사는 효율적으로 선택적으로 참조 마커의 분석은 인구의 동질성과 정체성을 평가하기 위해 유동 세포 계측법 다음에 대한 Wnt 경로를 변조하여 심근에 인간 만능 줄기 세포를 구별하는 자세한 방법을 설명합니다.

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 ...

high-efficiency-differentiation-human-pluripotent-stem-cells-to

Research

JoVE Journal

Biology

30.0K 조회수.  Medical College of Wisconsin.  이 기사는 효율적으로 선택적으로 참조 마커의 분석은 인구의 동질성과 정체성을 평가하기 위해 유동 세포 계측법 다음에 대한 Wnt 경로를 변조하여 심근에 인간 만능 줄기 세포를 구별하는 자세한 방법을 설명합니다. 

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

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocyte 엽 성의 전구 물질로 배아 줄기 세포의 분화

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

연구

생물학

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

마우스 Meiotic 셀 유동세포계측법의 정제

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

인간 유도 만능의 줄기 세포를 Transfecting 및 Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

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 ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

스펙트럼 유동 세포 계측법에 의해 고체 조직에서 분리 한 세포 현탁액의 분석

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

유세포측정에 의한 조혈줄기세포 및 T세포의 미토콘드리아 질량 및 막 전위 측정

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

나노유세포분석을 통한 단일 세포외 소포 막횡단 단백질 특성 분석

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

hiPSC-CM의 칼슘 및 수축성 파라미터의 실시간 측정

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. In this study, it is demonstrated that this optics-based system is a powerful tool to assess the functional parameters of hiPSC-CMs in 2D. By using this platform, it is possible to perform paired measurements in a well-preserved temperature environment on different plate layouts. Moreover, this system provides researchers with instant data analysis.
This paper describes a method for measuring the contractility of unmodified hiPSC-CMs. Contraction kinetics are measured at 37 &#176;C based on pixel correlation changes relative to a reference frame taken at relaxation at a 250 Hz sampling frequency. Additionally, simultaneous measurements of intracellular calcium transients can be acquired by loading the cell with a calcium-sensitive fluorophore, such as Fura-2. Using a hyperswitch, ratiometric calcium measurements can be performed on a 50 &#181;m diameter illumination spot, corresponding to the area of the contractility measurements.

저자 스포트라이트: 인간 유도 만능 줄기세포 유래 심근세포의 칼슘 및 수축성 매개변수의 실시간 측정  

Here, an established method is described to perform contractility and calcium measurements in human induced pluripotent stem cell-derived cardiomyocytes using an optics-based platform. This platform enables researchers to study the effect of mutations and the response to various stimuli in a rapid and reproducible manner.

인간 유도 만능 줄기 세포 유래 심근 세포에서 칼슘 및 수축성 매개변수의 실시간 측정

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte ...