1. 권장 사항 시작하기 전에 <ol><li> 그들은 동방 결절 세포로 제대로 구분하지 않기 때문에 마이코 플라즈마 오염 PSC를 사용하지 마십시오. 프로토콜을 시작하기 전에 마이코 플라스마 오염에 대한 테스트합니다. 마이코 플라즈마 신속, 고감도 검출을위한 PCR 키트를 사용하여이 작업을 수행하고 제조자의 프로토콜을 따르십시오. </li><li> 각 페트리 접시 (단계 2.3.4), 코트 한 10cm 37 ° C에서 1 시간 동안 차가운 물에 물고기 피부에서 멸균 7 ml의 0.1 % 젤라틴 2 세포 배양 접시하십시오. 멸균 벤치에서 무균 조건에서 요리 건조 젤라틴을 제거 할 수 있습니다. </li><li> 포유 동물 발현 벡터를 사용하여 TBX3의 난) 구성 적 과발현 : 당신이 분화 프로토콜을 시작하기 전에 다음과 같은 기능을 포함하는 이중 형질 전환 안정된 마우스 ES 세포주 클론이 필요합니다. ⅱ) Myh6 촉진제 (5)의 제어하에 G418 내성 유전자. </li><li> PSC를 미분화 세포 공동 배양되어야설명 1 표준 조건에서 조사 피더 세포와 D. </li></ol> 2. 차별화 절차 <ol><li> 두 일 분화하기 전에 - 피더 세포에서 PSC를 제거합니다. <ol><li> 10 ml의 포스페이트 완충 식염수 PBS로 세포를 세척 매체 기음 7 ㎖의 콜라게나 제 IV의 용액을 첨가하고, 37 ° C에서 10 분 동안 세포를 배양한다. 50 ML 튜브에 멸균 40 μm의 필터를 놓습니다. </li><li> 조심스럽게 PSC 식민지를 씻어 위아래로 5 번 콜라겐 솔루션을 피펫 팅 (피더 세포를 제거하는 방지). </li><li> , 40 μm의 필터에 세포 현탁액을 전송 8 ml의 PBS로 세 번 필터를 헹군다. 필터 주변에 돌고 거꾸로 페트리 접시에 놓습니다. 필터의 바닥에 10 ml의 PBS를 피펫 팅하여 PSC 세포 식민지를 제거합니다. </li><li> 300 x g에서 5 분 동안 15 ml의 원심 분리 튜브에 세포 현탁액을 전송. </li><li> 1 ml의 Accutase 그리고 난에 세포를 일시 중지, PBS를 제거5 분 37 ° C에서 ncubate. </li><li> 10 ml의 PBS를 추가 X g 300에서 5 분 동안 아래로 5 회 원심 분리기를 그것을 피펫 팅에 의해 셀 솔루션을 섞는다. </li><li> 상기 PBS를 제거 10 ㎖ 배양 배지에서 세포를 중단하고 세포 수를 결정한다. </li><li> 종자 15,000 세포 / 75cm 2 필터 플라스크에 2 cm, 37 ° C, 5 % CO2에서 2 일간 배양 그들을. 이일 후 플라스크에 50-70 %의 합류해야합니다. </li></ol></li><li> 0 일 - 차별화의 시작 <ol><li> 매체를 제거하고 10 ml의 PBS로 세포를 씻어. </li><li> , PBS 대기음 2ml의 Accutase을 추가 5 분 동안 37 ℃에서 세포를 배양한다. 50 ML 튜브에 멸균 40 μm의 필터를 놓습니다. </li><li> , 40 μm의 필터에 세포 현탁액을 전송하고, 10 ㎖의 PBS를 추가 10 ㎖의 PBS를 첨가하고 원심 분리기를 통해 필터 린스 관류 300 x g에서 5 분간. </li><li> 10 ㎖ 분화 배지에서 세포를 일시 중단하고 세포 수를 결정한다. </li><li> t을 희석그는 20,000 세포 / ml의 최종 농도로 분화 배지 현탁액 전지. </li><li> 피펫 20 ml의 물과 HDS는 건조 아웃을 피하기 위해 차 배양 접시에서 드롭 (HD) 솔루션을 걸려 5 ml의. 참고 : 각 24 웰 플레이트 포함 iSABs 16 페트리 접시로 시작 (섹션 2.8.6.4/2.8.7 참조​​). </li><li> 트레이에 50 ml의 세포 현탁액까지 피펫. </li><li> 페트리 접시의 뚜껑 주위를 돌립니다. 12 채널 피펫을 사용하여 뚜껑에 180 HDS는 각각의 포함 20 μL (400 셀 / HD) 세포 현탁액을 놓습니다. </li><li> 조심스럽게 뚜껑 돌아서과 페트리 접시에 놓습니다. 주 : 덮개의 주위를 선회 속도가 매우 중요하다. 뚜껑이 너무 느리거나 너무 빠른 바꾸게되면, 세포 현탁액의 표면 장력이 걸려 뚜껑에 삭제 유지할만큼 충분히 높지 않다. 매체의 20 μl의 방울과 뚜껑 주위를 선회 처음 연습 HDS는 생산하기 전에. </li><li> (3)에 2 일 동안 세포 배양7 ° C, 5 % CO 2는 그들이 사채를 형성 할 수있다. 참고 : 5 페트리 접시의 스택의 상단에 물이 가득 배치 한 페트리 접시. 그렇지 않으면 상위 페트리 접시에 HD 건조합니다. </li></ol></li><li> 2 일 <ol><li> 조심스럽게 차 페트리 접시의 뚜껑 주위를 켜고 50 ㎖ 튜브에 두 개의 배양 접시 (360 사채)에서 파생 된 사채를 전송합니다. </li><li> 사채는 튜브의 아래쪽에 정착하자 10 분을 기다립니다 (EB를 원심 분리하지 마십시오). </li><li> 대기음 극력 매체로서, 10 ㎖ 분화 배지에서 EB를 중단하고 10cm 페트리 접시에 현탁액을 전송. </li></ol></li><li> 하루 2-6 서스펜션 문화 <ol><li> 37 ° C에서 4 일 동안 정학의 사채를 양성, 5 % CO 2는 2 일 후 매체를 변경합니다. 주 : EBS는 종종 표면에 부착 (세포 배양 접시는 대조적으로) 배양 접시에 도포되지 않더라도. EB를 부착 사채 매일 매일 필요한 경우 분리 배양 접시를 제어부드러운 피펫 팅에 의해 표면에서. 옵션 : 현탁 배양 기간 동안 지속적으로 배양 접시를 흔들어. 셰이커의 속도에주의하십시오. 사채는 어느 쪽도 중간에 축적되지도 배양 접시의 테두리에 떠 있어야합니다. </li></ol></li><li> 6 일 <ol><li> 한 젤라틴 코팅 10cm 2 세포 배양 접시에 하나의 배양 접시에서 사채를 전송합니다. </li></ol></li><li> 하루 7-12 <ol><li> 세포 8-12 일 후에 이길 시작해야합니다. 매일 현미경으로 세포의 초점을 치고 있는지 확인합니다. 세포는 층을 형성하기 시작한다. </li><li> 매체 매일을 변경합니다. 주 : 매체를 변경 한 후, 세포를 복구하고 다시 박동 3-4 시간을 필요로한다. </li></ol></li><li> 동 노드 세포의 날 12 ~ 15 선택. <ol><li> 세포 (12 ~ 15 일 정도) 꺾고 사흘 시작한 후, 세포에 400 ㎍ / ㎖의 G418을 함유하는 신선한 배지 분화 배지 10 ml의 피펫 대기음. </li></ol></li><l난&gt; 나흘 선택의 시작 후 전지 층을 해리. 주 : 세포층 이미 표면으로부터 분리되는 것이 가능하다. <ol><li> 셀 층을 흡입하지 않고 신중하게 매체를 제거합니다. </li><li> PBS의 10 ML을 추가하고 세포 층을 흡입하지 않고 조심스럽게 PBS를 제거합니다. </li><li> 50 ㎖ 튜브에 세포를 전송하고, 5 분 동안 37 ° C에서 배양 한 세포를 콜라게나 제 IV 용액 10 ㎖를 추가한다. </li><li> 격렬하게 피펫 팅하여 10 배의 아래 현탁액을 혼합하고, 5 분 동안 37 ℃에서 세포를 배양한다. 작은 클러스터의 정지가 발생한다. 여전히 남아 층의 큰 부분이 있으면, 단계 2.8.4을 두 번 더 반복한다. </li><li> 300 x g에서 10 분 동안 20 ㎖의 PBS와 원심 분리기를 추가합니다. </li><li> PBS를주의 깊게 기음. 참고 : iSABs 단계 2.8.7을 계속 생성 될 경우. ISAB 파생 동 마디 하나의 세포가 생성 될 경우 단계 2.9에 이동합니다. </li><li> 세포를 일시12 ㎖에 분화 배지는 400 ㎍ / ㎖의 G418을 함유하는 코팅 된 젤라틴과 6 웰 24 웰 (/ 웰 2 ㎖)에 그들을 시드. </li></ol></li><li> 단일 노드 세포의 생성. <ol><li> 5 ㎖ Accutase 단계 2.8.6에서 세포를 일시 중단하고, 5 분 동안 37 ℃에서 배양한다. </li><li> 격렬하게 피펫 팅하여 10 배의 아래 현탁액을 혼합하고, 5 분 동안 37 ℃에서 세포를 배양한다. </li><li> 300 x g에서 5 분 동안 20 ㎖의 PBS와 원심 분리기를 추가합니다. </li><li> 400 ㎍ / ㎖의 G418을 함유하는 12 ml의 분화 배지에서 세포를 일시 중단하고 24 웰 (2 ㎖ / 웰) 코팅 젤라틴 6 웰 그들을 시드. </li></ol></li><li> 분리 후 제 2 일, 매체를 대기음하고 PBS로 세포를 세 번 씻는다. 잘 / 1 ml의 차별화 매체를 추가합니다. </li><li> 하루 4-8 분리 후, 매 2 차 일 중간을 변경합니다. 해리 사용 iSABs 또는 ISAB 파생 동 후 10 일에 마디 하나의 세포는 추가 분석을 위해 사용할 수 있습니다. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+a+human+potassium+channel+suppresses+cardiac+hyperexcitability+in+rabbit+ventricular+myocytes.">Overexpression of a human potassium channel suppresses cardiac hyperexcitability in rabbit ventricular myocytes.</a> J Clin Invest. 103 (6), 889-896 (1999).</li></ol>

저자가 공개하는 게 없다.

능력 &quot;생물학적 박동기&quot;의 의미로 적절한 심박동의 재구성을 허용 할 수있다 세포 유래 심장 페이스 메이커의 줄기 세포를 생성한다. 마찬가지로, 시험관 내에서 약물 검사는 가용성 도움이됩니다. PSC를 페이스 메이커의 셀 속성 8,9,10,11,12,13와 심근을 포함한 포유 동물의 신체의 세포 유형을 야기 할 수 있습니다. 그러나, 전형적으로 &quot;배양체&quot;내의 세포 집단은 필연적으로 ?...

용어 &quot;아픈 부비동 증후군은&quot;심장 맥박 조정기 시스템의 악화로 이어지는 여러 질병을 요약 한 것입니다. 그것은 병적 인 증상이 동 서맥, 동방 블록, 부비동 체포뿐만 아니라 빈맥 - 서맥 증후군을 포함한다. 이로써, &quot;아픈 부비동 증후군&quot;은 종종 심근 허혈, 심근 병증 심근염 또는 일반적으로 심장 질환을 수반한다. 현재 치료 방법은 전기 맥박 조정기의 주입을 기반으로합니다. 그러나,이 같은 감염과 배터리 고장 등의 위험 번호와 함께 간다. 전반적으로, 합병증의 발생률은 여전히​​ 환자가 인공 심장 박동기를 이식하는 데에 매우 높습니다. 내인성 박동기 반대로 더욱이, 이러한 장치는 호르몬 자극에 반응하지 않는다. 미래의 대안이되는 PSC를가 봉사 할 수 있었다 &quot;생물학적 페이스 메이커&quot;의 가용성에 의존 할 수있다적합한 셀룰러 소스와 어떤으로 또한 체외 약물 검사에 매우 유용 할 것입니다. 그러나, 큰 문제가 배아 체 (사채) 내에서 동 노드 세포의 매우 드문 모양에있다 - 이것은 일반적으로 ~ 0.5 % 1을 초과하지 않는다. 이전에는 특정 심근 서브 타입을 향해 &quot;앞으로 프로그래밍&quot;등 관련 중배엽 - 별 후방 1 (MesP1) 및 NK2 전사 인자 궤적 5 (Nkx2.5) 2와 별개의 조기 심장 혈관 전사 인자의 과발현을 통해 가능하다 것으로 나타났다, 3. 보통 크기와 동방 노드 (SAN)의 기능을 위해, T-박스 전사 인자 TBX3는 페이스 메이커 유전자 프로그램을 개시하고 SAN (4)의 분화를 제어하기 위해 도시 된, 결정적이다. 이는 기능상 박동기 세포의 모양을 개선하면서, 콘텐츠는 여전히 전체 cardiomyocytic 세포 집단 내에서 ~ 40 %를 초과하지 않았다. 따라서, 추가 Myh6 발기인 기반 항생제 선택 5 단계를 우리가 도입되었다. 체외 배양에 쥐 심장의 사람들과 비교를 근사 처음으로 체외에서 높은 증가 박동 주파수 (&gt; 400 BPM)를 전시; 이것은 궁극적으로 아직 관찰되지 심근 집계 (iSABs &quot;&quot;유도 된 중국 - 심방 기관 &quot;)에 리드 쥐의 심장 (6)에서 분리 된 동 결절 세포. 이소 프레 날린 관리에서 550 BPM 심지어 구타 주파수를 얻을 수있다. 특히, iSABs 7을 분석하여 광범위한 생리에서 명백한 바와 같이 80 % 이상 기능 림프절 세포로 구성되어 있습니다. 최근, 다양한 접근이 직접 리 프로그래밍 (19)을 이용하여 상악동에게 결절 세포를 생성하기 위해, 표면 (16, 17)에 대하여 설명 하였다 소분자 약물 치료 14 또는 마커. 그러나, 이러한 방법 중 어느 것도 페이스 메이커 세포의 이러한 높은 순도 주도하지 않고 쥐에 가까운 주파수를 치고 그예술 iSABs에서 관찰. 또한, 자신의 자율 박동 활동을 잃었다 재배 성인 마우스 심실 조각의 생체 모델, iSABs는 슬라이스 조직으로 통합하여 자발적으로 활성 남아있는 견고하게 수축 7 심장 조각을 페이싱 할 수있다. 이 iSABs의 생성에 대한 자세한 프로토콜은이 문서에 설명되어 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Name of the Material/Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:131px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#39;s liquid medium with stable glutamine</td>
			<td>Biochrom AG</td>
			<td>FG 0465</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM liquid medium without Na-pyruvate, with stable glutamine</td>
			<td>Biochrom AG</td>
			<td>FG 0435</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CLS type IV, CLS IV</td>
			<td>Biochrom AG</td>
			<td>C4-22</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Non-essential amino acids</td>
			<td>Biochrom AG</td>
			<td>K 0293</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS Superior</td>
			<td>Biochrom AG</td>
			<td>S 0615</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium pyruvate (100 mM)</td>
			<td>Biochrom AG</td>
			<td>L 0473</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G 418-BC liquid (ready-to-use solution), sterile</td>
			<td>Biochrom AG</td>
			<td>A 2912</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagent reservoir PP f.multichannel pip. 60ml,sterile</td>
			<td>Brand</td>
			<td>703409</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accutase</td>
			<td>eBioscience,Inc.</td>
			<td>00-4555-56</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf Xplorer/Xplorer plus, electronic pipette</td>
			<td>Eppendorf</td>
			<td>4861000155</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon Cell Strainer 40&#181;m</td>
			<td>Falcon</td>
			<td>352340</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin&#160;</td>
			<td>GE Healthcare</td>
			<td>P11-010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dishes</td>
			<td>Greiner BioOne</td>
			<td>663102</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS without Ca and Mg</td>
			<td>PAN-Biotech</td>
			<td>P04-36500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>PAN-Biotech</td>
			<td>P30-3302</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leukemia inhibitory factor</td>
			<td>Phoenix Europe GmbH</td>
			<td>LIF-250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quadratic petri dishes</td>
			<td>Roth</td>
			<td>PX67.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin from cold water fish skin</td>
			<td>SigmaAldrich</td>
			<td>G7765</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-Mercaptoethanol</td>
			<td>SigmaAldrich</td>
			<td>M3148&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Thioglycerol</td>
			<td>SigmaAldrich</td>
			<td>M6145</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture dishes</td>
			<td>TPP</td>
			<td>93100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture flask</td>
			<td>TPP</td>
			<td>90076</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture test plates (24 well)</td>
			<td>TPP</td>
			<td>92424</td>
		</tr>
	</tbody>
</table>

generation of murine cardiac pacemaker cell aggregates based on es-cell-programming in combination with myh6-promoter-selection

&quot;아픈 부비동 증후군 &#39;의 치료는 인공 심장 박동기를 기반으로합니다. 여기에는 배터리 오류 및 감염 등의 위험을 부담. 또한, 그들은 호르몬 응답이 부족하고 전반적인 절차는 비용 집약적이다. PSC를에서 생성 된 &quot;생물 페이스 메이커&quot;는 대안이 될 수있다, 아직 배아의 몸에서 심장 박동 조절 세포의 일반적인 내용 (사채)는 매우 낮다. 설명 프로토콜은 Myh6 발기인 기반 항생제 선택과 부비동 노드 유도의 TBX3을 통해 쥐의 PSC를의 &quot;앞으로 프로그래밍&quot;을 결합합니다. 이&gt; 80 % 생리 학적 기능성 페이스 메이커 세포의 일관된 심근 집계를 산출한다. 이러한 &quot;유도 동방-몸은&quot;( &quot;iSABs&quot;) 자발적으로 마우스 마음에서 고립 마디 세포에 해당 아직 미전도 주파수 (400 ~ 500 BPM)에서 계약과 쥐의 심근 생체 조절이 할 수 있습니다. 설명 프로토콜 고순도의 동을 사용하여절점 단셀 약물은 시험 관내 시험을 위해 사용될 수있는 예를 생성 할 수있다. 또한,이 프로토콜에 따라 생성 된 iSABs 심장 조직 공학을 향한 중요한 단계가 될 수 있습니다.

설명 프로토콜은 마우스의 심장 박동 주파수에 가까운 (동영상 참조) PSC를에서 약 450 BPM의 구타 주파수 iSABs의 생성을 할 수 있습니다. 도 1에 도시 된 바와 같이 iSABs 해리 (단계 2.8.8) 후에 관찰 된 단일 세포는 동방 결절 (주축 및 거미 세포)의 세포의 일반적인 형태를 나타낸다. 이들 세포를 알려진 고도로 익스프레스 단백질 기능에 필수적인 것으로 과분극 활성화 주기적 뉴클레오티?...

Watch this Scientific Journal Video about Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection at JoVE.com

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

이 프로토콜은 쥐의 다 능성 줄기 세포 (PSC)에서 기능 동 노드 조직을 생성하는 방법에 대해 설명합니다. T-Box3 (TBX3) 과발현 플러스 심장 미오신 헤비 체인 (My​​h6) 발기인 항생제 선택은 고순도의 페이스 메이커 세포 집합체로 연결됩니다. 이러한 &quot;유도 - 동방 - 몸&quot;( &quot;iSABs&quot;)는, 고도의 박동 속도를 증가 보여 80 %의 심장 박동 세포를 포함하고 심근 생체 조절이 할 수 있습니다.

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

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

generation-murine-cardiac-pacemaker-cell-aggregates-based-on-es-cell

Research

JoVE Journal

Developmental Biology

9.6K 조회수.  University of Rostock.  이 프로토콜은 쥐의 다 능성 줄기 세포 (PSC)에서 기능 동 노드 조직을 생성하는 방법에 대해 설명합니다. T-Box3 (TBX3) 과발현 플러스 심장 미오신 헤비 체인 (My​​h6) 발기인 항생제 선택은 고순도의 페이스 메이커 세포 집합체로 연결됩니다. 이러한 "유도 - 동방 - 몸"( "iSABs")는, 고도의 박동 속도를 증가 보여 80 %의 심장 박동 세포를 포함하고 심?...

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

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

화학 물질 안전 검사 및 시스템 생물학 데이터 생성을 위해 발생 독성 시험 법을 바탕으로 인간 다 능성 줄기 세포

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

연구

발생학

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/&#946;-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

마우스 배아 줄기 세포의 집계의 세대 대칭 속보, 양극화 및 응급 집단 행동은보기가<em&gt; 체외</em

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

인간 다 능성 줄기 세포의 서스펜션 기반의 심장 차별화를위한 마이크로 웰의 집계 크기 최적화

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

혈관 내피 줄기 세포의 혈관 생성을 시각화하기위한 유방 팻 패드 이식 기술

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

마우스 배아에서 심장 조상 인구의 양적 전체 마운트 면역 형광 분석

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

비 계, 3 차원 인슐린 표현 Pancreatoids 마우스 췌 장 창시자에 체 외에서 의 세대

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

전체 산 Epifluorescence를 사용 하 여 마우스 Embryogenesis 동안 심장 챔버의 분석

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

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 ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

차동 시도에 의한 모자이크 유방 오르가노이드 생성

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

인간 iPSC 기반 혈액-뇌 장벽 칩 생성

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...