그림 준비에 도움에 감사 Suwei가 오 하 고. 이 작품은 국립 자연 과학 재단의 중국 (81530004, 31425016) 사역의 과학 및 중국의 기술 (2016YFA0100500)에서 교부 금에 의해 지원 되었다. : J.L. 실험 수행 및 초안 원고; 플로리다는 원고 편집. 두 저자는 읽기와 최종 원고를 승인.

동물 주제를 포함 하 여 모든 절차 동물학 연구소, 중국 과학원, 베이징, 중국에서 윤리 검토 위원회에 의해 승인 되었습니다.
 1. 재료 준비 <ol><li>Sterilize는 0.65 μ m 필터 자외선 자외선 아래 생산 4 h에 대 한 깨끗 한 벤치에 램프를 레이 2 h 마크 후 필터를 넘겨. 
	참고: UV 라이트를 사용할 경우 적절 한 보호를 착용.</li>
	<li>소독 30 분 동안 121 ° C에 고압 소독 기와 스테인레스 스틸 메쉬. 
	참고: 스테인레스 스틸 메시의 특정 높이 공기 액체 인터페이스에서 필터를 지원 하기 위해 필요 하다 고이 강철 메시 ( 그림 1 A)에 와이어로 실현 될 수 있다.</li>
	<li>M5300 장기로 10 µ M의 최종 농도에 희석 부정맥 문화 매체와 혼합 균등 하 게. 
	참고: 6-잘 접시 2 mL M5300 장기 배양은 각 잘 적합 합니다. 10 -6 M에 날린 선택적으로 매체에 희석 수 있습니다.</li>
	<li>6 잘 플레이트 나 요리에 10 µ M에서 부정맥으로 보충 하는 매체를 전송 하 고 매체에 넣고 멸 균된 스테인레스 스틸 메쉬.</li>
	<li>때마다 살 균에 대 한 유리 셀 문화 접시에 두 번 3 분 동안 끓는 물으로 0.65 μ m 필터 세척. 각 세척, 염 인산 염 버퍼에 필터를 넣어 (PBS) 2 분 및 장소는 스테인리스에 젖은 필터 메쉬 공기 액체 인터페이스에 서 보듯이 그림 1 A [도식 후 센터]. 
	참고: 끓는 물 생성 될 수 있습니다와 무 균 상태를 달성 하기 위해 고압 소독 기 및 장비 온도 감소를 피하기 위해 적시에 고압 소독 기에서 취해야 한다.</li>
</ol> 2. AGM Explant 문화 <ol><li>신중 하 게 집게와 E10.5 또는 E11 배아에서 AGM 영역을 분리.
	<ol><li>해가 위로 임신한 쥐에서 uteruses를 벗기고 배아는 uteruses에서 분리.</li>
		<li>노른자위 낭, 탯 줄, 및 태아 몸에서 내장 닦아.</li>
		<li>신중 하 게 등 쪽 신경 조직 및 주변 근육 조직 제거.</li>
		<li>컷 앞쪽에 및 후방 사지의 사이트에 집게와 조직와 관련해 지역 태아 몸에서 분리. 그림 1 B C 표현식 및 해 부 AGM의 형태 표시.</li>
	</ol></li> <li>2-3 일 동안 37 ° C에서 5% CO 2 문화와 공기 액체 인터페이스 ( 그림 1 A)에서 필터에 별도로 해 부 AGMs explant. 
	참고: 않습니다 하지 강철 메시의 와이어에 해 부 관련해 놓고 필터 문화 중 공기 액체 인터페이스에는 다는 것을 확인 합니다. AGM, 외 노 른 자 sac와 태아 간 수 또한 될 교양이 시스템 7.</li>
</ol> 3. 식 분석 <ol><li>mRNA 수준 <li>1 mL PBS에 교양된 AGMs 수집 및 4 ° c, 310 x g 6 분에 대 한 원심</li> <ol>
		<li>제거한 후에 상쾌한, 총 RNA 추출에서 monophasic 솔루션 페 놀의 수집된 AGMs 제조 업체에 따르면 &#39; s 지침.</li>
		<li>총 RNA (2 µ g)은 역방향 다음 템플릿으로 cDNA를 M MLV 역전사를 사용 하 여 복사할. 양적 실시간 PCR 분석 실험 SYBR 녹색 공연과 Gapdh 내부 통제로 사용 됩니다.</li>
	</ol></li> <li>단백질 수준 <ol><li>단계 3.1.1에서에서 위에서 설명한 대로 교양된 관련해 수집.</li>
		<li>세포 세포의 용 해 버퍼와 교양된 AGM에서 단백질을 준비 (10 mM Tris HCl, 10 mM NaCl, 그리고 0.5 %NP-40) 포함 하는 효소 억제제와 단백질 수준으로 이전 검색 서양 blotting에 대 한 사용 설명 13.</li>
	</ol></li></ol> 4. 식민지 형성 단위 (CFU-C) 문화 분석 결과에 <ol><li>2-3 일 동안 배양 후 1 mL 버퍼링 하는 인산 염 (PBS) 및 4 ° C, 6 분 310 x g에서 원심 분리기에 관련해 지역 수집 하 고 37에서 200 µ L 콜라 (PBS에 0.1%)와 해리 ° C. 
	참고: 단일 셀-정지를 생성 하는 콜라에 필요한 시간은 약 20 분입니다. 관련해 포함 된 튜브를 떨고 5 분 마다 소화 과정을 가속화할 수 있습니다. 200 µ L 0.1% 콜라 각 관련해 사용 되 고 200 µ L 10% 혈 청 반응을 중지 하는 데 사용 됩니다.</li>
	<li>문화 초 저 첨부 파일 24-잘 접시에서 M3434 매체에 단일 셀 정지. 
	참고: 오염을 피하기 위해, 페니실린 스 솔루션 추가할 수 있습니다 선택적으로 매체에. M3434 매체 점성 때문에 1.0 cc 주사기 바늘 없이 세포와 매체는.</li>
	<li>후 7-10 일 식민지 형성 단위-erythroid (BFU-E), CFU-granulo-monocyte (CFU-GM) 및 CFU granulocyte, 버스트를 포함 한 적혈구, 대 식 세포, megakaryocyte (CFU-GEMM)의 각 유형에 대 한 수, 5% CO 2에서 37 ° C에서 배양 고유 형태에 따라 하 고 ( 그림 2 C) 거꾸로 현미경으로.</li>
</ol> 5. Vivo에서 분석 결과 이식 explant 경작의 <ol><li>식민지 형성 단위-비장 (CFU-S) 분석 결과 <ol><li>후 2-3 일은 AGMs 수집 하 고 준비 20 200 µ L 콜라 (PBS에 0.1%)와 함께 배양 하 여 단일 셀-정지 분</li>
		<li>8에 10 주 된 남성 C57BL/6 마우스의 치명적인 방사선 (x-레이의 9 Gy)을 수행 셀 주입 하기 전에 4-5 h.</li>
		<li>Restrainer의 활동을 제한 하에 비친된 마우스 넣어.</li>
		<li>꼬리 정 맥의 vasodilatation를 홍보 하기 위해 75% 알코올에 면봉을 사용 하 여 닦아.</li>
		<li>0.5 배아 상응 (ee) 또는 1 ee AGM의 단일 셀-정지 정 맥에 주사 방사능된 쥐의 꼬리 정 맥 1.0 cc 주사기. 
		참고: 세포는 PBS로 일시 중단 하 고 받는 당 0.5 mL PBS는 적합 한 볼륨. 25 게이지 또는 26 게이지 크기 바늘 1.0 cc 주사기 주입에 사용 됩니다. 공기는 주사기에 도입 될 수 있습니다. 눌러 출혈을 멈추게 하는 살 균 면봉으로 사출 사이트.</li>
		<li>11 일 이식, 수집 게시 하 고 받는 사람 마우스의 spleens Bouin 수정 &#39; s 솔루션 (15 mL 1.22 %Picric 산 포화 용액 2 mL 40% 메탄올, 아세트산 1 mL) 1-2 일 및 또 다른 1-2 일 동안 80% 알코올로 세척. 거시적 비장에 보이는 식민지를 계산 하 고 ee 당 식민지의 수를 결정. 
		&#8203; 참고: Bouin &#39; s 정착 액, 마모 보호.</li>
	</ol></li> <li>장기 이식 분석 결과 <ol><li>경작 explant는 AGMs를 수집 하 고 준비 하는 20 분에 대 한 200 µ L 콜라 (PBS에 0.1%)와 함께 배양 하 여 단일 셀 정지 후 2-3 일</li>
		<li>수행 8에 10 주 된 남성 C57BL/6 마우스의 치명적인 방사선 (x-레이의 9 Gy) 셀 주입 하기 전에 4-5 시간.</li>
		<li>Restrainer의 활동을 제한 하에 비친된 마우스 넣어.</li>
		<li>꼬리 정 맥의 vasodilatation를 홍보 하기 위해 75% 알코올에 면봉을 사용 하 여 닦아.</li>
		< l나 > 0.5 배아 상응 (ee)를 주입 또는 1 ee 1.0 cc 주사기 비친된 마우스의 꼬리 정 맥에 정 맥 AGM의 셀 정지를 단일. AGM의 2 × 10 5 골 수 세포 (CD45.1 배경)와 받는 사람 당 1 ee 복용량 주입. 
		참고: 공기는 주사기에 도입 수 있습니다. 눌러 출혈을 멈추게 하는 살 균 면봉으로 사출 사이트.</li>
		<li>4 개월 후 이식 받는 사람의 골 수를 수집 하 고 FACS에 의해 재구성 분석 결과 대 한 그것을 사용 하 여. 함께 받는 &#8805; 10% 기증자 파생 된 chimerism 성공적인 재구성 전시 여겨진다.</li>
	</ol>
	</li>
</ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bringing+together+the+organoid+field:+from+early+beginnings+to+the+road+ahead.">Bringing together the organoid field: from early beginnings to the road ahead.</a> Development. 144, 963-967 (2017).</li></ol>

저자 아무 충돌 금융 관심 있다.

그것은 잘 알려진 골 수에서 성숙 HSCs 조사 받는 사람 혈액 시스템을 다시 수 있습니다. 이러한 기능 HSCs 달리 마우스 배아의 AGM에 신흥 HSCs 성숙 하지 않습니다. 직접 이식 결과 유형을 보여 난 (VE-cad+ CD45- CD41+) 유형 II와 (VE-cad+ CD45+) 사전 HSCs 없이 재구성 능력20소유. AGM explant 문화 시스템 관련해 지역에서 초기 HSCs의 활용 이식 분석 결과<su...

조 혈 줄기 세포 (HSCs)는 자체 갱신 하는 기능 뿐만 아니라 erythroid, 골수성를 포함 하는 multilineage 잠재력 및 림프 세포 조직 관련 성인 줄기 세포 이다. 최근 연구 초기 HSCs 조 혈 등 쪽 대동맥1,2의 복 부 벽에 전환 (EHT) 내 피를 통해 (그는), hemogenic endothelium로 알려진 전문된 내 피 인구에서 발생 ,,34. HSCs 확장을 위한 태아 간으로 이동 되며 마지막으로 개인의 생활 내내 성인 조를 유지 하 골 식민지 대동맥-생식-mesonephros (AGM) 지역에 배아 (E) 10.5에서에서 E12.5 마우스 배아 형성, 면 5 , 6.이 몇 년 동안 공부 하고있다, 비록 HSC 출현 그리고 발달의 기본 메커니즘 남아 불완전 하 게 이해.
체 외에서 수정 및 zebrafish 태아의 개발와 달리 마우스 배아의 자궁내 개발은 확실 한 조 연구 embryogenesis 동안 훨씬 더 불편. 녹아웃 (KO) 마우스를 사용 하 여 유전 실험은 일반적으로 활용 하 고, 비록 특정 KO 마우스의 부족 또한 조 혈 연구 분야에서 그들의 사용을 제한 합니다. 또한, 구조 실험 vivo에서 KO 생쥐에서 쉽게 수행 하지 않습니다. 1996 년 이래로, AGM explant 문화 분야7에 개척자에 의해 조 혈 연구 개발 되었습니다. 이 문화 시스템의 도움으로 관련해 지역의 복 부 조직 등 조직 반대 효과8,9발휘 하는 동안 HSC 활동, 홍보 확인 되었습니다. AGM explant 문화 시스템 또한 세로토닌, Mpl, SCF, BMP, HSC 개발10,,1112,13, 신호 고슴도치의 역할 결정에 적용 된 14. 중요 한 것은, 그것은 또한 돌연변이 배아13,15조 혈 결함을 구출 하는 데 사용 하는 인기 있는 방법.

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			<td class="xl67" height="21" style="width:162pt;height:15.75pt" width="216">durapore 0.65 &#181;m filters</td>
			<td class="xl68" style="width:81pt" width="108">Millipore</td>
			<td class="xl69" style="width:89pt" width="119">R5BA63787</td>
			<td class="xl67" style="width:125pt" width="167">AGM explant culture</td>
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			<td class="xl67" height="42" style="height:31.5pt">M5300 long-term culture medium</td>
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			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">Trizol&#160;</td>
			<td class="xl68" style="width:81pt" width="108">Tiangen</td>
			<td class="xl69" style="width:89pt" width="119">5829</td>
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			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">M-MLV reverse transcriptase</td>
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			<td class="xl69" style="width:89pt" width="119">90694</td>
			<td class="xl67">reverse transcription</td>
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			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">SYBR Green</td>
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			<td class="xl69" style="width:89pt" width="119">A6002</td>
			<td class="xl67">quantatitive real-time PCR</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">protease inhibitor</td>
			<td class="xl68" style="width:81pt" width="108">Roche</td>
			<td class="xl69" style="width:89pt" width="119">55622</td>
			<td class="xl67">Protein extration</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">collagenase</td>
			<td class="xl68" style="width:81pt" width="108">Sigma</td>
			<td class="xl69" style="width:89pt" width="119">C2674</td>
			<td class="xl67">digestion of AGM tissues</td>
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		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">MethoCult GF M3434 medium</td>
			<td class="xl68" style="width:81pt" width="108">Stem Cell Technologies</td>
			<td class="xl69" style="width:89pt" width="119">3434</td>
			<td class="xl67">CFU-C assay</td>
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			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">ultra-low attachment 24-well plates</td>
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			<td class="xl69" style="width:89pt" width="119">3473</td>
			<td class="xl67">CFU-C assay</td>
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			<td class="xl67" height="63" style="height:47.25pt">C57BL/6 CD45.2 mice</td>
			<td class="xl68" style="width:81pt" width="108">Beijing HFK Bioscience Co. Ltd</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl67">CFU-S assay</td>
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		<tr height="63" style="height:47.25pt">
			<td class="xl68" height="63" style="width:162pt;height:47.25pt" width="216">C57BL/6 CD45.1 mice</td>
			<td class="xl68" style="width:81pt" width="108">Beijing HFK Bioscience Co. Ltd</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl67">Long-term transplantation</td>
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		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">phosphate buffered saline</td>
			<td class="xl68" style="width:81pt" width="108">Life Technologines</td>
			<td class="xl69" style="width:89pt" width="119">8115284</td>
			<td class="xl68" style="width:125pt" width="167">AGM Collection</td>
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			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">Penicillin-Streptomycin solution</td>
			<td class="xl68" style="width:81pt" width="108">HyClone</td>
			<td class="xl68" style="width:89pt" width="119">SV30010</td>
			<td class="xl68" style="width:125pt" width="167">AGM explant culture</td>
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			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">anti-CD45.2-PE-CY7</td>
			<td class="xl68" style="width:81pt" width="108">eBioscience</td>
			<td class="xl68" style="width:89pt" width="119">25-0454-80</td>
			<td class="xl68" style="width:125pt" width="167">Long-term transplantation</td>
		</tr>
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			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">anti-CD45-FITC</td>
			<td class="xl68" style="width:81pt" width="108">eBioscience</td>
			<td class="xl68" style="width:89pt" width="119">11-0451-81</td>
			<td class="xl68" style="width:125pt" width="167">Long-term transplantation</td>
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		<tr height="105" style="height:78.75pt">
			<td class="xl68" height="105" style="width:162pt;height:78.75pt" width="216">UV lamp</td>
			<td class="xl68" style="width:81pt" width="108">Beijing jiangmorning yuan bio-technology co., LTD</td>
			<td class="xl68" style="width:89pt" width="119">039-14402</td>
			<td class="xl68" style="width:125pt" width="167">power: 30W</td>
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		<tr height="63" style="height:47.25pt">
			<td class="xl68" height="63" style="width:162pt;height:47.25pt" width="216">stainless steel mesh</td>
			<td class="xl68" style="width:81pt" width="108">AS ONE SHANGHAI Corporation</td>
			<td class="xl68" style="width:89pt" width="119">2-9817-10&#160;</td>
			<td class="xl68" style="width:125pt" width="167">aperture diameter: 2.5 mm</td>
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			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">hydrocortisone</td>
			<td class="xl68" style="width:81pt" width="108">Sigma</td>
			<td class="xl68" style="width:89pt" width="119">H0396</td>
			<td class="xl68" style="width:125pt" width="167">selectively diluted into M5300 medium</td>
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application of aorta-gonad-mesonephros explant culture system in developmental hematopoiesis

조 연구에 대 한 마우스 배아를 사용 하 여 제한 주로 태아의 자궁내 개발 하는 작업에 추가 불편입니다. 녹아웃 (KO) 마우스에서 유전자 데이터를 설득 하지만 필요에 따라 모든 유전자에 대 한 코 쥐를 생성할 비현실적 이다. 또한, vivo에서 수행 코 쥐에서 얻은 데이터를 통합 하는 구조 실험은 하지 편리. 이러한 한계를 극복 하기 위해 대동맥-생식-Mesonephros (AGM) explant 문화는 조 혈 줄기 세포 (HSC) 개발을 공부 하는 적절 한 시스템으로 개발 되었다. 구조 실험을 위해 특히 코 쥐에 장애인된 조 복구 사용할 수 있습니다. 매체에 적합 한 화학 물질을 추가 하 여 장애인 신호 재 활성화할 수 있습니다 또는 위로 경로 저해 될 수 있습니다. 이 메서드를 사용 하 여, 많은 실험 HSC 개발의 중요 한 레 귤 레이 터를 식별 하기 위해 수행할 수 있습니다, 그리고 HSC를 포함 하 여 관련 된 mRNA와 단백질 수준, 식민지 형성 능력, 및 재구성 능력에서 유전자 발현. 이 일련의 실험 포유류에 HSC 개발에 필수적인 기본 메커니즘을 정의 하는 데 도움이 될 것 이다.

최근 간행물 보고 내 피 세포 유래 세로토닌 관련해13프로 apoptotic 통로 억제 함으로써 HSCs의 생존을 촉진. HSC 개발에 세로토닌의 홍보 효과 확인 하려면 팩 포함 되었다. 선택적 세로토닌 재 이해 억제제 (SSRI)로 부정맥 주변 조직16,,1718세로토닌 재 흡수를 억제 하기 위해 입증 되었습니...

Watch this Scientific Journal Video about Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis at JoVE.com

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

조 혈 줄기에 규제 요인 및 신호 통로의 효과 결정 하 식 분석, 문화와 비장, 장기 재구성에 콜로 니 형성 단위에 대 한 교양된 대동맥-생식-Mesonephros를 사용 하 여이 프로토콜에 설명 합니다. 셀 개발입니다. 이 조 혈 줄기 세포 생물학을 연구 하 고 기능에 대 한 효과적인 시스템으로 입증 되었습니다.

대동맥-생식-mesonephros Explant 문화 시스템 개발 조에의 응용

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

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JoVE Journal

Developmental Biology

8.3K 조회수.  Chinese Academy of Sciences. 조 혈 줄기에 규제 요인 및 신호 통로의 효과 결정 하 식 분석, 문화와 비장, 장기 재구성에 콜로 니 형성 단위에 대 한 교양된 대동맥-생식-Mesonephros를 사용 하 여이 프로토콜에 설명 합니다. 셀 개발입니다. 이 조 혈 줄기 세포 생물학을 연구 하 고 기능에 대 한 효과적인 시스템으로 입증 되었습니다.

비디오: Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

형광 단백질의 발현에<em&gt; 창고 기</em&gt; 미 수정 난자에 mRNA의 주입에 의한

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

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An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

공동 문화 시스템에 신경 연결의 형성을 평가하기위한 Optogenetic 접근

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

고도의 재현 작은 분자 기반의 차별화 프로토콜을 사용하여 인간 다 능성 줄기 세포로부터 심근 세포의 대규모 생산

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

C. 선 충 에서 신경 세포를 세균 세포를 재 설 정할 RNAi 및 열 충격 유도 하는 전사 인자 식의 적용

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

효율적인 세대의 췌 장/십이지 Homeobox 단백질 1+ 뒤 Foregut/췌 장 창시자 접착 문화에 hPSCs에서

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

인간 유도에서 Somite 파생 상품의 체 외 발생에서 만능 줄기 세포

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

피더 및 제노 프리 정의 상태에서 인간 다능성 줄기 세포에서 혈생 내피 분화

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

인체 유발 만능 줄기 세포에서 유도 된 체외 신경 근육 정션

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

얼룩말피에서 척추동물 세분화를 연구하는 3-D 꼬리 이식 문화

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

각질 줄기 세포 가소성을 모델링하기 위해 높은 처리량 표피 스페로이드 배양 시스템 구축

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...