이 연구 (KWF; 네덜란드 암 학회 로부터 자금에 의해 가능 하 게 되었다 EMCR 2012-5473)와 세계 암 연구 기금 국제 (WCRF; 프로젝트 번호 2014-1181)

모든 절차는 로컬 동물 복지 법률과 지침에 따라 수행 되었다.
1. 악기, 문화 미디어, 및 요리 준비
<ol><li>장가 위, 일반가 위, 및 살 균 컨테이너에 집게의 압력솥 1 세트.</li>
	<li>(플랫 바닥) 96 잘 접시 37 ° c.에는 인큐베이터에 배치</li>
	<li>시 약 재료의 테이블에 나열 된 완전 한 문화 매체의 10 mL를 준비 합니다.</li>
	<li>물 욕조에 37 ° C에서 완전 한 매체를 품 어.</li>
	<li>해 동 한 얼음 양동이에 그것을 배치 하 여 지하실 멤브레인을 재구성 된다. 재구성 된 지하실 막 4 ° C에 액체 될 것입니다.</li>
	<li>인산 염 버퍼 차가운 식 염 수 또는 약식된 PBS (4 ° C)로 4 접시를 채우십시오.</li>
</ol>2입니다. 작은 창 자 지하실의 격리
<ol><li>Lgr5-eGFP IRES 있다t2 마우스 C57BL6/J 배경에 CO2 흡입에 의해 희생. 가 위 쌍 복 경도 해 부.</li>
	<li>집게와 위를 누른 종횡 반으로 잘라.</li>
	<li>이제 장가 위를 사용 하 여, 내장을 꺼내 고 PBS를 포함 하는 페 트리 접시에 놓습니다. 참고: 장가 위는 샤 프와 무딘 팁 있다. 무뚝뚝한 끝이가이 위 소장을 여는 동안 토 굴 모 건축을 손상 하지 의미입니다.</li>
	<li>복 부에 무뚝뚝한 끝을 배치 하 여 시작 하 고 부드럽게는 pylorus 통해 밀어. 여는 위로 절단 하 고 당기는 집게와 함께 진행 합니다.</li>
	<li>일단 전체 소장 경도 열는 집게로 잡고 차가운 PBS에서 그것을 세척 하 고 부드럽게 U 모양 움직임으로 PBS 솔루션에 그것을 씻어.</li>
	<li>모든의 자 나머지는 들어가면 절단 보드, luminal 쪽에 내장을 평평 하 게 진행 합니다. Luminal 측면은 쉽게 인식할 수 있는 혈관의 부재 및 외부 부품과 비교 될 때 창백한 모습.</li>
	<li>유리 슬라이드, 부드럽게 제거는 villi 평평된 내장 된다고 하 여. 이 단계는 조직의 전체 길이 따라 두 번 수행 합니다.</li>
	<li>2-5 밀리미터 조각으로 메 마른 외과 블레이드와 소장을 잘라.</li>
	<li>포함 하는 얼음 차가운 PBS의 10 mL 50 mL 튜브에 작은 창 자 조각 놓습니다.</li>
	<li>PBS에서 그들을 아래로 pipetting으로 모든 남아 있는 불순물을 제거 조직 파편을 청소. 상쾌한 삭제 하 고 PBS 완전히 취소 될 때까지이 단계를 반복 합니다.</li>
	<li>25 ml PBS의 총 최종 볼륨을가지고 15 mL 차가운 PBS를 추가 합니다. 2 mM ethylenediaminetetraacetic 산 (EDTA)를 추가 하 고 4 ° c.에 롤러에 45 분 동안 품 어</li>
	<li>PBS/EDTA를 삭제 합니다.</li>
	<li>PBS의 10 mL을 추가 하 고 거칠게 pipetting 조직 조각을 아래로 (세 번 이상)는 지하실을 분리. 상쾌한을 수집 합니다.</li>
	<li>반복 단계 2.13 4 시간. 문화 매체 도달 50 mL의 최종 볼륨을 추가 합니다.</li>
	<li>원심 (300 x g 5 분)에 의해 셀을 펠 렛.</li>
	<li>문화 매체의 10 mL에 펠 릿을 resuspend 하 고 원심 (80 x g 3 분)으로 지하실을 작은.</li>
</ol>3. 단일 셀 준비
<ol><li>수송과 지하실의 상쾌한 무시 하 고 1 mL의 트립 신 같은-효소 50 µ L 함께 resuspend DNAse (50 µ g/mL).</li>
	<li>2 분 동안 32 ° C에 물 욕조에 셀을 품 어.</li>
	<li>문화 매체의 10 mL를 추가 합니다. 가혹 하 게 아래로 5 번 이상 pipetting으로 단일 셀에는 지하실을 해리.</li>
	<li>40 µ m 여과기 (덩어리 및 기타 불순물 제거)를 통해 솔루션을 필터링 및 5 분에 대 한 300 x g에서 단일 셀을 작은.</li>
</ol>4. flow Cytometry 및 도금
<ol><li>1 x 행 크의 버퍼링 된 소금 솔루션 또는 HBSS/2% 소 태아 혈 청 (FCS) 솔루션 50 mL를 준비 하 고 모든 106 셀 1 mL에 펠 릿 resuspend</li>
	<li>세포 현 탁 액 BV421 린- (CD31, CD45, TER119) 30 분 1: 250 항 체의 농도에서 1: 100, 및 CD24-APC CD117-PE 모두의 농도에 추가 합니다.</li>
	<li>정렬 Lgr5+ 줄기 세포 그리고 Paneth 세포로 별도 낮은 바인딩 튜브. 정렬에 대 한 프로토콜에 대 한 내용은 Lgr5+ 그리고 Paneth 세포, 보십시오 로스 외. 7 과 Schewe 외. 6<ol>
<li>5 분에 대 한 300 x g에서 정렬된 셀 1.3에서 설명 하는 구성 요소와 100 µ L 매체에 셀 Resuspend 원심 분리기 ( 재료의 표참조).</li>
	</ol>
</li>
	<li>(예를 들어, 화학 또는 유전 수정에 의해) Paneth 세포 치료 (n = 2000) 또는 Lgr5+ 줄기 세포 (n = 2000).<ol>
<li>세포 치료를 사용 하 여 transfection liposome 중재 시 약의 5 µ L 100 µ L 문화 매체와 100의 농도에 작은 간섭 RNA (siRNA)의 총 볼륨에 nM. SiRNA 또는 선택한 수정에 필요한 시간 동안 37 ° C에서 30 분 동안 품 어.</li>
	</ol>
</li>
	<li>워시 500 µ L 문화 매체에 두 번 셀.</li>
	<li>5 분 x 300g에 centrifuge 고 10 µ L 문화 매체에 셀 resuspend.</li>
	<li>수영장 2 개의 세포 구성 성분 (Paneth 또는 Lgr5+ 세포)는 20 µ l 총 볼륨에 실시간에 5 분에 대 한 300 x g에서 원심 분리기</li>
	<li>공동은 Paneth 품 어 및 Lgr5+ 셀 실시간에서 10 분</li>
	<li>상쾌한의 10 µ L을 제거 하 고 반드시 셀 펠 릿을 발음 하지 액체의 초승달 모양을 두고.</li>
	<li>40 µ L의 전체 볼륨에 대 한 셀을 재구성된 지하실 막의 30 µ L을 추가 하 고 셀 resuspend는 미리 데워 진 96 잘 접시 접시. 10 분 후 완전 한 매체의 200 µ L 추가 ( 재료의 표참조). 변화 중간 마다 48 h입니다.</li>
	<li>날 5에 organoid 다양성을 계산 합니다.</li>
</ol>

<ol>
	<li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5.">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449, 1003-1007 (2007).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paneth+cells+constitute+the+niche+for+Lgr5+stem+cells+in+intestinal+crypts.">Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.</a> Nature. 469, 415-418 (2011).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459, 262-265 (2009).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Development+and+Disease+with+Organoids.">Modeling Development and Disease with Organoids.</a> Cell. 165, 1586-1597 (2016).</li><li>Cantrell, M. A., Kuo, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+modeling+for+cancer+precision+medicine.">Organoid modeling for cancer precision medicine.</a> Genome Med. 7, 32 (2015).</li><li>Schewe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+Phospholipases+A2+Are+Intestinal+Stem+Cell+Niche+Factors+with+Distinct+Roles+in+Homeostasis,+Inflammation,+and+Cancer.">Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer.</a> Cell Stem Cell. 19, 38-51 (2016).</li><li>Roth, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paneth+cells+in+intestinal+homeostasis+and+tissue+injury.">Paneth cells in intestinal homeostasis and tissue injury.</a> PLoS One. 7, e38965 (2012).</li><li>Rodriguez-Colman, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+metabolic+identities+in+the+intestinal+crypt+supports+stem+cell+function.">Interplay between metabolic identities in the intestinal crypt supports stem cell function.</a> Nature. 543, 424-427 (2017).</li><li>Dow, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apc+Restoration+Promotes+Cellular+Differentiation+and+Reestablishes+Crypt+Homeostasis+in+Colorectal+Cancer.">Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer.</a> Cell. 161, 1539-1552 (2015).</li><li>Yilmaz, O. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1+in+the+Paneth+cell+niche+couples+intestinal+stem-cell+function+to+calorie+intake.">mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake.</a> Nature. 486, 490-495 (2012).</li><li>Igarashi, M., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1+and+SIRT1+Cooperate+to+Foster+Expansion+of+Gut+Adult+Stem+Cells+during+Calorie+Restriction.">mTORC1 and SIRT1 Cooperate to Foster Expansion of Gut Adult Stem Cells during Calorie Restriction.</a> Cell. , 436-450 (2016).</li></ol>

저자는 경쟁 금융 관심 또는 다른 충돌

오 라는 두 가지 필수 구성 요소 장 줄기 세포 틈새의 즉 Lgr5+ 그리고 Paneth 세포의 세련 된 기능 분석을 허용 한다. 이 방법은 이전 우리와 다른 사람에 의해 약간의 수정6,,1011를 고용 하고있다. 여기, 재현할 수 및 표준화 된 실험실 프로토콜 또는 프로시저 선물이. 또한, 우리 유전자 구현의 가능성의 예를 들어 <e...

장 상피 포유류 몸에 있는 가장 빠르게 자기 갱신 조직 그리고, 따라서, 식별 및 크립 트의 하단에는 성인 줄기 세포의 기능적 특성을 겨냥 하는 연구의 과다의 개체 Lieberkühn, Lgr5 유전자의 표현에 의해 갑니다 고 정식 Wnt 신호1의 특히, Lgr5+ 줄기 세포 형벌 이며 전문된 틈새 세포, 즉 Paneth 세포, 또한 그들의 성숙2Wnt 신호에 의존 하는 의해 지원. 함께, 이러한 두 셀 형식 자체-갱신 창 자 상피 안 대기의 기초 및 일일 homeostatic 균형 유지: Lgr5+ 줄기 세포는 급속 하 게 분할 하 고 뿌리를 및 더 많은 전문 장 상피 셀; Paneth 세포 제공 필수 틈새 요인 (예를 들어, Dll1, Wnt3, EGF) Lgr5+ 줄기 세포2. 장 지하실 때 도금 비보 전 조직과 자체 갱신 구조를 형성 하기 위하여 organoids, 또는 "미니-배 짱", 용량 자체 갱신 등 프로세스에 대 한 통찰력을 제공 하는 실험 도구로 이용 되 고 암4를 포함 하 여 정상 및 병 적인 조건에서 차별화. Organoid 문화 소장, 췌 장, 간, 그리고 신장, 마우스 및 인간의 샘플4에서 포함 하 여 여러 조직에서 설립 되었습니다. 추출 방법 및 성장 요인이 organoid 문화를 개발 하는 조직 및 다중 계보 차별화를 운전 하 고 최대한 원래 줄기 세포 틈새를 모방 하도록 설계 된 vivo에서. Organoids는 유전 질환의 치료, 암에서 치료 효능, 약물 독성의 분석 또는 생체 외에서organogenesis5의 연구의 평가 포함 하 여 잠재적인 응용 프로그램을 할 수 있습니다.
전반적으로, organoid 문화 조직 샘플에서 설립 하는 때의 주요 제한은 세포 특이성의 부족입니다. 예를 들어 장 organoids 전체 장 지하실에서 설립 조직 소스 내 포함 개별 세포 구성 성분의 분석을 허용 하지 않습니다 (예를 들어, 전체 지하실 포함 Lgr5+, Paneth, 그리고 조상 세포)입니다.
여기, 우리 소설 메서드, 오 라로 그것의 가장 기본적인 구성 요소, 즉 줄기 (Lgr5+)와 틈새 (Paneth) 세포의 기능 분석 장 organoid 문화의 장점을 결합 하 여 설명 합니다. 이 Paneth의 독특한 능력을 통해 이루어집니다 그리고 실제로 공동 인 큐베이 팅 및 organoids2,,610을 때 서로 연관 Lgr5+ 세포. 우리는이 기능 활용 하 고 미리 organoids를 다시 구성 하도록 허용 하기 전에 개별적으로 2 개의 세포 유형 취급. 때 이렇게, 특정된 약물, 성장 인자, 생화학 억제제, 유전자 조작, 또는 재구성 및 organoid 형성 전에 화학적 치료 각 셀 구성 요소를 노출 될 수 있습니다. 따라서, 지금 분석 결과 사용 하 여 특정 약물 치료 또는 유전 수정에 줄기 세포 또는 그들의 틈새에 특정 효과 있는지 여부의 결정을 허용할 것 이다.

<table>
	<tbody>
		<tr>
			<td>Advanced DMEM F12 *</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax *</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Final concentration: 10 mM</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin *</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Final concentration: 1%</td>
		</tr>
		<tr>
			<td>Hepes *</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td>Final concentration: 10 mM</td>
		</tr>
		<tr>
			<td>Recombinant murine EGF *</td>
			<td>Thermo Fisher Scientific</td>
			<td>PMG8041</td>
			<td>Final concentration: 50 ng/ml</td>
		</tr>
		<tr>
			<td>Recombinant murine Noggin *</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>Final concentration: 100 ng/ml</td>
		</tr>
		<tr>
			<td>y27632 *</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Final concentration: 10 &#181;M</td>
		</tr>
		<tr>
			<td>Jagged-1 *</td>
			<td>Anaspec Bio</td>
			<td>AS-61298</td>
			<td>Final concentration: 1 &#181;M</td>
		</tr>
		<tr>
			<td>Recombinant murine R-spondin *</td>
			<td>R&#38;D systems</td>
			<td>3474-RS-050</td>
			<td>Final concentration: 1 mg/ml</td>
		</tr>
		<tr>
			<td>Flexitube Gene solution siRNA APC</td>
			<td>Qiagen</td>
			<td>GS11789</td>
			<td>Final concentration: 100 nM</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>CD24 APC</td>
			<td>Biolegend</td>
			<td>101814</td>
			<td></td>
		</tr>
		<tr>
			<td>c-kit PE</td>
			<td>Biolegend</td>
			<td>105808</td>
			<td></td>
		</tr>
		<tr>
			<td>BV 421 CD31</td>
			<td>Biolegend</td>
			<td>102424</td>
			<td></td>
		</tr>
		<tr>
			<td>BV 421 CD45</td>
			<td>Biolegend</td>
			<td>103134</td>
			<td></td>
		</tr>
		<tr>
			<td>BV421 TER119</td>
			<td>Biolegend</td>
			<td>116234</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14180046</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.129P2-Lgr5tm1(cre/ERT2)Cle/J</td>
			<td>Jackson Laboratories</td>
			<td>008875</td>
			<td></td>
		</tr>
		<tr>
			<td>Low binding tubes</td>
			<td>Eppendorf</td>
			<td>Z666548-250EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>* The combination of these reagents constitutes the complete culture medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the organoid reconstitution assay (ora) for the functional analysis of intestinal stem and niche cells

장 상피는 매우 빠른 회전 속도 의해 특징입니다. 포유류에서 전체 상피 안 대기는 4-5 일 이내에 갱신 됩니다. 성인 장 줄기 세포 Lieberkühn의 지하실의 하단에, Lgr5 유전자의 표현에 의해 배정 있으며 그들의 특성 높은 증식 속도1통해 항상성 유지. 작은 창 자에 걸쳐 Lgr5+ 줄기 세포 Paneth 세포 라고 하는 특수 분 비 세포와 혼합 된. Paneth 세포 항균 화합물 (즉, lysozyme과 cryptdins/defensins) 분 비와 창 자 식물에 제어 역할을 발휘. 더 최근에, 소설 함수 Paneth 세포, 즉 그들의 용량을 Wnt3, EGF, 및 Dll12로 몇 가지 주요 ligands 통해 Lgr5+ 줄기 세포 틈새 지원 제공에 대 한 발견 되었습니다.
전 비보 를 격리 하 고 특정 성장 인자와 세포 외 기질 구성 요소 존재 양식, 전체 장 지하실을 일으키 긴와 매우 유사한 크립 트 모 organoids 라는 자체 3D 구조를 갱신 성인 소장3의 상피 아키텍처입니다. Organoid 문화, 전체 지하실에서 설치 될 때 비록 그것의 개별 구성 요소, 즉는 Lgr5+ 의 기여를 해결 하지 않고 자기 갱신의 연구와 장 줄기 세포 틈새의 허용 및 Paneth 세포입니다.
여기, organoid 분석 결과 Paneth의 능력을 활용 하는 새로운 접근 방식을 설명 및 Lgr5+ 연결 하 고 organoids 형성을 셀 때 공동 경작. 이 이렇게, 여기 "organoid 재구성 분석 결과" 라고 (오 라), Paneth 유전 그리고 생 화 확 적인 수정할 수 있게 또는 Lgr5+ organoids로 재구성 뒤 줄기 세포. 따라서, 장 줄기 세포 틈새의 두 가지 주요 구성 요소 기능 분석을 수 있습니다.

Organoid 재구성 분석 결과 Apc 유전자의 작은 간섭 RNA (siRNA)에 의해 증명 여기 장 상피의 필수 틈새 및 줄기 세포 구성 요소의 별도 기능 분석을 수 있습니다.
이 목표를 달성 하기 위해 우리는 먼저 형광 활성화 된 세포 분류 (FACS) 타고 난된 C57BL6/J 쥐에서 8000 Lgr5+ 세포와 6000 Paneth 세포를 분리 하 활용. <strong c...

Watch this Scientific Journal Video about The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells at JoVE.com

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

장 줄기와 틈새 세포의 기능 분석을 위한 Organoid 재구성 분석 결과 (오 라)

장 organoid 문화 전체 지하실에서 설정 되 고 셀 전용 패션에 자기 갱신과 차별화의 분석을 허용 하지 않습니다. 이 프로토콜에 설명 합니다 정렬 된 줄기 (Lgr5+)의 재구성, 틈새 (Paneth) 세포, 그들의 이전 생 화 확 및 유전 수정 및 기능 분석 하는 동안 organoids을 일으키.

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

the-organoid-reconstitution-assay-ora-for-functional-analysis

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Bioengineering

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

Unit 1

Stem Cell Biology And Renewal in Epithelial Tissue

SCIENTISTS IN ACTION

10.4K 조회수.  Erasmus University Medical Center. 장 organoid 문화 전체 지하실에서 설정 되 고 셀 전용 패션에 자기 갱신과 차별화의 분석을 허용 하지 않습니다. 이 프로토콜에 설명 합니다 정렬 된 줄기 (Lgr5+)의 재구성, 틈새 (Paneth) 세포, 그들의 이전 생 화 확 및 유전 수정 및 기능 분석 하는 동안 organoids을 일으키.

비디오: The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

뼈 재생을위한 인적 Perivascular 줄기 세포의 이용

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

연구

생체공학

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

다른 Lineages로 인간 배아 줄기 세포 (hESC)의 전파 및 차별 화를위한 3D 플랫폼으로서 알긴산의 Microcapsule

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

단일 세포의 다목적 화학 분석을위한 미세 유체 칩

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

의 리그닌 아래로 규제<em&gt; ZEA 메이스</em&gt; dsRNAi 및 Klason 리그닌 분석을 통해

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

개별 리포좀 간의 조성 불균질성을 검출하기 위한 정량적 형광 현미경 검사법 기반 단일 리포좀 분석

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

생체 외에서 인간 호중구 떼의 화학 및 분자 분석을 위한 생체 입자 미세어레이

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

염소 스티플 관절의 슬개골 하 지방 패드에서 중간엽 줄기 세포의 분리, 확장 및 분화

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

말초 신경 공학을 위한 그래핀 기반 3D 바이오하이브리드 하이드로겔 바이오잉크의 준비 및 특성화

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

Formulating and Characterizing an Exosome-based Dopamine Carrier System

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an active role in intercellular cargo and communication. Exosomes are mostly found in body fluids such as plasma, cerebrospinal fluid, urine, saliva, amniotic fluid, colostrum, breast milk, joint fluid, semen, and pleural acid. Considering the size of exosomes, it is thought that they may play an important role in central nervous system diseases because they can pass through the blood-brain barrier (BBB). Hence, this study aimed to develop an exosome-based nanocarrier system by encapsulating dopamine into exosomes isolated from Wharton's jelly mesenchymal stem cells (WJ-MSCs). Exosomes that passed the characterization process were incubated with dopamine. The dopamine-loaded exosomes were recharacterized at the end of incubation. Dopamine-loaded exosomes were investigated in drug release and cytotoxicity assays. The results showed that dopamine could be successfully encapsulated within the exosomes and that the dopamine-loaded exosomes did not affect fibroblast viability.

Here, we aimed to obtain a formulation by dopamine loading of the isolated exosomes of stem cells from Wharton's jelly mesenchymal stem cells. Exosome isolation and characterization, drug loading into the resulting exosomes, and the cytotoxic activity of the developed formulation are described in this protocol.

엑소좀 기반 도파민 운반체 시스템의 공식화 및 특성화

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an ...

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

인간 오가노이드와 장기 온 칩 기술을 결합하여 장 영역별 기능 모델링

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...