저자는 훌륭한 기술 지원 pHPL와 Margaretha Frühwirth와 다니엘라 탈러를 제공 박사 Katharina Schallmoser 감사합니다.

A.) ECFC ISOLATON DAY 1 : <ol><li> 당신이 시작하기 전에 실험은 세포 배양 매체 내피 성장 중간 2 (EGM - 2)를 준비합니다. (; 모든 시그마, 세인트 루이스, MO 500ml 내피 기초 헤파린 (1000 U / ML의 1ml)로 중간 2 페니실린 / 스트렙토 마이신의 5ml 100x 솔루션을 보완 <a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a> ), L - 글루타민 (2 MM 최종 농도 ) 50 ML 풀링된 인간의 혈소판의 lysate (pHPL). ; 다음 0.2 ML의 하이드로코티손, 2 ML hFGF - B, 0.5 ML VEGF, 0.5 ML EGF, 0.5 ML IGF - 1과 0.5 ML의 아스코르비 산 (SingleQuots로 제공, 모든 Lonza, 워커스빌, MD 추가 <a href="http://www.lonzabioscience.com/">http://www.lonzabioscience.을 COM /</a> ). 무균 여과액 0.2 m - 기공 크기의 500 ML Millipore 진공 필터를 통해 중간. pHPL 작은 혈전을 형성하고 있기 때문에 하나의 매체의 두 가지 필터가 필요할 수 있습니다. 사전 따뜻한 필터링 EGM - 2 37 ° C 물 목욕으로 </li><li> 6ml 방부제 무료 나트륨 - 헤파린 혈액 vacutainer 관에 cubital 정맥에서 어른이 인간의 말초 혈액의 5 ML를 그림으로써 혈액 샘플을 수집합니다. 당신은 2 시간 최대 내에서 혈액 샘플을 처리한다. </li><li> 통풍 모자, 한 25 ML의 피펫과 층류 벤치에 두 5 ML pipettes와 플라스크, 한 75cm 2 (코스타 T75) 준비하여 시작하십시오. </li><li> 사전 채우기 15 ML 사전 예열 25 ML의 피펫으로 플라스크에 EGM - 2 보충. 이제 혈액 약병 및 샘플 술병에 5 ML의 피펫과 함께 모든 5 ML을 엽니다. 두 번째 5 ML의 피펫과 함께 추가 EGM - 2 5 ML과 빈 혈액 병을 린스. T75 내에서 총 판매량은 25 ML이다. 37 인큐베이터에있는 술병과 장소의 통풍 - 뚜껑을 닫기 ° C 5 % CO 2, 24 시간 동안 95 %의 습도. </li></ol> DAY 2 : <ol><li> 약 24 시간 (야간 이상) 후 비 점착 성의 세포의 제거에 대한 중간 혈액 혼합물을 제거합니다. 층류 흐름에 준비이 25 ML pipettes 세 5 ML pipettes 경우, 사전 따뜻한 20 ML ° C 물 목욕에 37 EGM - 2 (전날에 준비) 30 ML PBS를 보충. </li><li> 인큐베이터에서 샘플로 술병을 제거하고 25 ML의 피펫 모든 뜨는을 제거합니다. 어떤 잠재적인 식민지 손상을 방지하기 위해 술병의 표면을 스크래치 않도록주의를 가져가라. 부드럽게 다른 한쪽에서 미리 예열 PBS 및 틸팅 술병 10 ML을 추가하여 내부 술병 세포 부착 표면에 세 번 씻으십시오. PBS 즉시 각 시간과 폐기를 제거합니다. </li><li> 플라스크에 신선한 사전 예열 EGM - 2 20 ML를 추가하고 인큐베이터에 다시 넣습니다. 이 과정은 분리하지 연결된 세포에 대한 신속하고 최대한 부드럽게으로 수행되어야합니다. 그들이 오래 위해 PBS 또는 상온에서 보관하는 경우 ECFCs 쉽게 분리합니다. </li><li> 술병을 통해 화면 systemically 매초마다 하루 ECFC 파생물을 찾고 가벼운 현미경의 낮은 배율을 사용하여 하루에 두세에서 시작. 식민지는 식민지의 위치를​​ 나타내는 술병의 바깥 쪽 상단에있는 마크를 발견하는 것입니다. flaks 이상 5 분 인큐베이터 밖되지해야합니다. </li></ol> DAY 5 : <ol><li> 5 일 시딩 이후 비 점착 성의 세포를 제거하는 완벽한 매체 변경을 수행합니다. 사전 따뜻한 20 ML 이러한 목적 EGM - 2 37 ° C 물 목욕으로 두 25 ML pipettes 준비. </li><li> 25 ML의 피펫을 사용하여 완전한 매체를 제거하고 가능한 빨리 신선한 사전 예열 보충 EGM - 2를 추가합니다. 전지는 건조하지 않도록 분 이상 매체없이 왼쪽해서는 안됩니다. 인큐베이터에 다시 플레이스와 매일 술병을 검사 반복합니다. </li><li> 신선한 보충 EGM - 2 회 주간의 중간의 30 %를 교체하여 세포를 공급. </li></ol> DAY 14-28 : <ol><li> 코블 스톤의 형태로 전형적인 식민지는 하루에 1,200 나타납니다. 각 식민지의 위치를​​ 나타내기 위해 술병의 맨 바깥쪽 면을 표시한다. 단일 세포 분리 시작하지 않는 한 식민지가 하루 28까지 성장할 수 있습니다. 일단, 기본 식민지들은 그들의 크기에 따라 일 14 28 사이에 수확한 수 확인되었습니다. </li></ol> B.) ECFC 하베스트 <ol><li> 사전 따뜻한 5 ML 트립신 0.05 % / EDTA (에틸렌 다이아 민 테트라 초산), 20 ML PBS 및 5ml 신선한 보충 EGM - 2. </li><li> 완전히 (잠재적인 식민지 손상을 방지하기 위해 술병의 표면을 만지지 않도록 돌봐) 팔콘 튜브에 술병에서 매체를 제거하고 10 ML preheated PBS로 세포를 신중하게 씻으십시오. </li><li> 인큐베이터 5 분 5 ML 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)과 부화를 추가합니다. 그것이 독성되는 이후 세포는 7 개 이상의 분 트립신에 노출해서는 안됩니다. 철저하게 플라스크의 측면을 감청하는 것은 세포를 분리하는 데 도움이됩니다. 보존된 사용하는 문화 매체의 약 10 ML을 추가하여 트립신 프로 테아제 활동을 끄다. </li><li> 50 ML 팔코에 세포 현탁액을 제거N 튜브. 이후 수확 세포 현탁액에 추가되면 10 ML PBS로 술병을 씻으십시오. 4 7 분에 대한 300g에 세포 현탁액을 원심 분리기 ° C는 세포를 수집하고 트립신을 제거합니다. 뜨는을 취소하고 1 ML PBS에서 즉시 세포 펠렛을 resuspend. 얼음 계속. </li><li> R. 리카르도와 K. Phelani하여 조브 프로토콜에서 설명한대로 휴대폰 번호를 결정 5. </li></ol> C.) ECFC 확장 <ol><li> 보충 500 ML EGM - 2 등) 1.1에서 설명했다. 37 사전 따뜻한 ° C 물 목욕 인치 </li><li> Nunc, 네이퍼빌, IL,, 약 2,500cm이 문화 공간 11 T220 (225cm 2) 15 T175 (175cm 2) 또는 하나에 안경 계층 세포 공장 (CF - 4 준비 <a href="http://www.nuncbrand.com/">http://www.nuncbrand . com</a> ). 또한 두 개의 새로운 통풍구 - 모자와 층류 벤치에서 특별한 살균 깔때기가 필요합니다. </li><li> 100 시작 세포 밀도의 씨앗 ECFCs에 휴대폰 번호의 C / cm 2 계산이 필요합니다. 한 CF - 4 252,800 전지 500 ML 신선한 EGM - 2 resuspended되고 멸균 깔때기를 사용하여 CF - 4에 부어. 환기 - 모자 하나의 통풍구를 닫고 모든 4 밀가루 사이에 매체의 평등한 분배를 가능하게 CF - 4 한 경도 측면을 기울. 다음 챔버를 뒤로 기울이기 및 통풍 - 캡의 뚜껑을 엽니다. 인큐베이터에서 CF - 4를 놓습니다. </li><li> CF - 4까지 적어도 한번 매주 매체의 20 %를 변경 합류합니다. </li><li> 수확 세포는 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) 70 ML과 약 200 ML PBS를 사용하여) B.에 설명되어. </li></ol> 대표 결과 이 격리 방법을 사용하여 우리는 하루에 12 주 콜로니 형성을 관찰했다. 이전 연구에서는 4.0 ± 의미 말초혈 각 ML에서 파생된 0,8 ECFC - 식민지를 복구할 수있었습니다. 4 ECFCs는 일반 코블 스톤의 모습을 보여주었다. 대규모 확장 후, 100 세포 / cm 2 시딩 밀도, 우리 3 남 1 여성 자원 봉사자 (26 ~ 50 세 사이의 연령)에서 약 30 일의 총&gt; 1 X 10 8 세포를 획득하였습니다. 이러한 조건 하에서 교양 ECFC은 proliferative 기능 및 genomically 안정 표시되었습니다. 4 나아가 세포가 추가로 사용하기 위해 액체 질소로 (10 % DMSO)를 저장할 수 있습니다. 3,4

<ol>
	<li>Solovey, A., Lin, Y., Browne, P., Choong, S., Wayner, E., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+activated+endothelial+cells+in+sickle+cell+anemia.">Circulating activated endothelial cells in sickle cell anemia.</a> N Engl J Med. 337, 1584-1590 (1997).</li><li>Yoder, M. C., Mead, L. E., Prater, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+large-scale+expansion+of+functional+mesenchymal+stem+cells+from+unmanipulated+bone+marrow+without+animal+serum.">Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum.</a> Tissue Eng Part C Methods. 14, 185-196 (2008).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. , (2009).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+and+Determining+the+Viability+of+Cultured+Cells.">Counting and Determining the Viability of Cultured Cells.</a> J Vis Exp. , (2008).</li></ol>

이 소설 절연 및 확장 방법이 더 효율적이고 더 적은 시간이 소요 모든 세포 분리합니다 (밀도 기울기가 필요 없습니다)을 피함으로써과 기존 기술보다 비싸 간단하고 효율적인 프로세스입니다. 보빈 항원과 putative 이후 xenoimmunization에 pHPL 연락처를 사용하여 제외됩니다. 문화 기간 동안 작은 혈전이 pHPL에 의한, 양식 수 있습니다. 우리의 경험을 바탕으로 이러한 혈전은 콜로니 형성과 세포 증식?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Endothelial basal Medium-2</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>500ml</td>
</tr>
<tr>
<td>EGM-2 SingleQuots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>EGF, hFGF-B, VEGF, IGF-1, Hydrocortisone, ascorbic acid</td>
</tr>
<tr>
<td>preservative-free Heparin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>&nbsp;</td>
<td>10U/ml per EGM-2</td>
</tr>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>&nbsp;</td>
<td>2mM per EGM-2</td>
</tr>
<tr>
<td>Penicillin and Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>&nbsp;</td>
<td>100U/mL Penicillin and 100&mu;g/mL Streptomycin per EGM-2</td>
</tr>
<tr>
<td>Trypsin/1mM EDTA </td>
<td>&nbsp;</td>
<td>Gibco Cell Culture, Invitrogen </td>
<td>&nbsp;</td>
<td>7ml for T75 flask 70ml for CF-4</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Four-layered cell factory (CF-4) </td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Funnel</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T75cm2 culture flask (with vented cap)</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 mL vacutainer tubes (preservative-free sodium heparin) </td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>preloaded with 108 IU/6ml of preservative-free sodium heparin</td>
</tr>
<tr>
<td>50ml Falcon tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>5ml Stripetts</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25ml Stripetts</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Squeezer</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0,2 pore size sterile filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>2x 500ml per EGM-2</td>
</tr>		</tbody>
		</table>
		</div>

isolation and large scale expansion of adult human endothelial colony forming progenitor cells

본 논문은 heparinized에서 내피 콜로니 형성 전구 세포 (ECFCs)에 대한 새로운 복구 전략을 도입하지만, 그렇지 12일의 의미 이내에 성인 인간의 말초 혈액을 unmanipulated. 대규모 확장 후&gt; 1x10 8 ECFCs는 추가적인 테스트를 위해 얻을 수 있습니다. Advantageously pHPL를 사용하여 소 혈청 항원과 인간 세포의 접촉은 제외시킬 수 있습니다. 유동세포계측법 및 immunohistochemistry으로 분리된 세포는 ECFC 및 체외 기능의 구조와 같은 혈관을 형성하는가 matrigel 분석에서 테스트 수있는 특징 수 있습니다. 또한이 세포는 생체내 subcutaneously에 기능, perfused 혈관을 형성하는 마우스 모델에서 주입 수 있습니다. 장기 확장 및 cryopreservation 확산 후, 기능 및 게놈의 안정성은 화석이 보존 되기에 나타납니다. 3,4가 이것은 동물 단백질 무료로 절연 및 확장 방법은 ECFCs 대량를 생성하기 위해 쉽게 적용됩니다.

Watch this Scientific Journal Video about Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells at JoVE.com

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

내피 콜로니 형성 전구 세포 (ECFCs)은 혈관 항상성 및 수리를 공부하는 유망 도구입니다.<sup&gt; 1,2</sup&gt;이 논문은 절연 및 안티 소 예방 접종의 위험이 떨어지면서 풀링된 인간의 혈소판 lysate (pHPL)와 heparinised 성인 인간의 말초 혈액에서 ECFC의 확장을위한 소설 동물 - 혈청 무료 방법을 소개합니다.

분리 및 성인 인간 내피 콜로니 형성 전구 세포의 대규모 확장

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

isolation-large-scale-expansion-adult-human-endothelial-colony

Research

JoVE Journal

Biology

10.6K 조회수.  Medical University of Graz, Austria. 내피 콜로니 형성 전구 세포 (ECFCs)은 혈관 항상성 및 수리를 공부하는 유망 도구입니다. 1,2이 논문은 절연 및 안티 소 예방 접종의 위험이 떨어지면서 풀링된 인간의 혈소판 lysate (pHPL)와 heparinised 성인 인간의 말초 혈액에서 ECFC의 확장을위한 소설 동물 - 혈청 무료 방법을 소개합니다.

비디오: Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

인간 제대 베인 내피 세포의 절연 (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

연구

생물학

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic 도서관 대규모 화면

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

인간 탯줄의 분리 및 동물 혈청 무료 확장 Mesenchymal Stromal 세포 (MSCS)와 내피 콜로니 형성 전구 세포 (ECFCs)을 유래

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

판막병 내피 세포의 분리

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

조류 배아 판막병 전구 세포의 분리 및 문화

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

인슐린을 표현 콜로니를 형성 Progenitors에 대한 정량 분석

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Clonal 확장 CD133 + 간 줄기 세포의 분리

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

내피 콜로니 형성 세포의 Phenotypic 및 기능성 특성은 인간 탯줄의 혈액에서 유래

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...