We would like to thank Andrea Portbury and Davin Townley-Tilson for critical reading of the manuscript and the NIH (grant # R01HL061656) for funding support.

참고 : 모든 실험 채플 힐 노스 캐롤라이나 대학의 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 1 소비세 대동맥판 <ol><li> 관심 배아 스테이지 안락사 승인 기술을 사용하여, 개발의 목적 배아 일 (E)에서의 새끼와 임신 마우스를 안락사. </li><li> 임신 여성의 복부를 소독하기 위해 70 % 에탄올을 사용합니다. 멀리 내부 장기에서 낮은 복부 위쪽의 피부와 근육을 들어 올리고 자궁 뿔에 액세스 할 수 있도록 열려있는 여성의 낮은 복강을 절개. </li><li> 자궁 경적을 검색하려면 집게에 집게로 자궁 경부 조심스럽게 잘라 꼬리를 개최합니다. 장소에 혼을 유지하는 결합 조직을 절단, 자궁 경적을 들어 올리고 난관과 자궁 뿔의 접합을 절단하여 제거를 완료합니다. </li><li> 페트리 접시 포함 COL에 해부 자궁 경적을 배치D 0.1 M 트리스 완충액 (pH 7.6) 및 필요에 따라 린스. 그런 다음, 오픈 길이 현명한 배아 주머니를 노출하는 자궁 뿔을 잘라. </li><li> 배아를 표시하려면, 태반 및 태아의 교차점에서 배아 주머니를 열고 잘라 배아를 무료로 제대 혈관을 잘라. 차가운 0.1 M 트리스 버퍼를 가지는 초 페트리 접시에서 해부 배아를 놓습니다. 다음 배아에 대한 준비가 될 때까지 얼음에 나머지 undissected 배아와 첫번째 페트리 접시를 돌려줍니다. </li><li> 배아 내의 흉벽 접근을 개선하기 위해 배아를 참살. 유전자형이 필요한 경우 제거하고 얼음에 배치 된 에펜 도르프 튜브에 여분의 조직 일부를 저장한다. </li><li> 그 뒷면에있는 배아를 놓고 앞다리 근처, 흉곽의 측면을 따라 수직으로 가슴 벽을 잘라 수평 격막 위의 마음을 시각화 할 수 있습니다. 그런 마음을 노출하는 가슴 벽을 엽니 다. </li><li> 대 혈관을 따라 높은 잡아 집게를 사용하여 집게를 사용하여 마음을 들어 올려 아래의 혈관을 잘라.N, 마음을 무료로 집게 위에 잘라. 폐 혈관 포경 남아 있으면, 폐 심장으로 제거 될 수 있고 계속하기 전에 제거되어야한다. </li><li> 잘라 내기 및 밸브의 수준의 위 폐동맥을 제거; 여기에 기술 된 배아 단계에서, 폐동맥 밸브 지역에서의 차별에 도움되는 약간 불투명하다. 그냥 폐동맥 판막 아래 잘라 trabeculated 심실의 심근을 방지하고이 지역이 관심 또한 경우 단계 1.10에 설명 된대로 수집합니다. 여기서 및 다음 단계에서, 종래의 용해 완충액에 수집 어웨이 관심 영역으로부터 과량의 트리스 완충액을 그리 모세관을 사용한다. </li><li> 조심스럽게 그냥 밸브의 수준 이상으로, 대동맥 판막을 대동맥 원위부를 제거; 폐동맥과 같은 대동맥의 제거에 도움을줌으로써 이러한 배아 단계에서 약간 불투명 남아있다. 다시 trabeculated 심실 심근을 피하고, 단지 대동맥판 이하 잘라 및 전송얼음에 단백질 분해 효소와 포스 파타 아제 억제제를 포함하는 (재료 표에 설명) 2 μL 용해 버퍼 에펜 도르프 튜브에 집게를 사용하여 밸브. </li><li> 밸브는 별도의 에펜 도르프 튜브에 각 밸브를 수집, 모든 배아에서 적출 될 때까지 반복 1.6-1.10 단계를 반복합니다. 모든 배아는 동일한 유전자형을 가지고 있다면, 모든 밸브는 단일 에펜 도르프 튜브에 수집 될 수있다. </li><li> 유전자형이 함께 풀에 밸브를 결정하기 위해 수행해야하는 경우, 즉시 더 사용할 때까지 -80 ° C에서 용해 버퍼에 샘플을 배치. 시료 (단계 2.1.1) 유전형 후 풀링 될 수있다. </li></ol> 2 단백질 추출 <ol><li> 1 분은 튜브의 바닥에 용해 완충액에서 조직을 수집하기위한 13100 XG에서 얼음과 원심 분리기에 수집 된 조직을 해동. <ol><li> 배아 유전자형 된 경우, 부드럽게 수집하고 풀 용해 버퍼 유사한 유전자형을 가진 배아에서 밸브 영역을 포함하는 피펫을 사용합니다. 강한 B를 확보하려면샘플이 좋습니다 당과, E13.5 배아에서 3-4 밸브 영역을 풀링. </li><li> 모든 배아는 동일한 유전자형의이었고 모든 밸브 영역은 1.10 단계에서 함께 회수하고 있으면 단계 2.2에 기재된 바와 같이, 전체 샘플을 용해. </li></ol></li><li> 풀링 얻어진 시료의 부피를 점검하고 40 ㎕의 총 부피에 용해 완충액을 추가; 그때, 중단하고 에펜 도르프 튜브에 5mm 스테인리스 비즈를 사용하여 샘​​플을 균질화. 제조업체의 지침에 따라, 50 Hz에서 2-4 분 동안 lyser을 사용하십시오. 간단히 스핀 튜브, 불용성 이물질 남기고 새로운 튜브로 (13100 ~ 1 분간 XG) 및 전송 샘플. </li><li> 준비 및 Eslami 외 9와 같은 프로토콜 다음 SDS-PAGE 이후 서양 얼룩 분석을 위해 전송을위한 별도의 샘플. 로드 제어 단백질 블로 팅하는 단백질 정량에 필수적이다. </li></ol>

<ol>
	<li>Wirrig, E. E., Hinton, R. B., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+cartilage+and+bone-related+proteins+in+pediatric+and+adult+diseased+aortic+valves.">Differential expression of cartilage and bone-related proteins in pediatric and adult diseased aortic valves.</a> J Mol Cell Cardiol. 50, 561-569 (2011).</li><li>Chakraborty, 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=Twist1+promotes+heart+valve+cell+proliferation+and+extracellular+matrix+gene+expression+during+development+in+vivo+and+is+expressed+in+human+diseased+aortic+valves.">Twist1 promotes heart valve cell proliferation and extracellular matrix gene expression during development in vivo and is expressed in human diseased aortic valves.</a> Dev Biol. 347, 167-179 (2010).</li><li>Lee, M. P., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twist1+directly+regulates+genes+that+promote+cell+proliferation+and+migration+in+developing+heart+valves.">Twist1 directly regulates genes that promote cell proliferation and migration in developing heart valves.</a> PLoS One. 6 (e29758), (2011).</li><li>Peacock, J. D., Huk, D. J., Ediriweera, H. N., Lincoln, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox9+transcriptionally+represses+Spp1+to+prevent+matrix+mineralization+in+maturing+heart+valves+and+chondrocytes.">Sox9 transcriptionally represses Spp1 to prevent matrix mineralization in maturing heart valves and chondrocytes.</a> PLoS One. 6 (e26769), (2011).</li><li>Chakraborty, S., Cheek, J., Sakthivel, B., Aronow, B. J., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shared+gene+expression+profiles+in+developing+heart+valves+and+osteoblast+progenitor+cells.">Shared gene expression profiles in developing heart valves and osteoblast progenitor cells.</a> Physiol Genomics. 35, 75-85 (2008).</li><li>Vogel, C., Marcotte, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+regulation+of+protein+abundance+from+proteomic+and+transcriptomic+analyses.">Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.</a> Nat Rev Genet. 13, 227-232 (2012).</li><li>Meng, X., 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=Expression+of+functional+Toll-like+receptors+2+and+4+in+human+aortic+valve+interstitial+cells:+potential+roles+in+aortic+valve+inflammation+and+stenosis.">Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells: potential roles in aortic valve inflammation and stenosis.</a> Am J Physiol Cell Physiol. 294, 29-35 (2008).</li><li>Dyer, L. A., Wu, Y., Moser, M., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPER-induced+BMP+signaling+promotes+coronary+artery+remodeling.">BMPER-induced BMP signaling promotes coronary artery remodeling.</a> Developmental Biology. 386, 385-394 (2014).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Garside, V. C., Chang, A. C., Karsan, A., Hoodless, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-ordinating+Notch,+BMP,+and+TGF-beta+signaling+during+heart+valve+development.">Co-ordinating Notch, BMP, and TGF-beta signaling during heart valve development.</a> Cell Mol Life Sci. 70, 2899-2917 (2013).</li><li>Jiang, X., Rowitch, D. H., Soriano, P., McMahon, A. P., Sucov, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+the+mammalian+cardiac+neural.">Fate of the mammalian cardiac neural.</a> Development. 127, 1607-1616 (2000).</li><li>Scherptong, R. W., 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=Morphogenesis+of+outflow+tract+rotation+during+cardiac+development:+the+pulmonary+push+concept.">Morphogenesis of outflow tract rotation during cardiac development: the pulmonary push concept.</a> Dev Dyn. 241, 1413-1422 (2012).</li><li>Mohler, E. R., Adam 3rd, L. P., McClelland, P., Graham, L., Hathaway, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+osteopontin+in+calcified+human+aortic+valves.">Detection of osteopontin in calcified human aortic valves.</a> Arterioscler Thromb Vasc Biol. 17, 547-552 (1997).</li></ol>

The authors have nothing to disclose.

심장 밸브 영역을 조기에 마우스 배아 단백질 수준을 정량화하고 병아리 능력 밸브 개발에 중요한 세포 신호 전달 사건을 이해하기위한 추가 툴을 제공한다. 여기에 설명 된 우리의 프로토콜은 표준 단백질 분리 과정에서 크게 차이가 없습니다. 그러나, 몇 가지 주요 단계를 수정하여, 우리는 성공적으로 매우 작은 샘플 크기에서 인산화 단백질을 얻을 수있다. 이 결과를 달성하기 위해, 다음 단?...

단백질 발현 수준을 확인하고 정량 할 수있는 것은 동물성 및 세포 기반 실험을위한 표준 기술이다. 그러나,에도 불구하고 오랜 현재 병아리와 마우스 1, 2 모두에서 면역 조직 화학 염색으로 제한됩니다 개발 과정이 특정 조직에서 단백질 발현을 평가, 초기 배아 심장 밸브 개발에 관심을. 대부분의 모델 유기체 (예, 마우스 및 병아리)의 현상 밸브에서 단백질 발현을 정량화하는 어려움의 일부를 얻을 수있다 단백질의 양을 제한하는 밸브의 작은 크기이다. 따라서, 정량 분석을 위해, 연구원은 일반적으로 다음 정량 PCR이나 마이크로 어레이 분석 2-5 RNA 추출 및 증폭에 의존하고 있습니다. 그러나, RNA 및 단백질 발현 수준은 6 전적으로 상관 아니므 RNA 발현에 집중하면 특정 신호 발생 수많은 변화 엄격한 계정을 제공 할 수 없다개발하는 동안 여러 번 통로입니다. 현재 가능한 방법이 한계에 기초하여,이 절차의 목적은 안정적으로 중요한 다양한 신호 경로에서 발생하는 변화의 정량적 분석을 위해 개발 배아 마우스 심장 밸브 영역에서 단백질의 충분한 양을 획득하기위한 프로토콜을 개발하는 것이었다 이 조직의 성숙. 배아 밸브 이미 일반적으로 RNA 분리 및 후속 유전자 발현 분석을위한 2-5 생쥐 해부된다. 그러나, 이들 연구는 하류 시그널링에 영향을 미칠 수있는 번역 후 단백질 변형을 검출의 검출을 허용하지 않는 경로를 활성화, 신호의 판독으로 유전자 발현을 사용하여 한정 하였다. 출발점으로 RNA 분리 기법을 사용하여, 관심 영역을 해부 시작되었다. 우리의 관심은 인산화 된 단백질을 검출 되었기 때문에 신호 확실 나타내는라고대동맥 판막 개발 (E13.5-14.5)의 특정 기간 동안 hway 활동, 우리는 포스페이트없는 트리스 완충액에서 모두 박리를 수행 프로테아제 및 포스파타제 억제제 트리스 계 용해 완충액에 밸브를 모았다. 우리의 특정 경우에만 대동맥판 영역은 회수하고 있지만, 폐동맥 판막 영역 용이 동시에 얻을 수 있었다. 밸브 영역은 균질화 현재 성인 심장 밸브 (7)의 단백질 발현을 연구하는 데 사용되는 샘플 완충액과 혼합 하였다. 작은 샘플 볼륨 (예를 들어, 2 μL)를 사용하고 동일한 유전자형 배아에서 밸브 영역 풀링함으로써 배아 일 13.5 8에서 인산화 및 비 인산화 된 단백질을 검출 할 수 있었다. 밸브 영역 동결 및 용해 버퍼에 저장 될 수 있기 때문에 풀링하기 전에, 필요한 경우, 배아는 유전자형을 할 수있다. 이 기술은 셀 signalin 평가에 사용할 수있는 툴 세트를 넓게g 개발 중에 경로와 특별히 개발 심장 밸브에 대한 면역 조직 화학 염색에 대한 정량적 칭찬을 제공합니다. 이 기술은 발달 심장병 전문의로뿐만 아니라 제한적인 조직을 포함하는 지역에 관심이있는 초기 단계의 배아에서 작동하는 모든 발달 생물 학자뿐만 아니라 도움이 될 것이다.

<table><tbody><tr><td>Timed-pregnant mouse</td><td></td><td></td><td>To be dissected at the embryonic stage of interest</td></tr><tr><td>Stereoscopic microscope</td><td>Nikon</td><td>SMZ645</td><td></td></tr><tr><td>0.1 M Tris, pH 7.6</td><td></td><td></td><td></td></tr><tr><td>Microscissors</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Fine forceps, #5</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Dissecting needle holders</td><td>Ted Pella Inc.</td><td>13560</td><td></td></tr><tr><td>Dissecting needles</td><td>Ted Pella Inc.</td><td>13561-10</td><td></td></tr><tr><td>Micropipette, 20 &amp;mu;l, with tips</td><td></td><td></td><td></td></tr><tr><td>Lysis buffer</td><td></td><td></td><td>50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton</td></tr><tr><td>PhosSTOP</td><td>Roche</td><td>4906845001</td><td>Add 1 tablet to 10 ml lysis buffer</td></tr><tr><td>TissueLyser LT</td><td>Qiagen</td><td>85600</td><td></td></tr><tr><td>Stainless steel beads</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr></tbody></table>

protein isolation from the developing embryonic mouse heart valve region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

이러한 준비의 기술을 사용하여, 우리는 E13.5 배아에서 하나의 대동맥 판막 지역에서 인산화 TGF 계통 1,5,8 (pSmad)을 감지 할 수 있었다. 도 1a에 도시 된 바와 같이, 심지어 단일 밸브 영역으로부터 분리 된 단백질은 희미한 pSmad 밴드를 검출하기에 충분하다. 신호 강도는 풀링 밸브 영역의 개수에 비례하여 증가한다. 중요한 것은, pSmad / β-액틴 비율은 서로 다른 샘플 크기 (...

Watch this Scientific Journal Video about Protein Isolation from the Developing Embryonic Mouse Heart Valve Region at JoVE.com

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

개발 배아 마우스 심장 밸브 지역에서 단백질 분리

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

protein-isolation-from-developing-embryonic-mouse-heart-valve

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.3K 조회수.  University of North Carolina at Chapel Hill. The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

비디오: Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

낮은 혈 청 조건 하에서 심장 조상 세포에서 내 피 분화의 유도

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

연구

발생학

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 ...

생물학

Single-Cell Resolution에서 Mouse Cardiac Progenitor Cells의 핵 분리(Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution)

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

저자 스포트라이트: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

단일 세포 분해능에서 후성유전체 및 유전자 발현 프로파일링을 위한 마우스 심장 전구 세포에서 핵 분리

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

마우스의 심장에서 차 멜라닌 세포와 같은 세포를 분리

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

절연, 문화, 그리고 성인 마우스 된 심근의 기능적 특성

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

배아 심실 내피 세포의 분리

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

관상 동맥 혈관 신생의 체외 모델

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

의학

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

쥐과 밸브 내피 세포의 분리

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

개발 배아 마우스 심장 밸브 지역에서 단백질 분리

요약

더 많은 비디오 탐색

이 비디오의 챕터

낮은 혈 청 조건 하에서 심장 조상 세포에서 내 피 분화의 유도

조류 배아 판막병 전구 세포의 분리 및 문화

단일 세포 분해능에서 후성유전체 및 유전자 발현 프로파일링을 위한 마우스 심장 전구 세포에서 핵 분리

단일 세포 분해능에서 후성유전체 및 유전자 발현 프로파일링을 위한 마우스 심장 전구 세포에서 핵 분리

단일 세포 분해능에서 후성유전체 및 유전자 발현 프로파일링을 위한 마우스 심장 전구 세포에서 핵 분리

마우스의 심장에서 차 멜라닌 세포와 같은 세포를 분리

절연, 문화, 그리고 성인 마우스 된 심근의 기능적 특성

배아 심실 내피 세포의 분리

관상 동맥 혈관 신생의 체외 모델

쥐과 밸브 내피 세포의 분리