Name: isolation, characterization, and differentiation of cardiac stem cells from the adult mouse heart
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart at JoVE.com

这项工作得到了国家卫生研究院向 paras kumar mishra 提供的 hl-113281 和 hl116205 赠款的部分支持。

小鼠的住房、麻醉和牺牲是按照内布拉斯加州大学医学中心批准的 iacuc 协议进行的。
1. 材料
<ol><li>使用在机构动物设施内部饲养的10至12周的 c57bl/6j 黑雄性小鼠, 对 csc 进行隔离, 还可以从非怀孕的雌性小鼠中分离。</li>
	<li>消毒所有必要的手术器械, 包括手术剪刀, 精细的手术剪刀, 弯曲的小腿钳, 和手术刀片, 通过高压灭菌他们之前, 鼠标安乐死的小鼠。</li>
	<li>在灭菌条件?...

<ol>
	<li>Benjamin, E. 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=Heart+Disease+and+Stroke+Statistics-2017+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association.</a> Circulation. 135, e146 (2017).</li><li>Nguyen, P. K., Rhee, J. W., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Stem+Cell+Therapy+and+Heart+Failure,+2000+to+2016:+A+Systematic+Review.">Adult Stem Cell Therapy and Heart Failure, 2000 to 2016: A Systematic Review.</a> The Journal of the American Medical Association Cardiology. 1, 831-841 (2000).</li><li>Emmert, M. 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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=Comparison+of+allogeneic+vs+autologous+bone+marrow-derived+mesenchymal+stem+cells+delivered+by+transendocardial+injection+in+patients+with+ischemic+cardiomyopathy:+the+POSEIDON+randomized+trial.">Comparison of allogeneic vs autologous bone marrow-derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial.</a> The Journal of American Medical Association. 308, 2369-2379 (2012).</li><li>Guijarro, D., 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=Intramyocardial+transplantation+of+mesenchymal+stromal+cells+for+chonic+myocardial+ischemia+and+impaired+left+ventricular+function:+Results+of+the+MESAMI+1+pilot+trial.">Intramyocardial transplantation of mesenchymal stromal cells for chonic myocardial ischemia and impaired left ventricular function: Results of the MESAMI 1 pilot trial.</a> International Journal of Cardiology. 209, 258-265 (2016).</li><li>Bobi, 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=Intracoronary+Administration+of+Allogeneic+Adipose+Tissue-Derived+Mesenchymal+Stem+Cells+Improves+Myocardial+Perfusion+But+Not+Left+Ventricle+Function,+in+a+Translational+Model+of+Acute+Myocardial+Infarction.">Intracoronary Administration of Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells Improves Myocardial Perfusion But Not Left Ventricle Function, in a Translational Model of Acute Myocardial Infarction.</a> Journal of the American Heart Association. 6, (2017).</li><li>Suzuki, E., Fujita, D., Takahashi, M., Oba, S., Nishimatsu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue-derived+stem+cells+as+a+therapeutic+tool+for+cardiovascular+disease.">Adipose tissue-derived stem cells as a therapeutic tool for cardiovascular disease.</a> World Journal of Cardiology. 7, 454-465 (2015).</li><li>Gao, L. R., 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=Intracoronary+infusion+of+Wharton's+jelly-derived+mesenchymal+stem+cells+in+acute+myocardial+infarction:+double-blind,+randomized+controlled+trial.">Intracoronary infusion of Wharton's jelly-derived mesenchymal stem cells in acute myocardial infarction: double-blind, randomized controlled trial.</a> BMC Medicine. 13, 162 (2015).</li><li>Simpson, D. L., 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=A+strong+regenerative+ability+of+cardiac+stem+cells+derived+from+neonatal+hearts.">A strong regenerative ability of cardiac stem cells derived from neonatal hearts.</a> Circulation. , S46-S53 (2012).</li><li>Kazakov, A., 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=C-kit(+)+resident+cardiac+stem+cells+improve+left+ventricular+fibrosis+in+pressure+overload.">C-kit(+) resident cardiac stem cells improve left ventricular fibrosis in pressure overload.</a> Stem Cell Research. 15, 700-711 (2015).</li><li>Ong, S. G., 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=Cross+talk+of+combined+gene+and+cell+therapy+in+ischemic+heart+disease:+role+of+exosomal+microRNA+transfer.">Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer.</a> Circulation. 130, S60-S69 (2014).</li><li>Sahoo, S., Losordo, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+cardiac+repair+after+myocardial+infarction.">Exosomes and cardiac repair after myocardial infarction.</a> Circulation Research. 114, 333-344 (2014).</li><li>Zhang, Z., 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=Pretreatment+of+Cardiac+Stem+Cells+With+Exosomes+Derived+From+Mesenchymal+Stem+Cells+Enhances+Myocardial+Repair.">Pretreatment of Cardiac Stem Cells With Exosomes Derived From Mesenchymal Stem Cells Enhances Myocardial Repair.</a> Journal of the American Heart Association. 5, (2016).</li><li>Ibrahim, A. G., Cheng, K., Marban, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+as+critical+agents+of+cardiac+regeneration+triggered+by+cell+therapy.">Exosomes as critical agents of cardiac regeneration triggered by cell therapy.</a> Stem Cell Reports. 2, 606-619 (2014).</li><li>Emanueli, C., Shearn, A. I., Angelini, G. D., Sahoo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+exosomal+miRNAs+in+cardiovascular+protection+and+repair.">Exosomes and exosomal miRNAs in cardiovascular protection and repair.</a> Vascular Pharmacology. 71, 24-30 (2015).</li><li>Menasche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+cell+therapy:+lessons+from+clinical+trials.">Cardiac cell therapy: lessons from clinical trials.</a> Journal of Molecular and Cellular Cardiology. 50, 258-265 (2011).</li><li>Trounson, A., McDonald, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Therapies+in+Clinical+Trials:+Progress+and+Challenges.">Stem Cell Therapies in Clinical Trials: Progress and Challenges.</a> Cell Stem Cell. 17, 11-22 (2015).</li><li>Takamiya, M., Haider, K. H., Ashaf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+multipotent+sub-population+of+Sca-1(+)+cardiac+progenitor+cells+for+myocardial+regeneration.">Identification and characterization of a novel multipotent sub-population of Sca-1(+) cardiac progenitor cells for myocardial regeneration.</a> PLoS One. 6, e25265 (2011).</li><li>Cambria, 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=Translational+cardiac+stem+cell+therapy:+advancing+from+first-generation+to+next-generation+cell+types.">Translational cardiac stem cell therapy: advancing from first-generation to next-generation cell types.</a> NPJ Regenerative Medicine. 2, 17 (2017).</li><li>Bruyneel, A. A., Sehgal, A., Malandraki-Miller, S., Carr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Therapy+for+the+Heart:+Blind+Alley+or+Magic+Bullet?.">Stem Cell Therapy for the Heart: Blind Alley or Magic Bullet?.</a> Journal of Cardiovascular Translational Research. 9, 405-418 (2016).</li><li>Garbern, J. C., Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+stem+cell+therapy+and+the+promise+of+heart+regeneration.">Cardiac stem cell therapy and the promise of heart regeneration.</a> Cell Stem Cell. 12, 689-698 (2013).</li><li>Oh, H., Ito, H., Sano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+to+success+in+heart+failure:+Cardiac+cell+therapies+in+patients+with+heart+diseases.">Challenges to success in heart failure: Cardiac cell therapies in patients with heart diseases.</a> Journal of Cardiology. 68, 361-367 (2016).</li><li>Smith, A. 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=Isolation+and+characterization+of+resident+endogenous+c-Kit++cardiac+stem+cells+from+the+adult+mouse+and+rat+heart.">Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart.</a> Nature Protocols. 9, 1662-1681 (2014).</li><li>Rutering, 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=Improved+Method+for+Isolation+of+Neonatal+Rat+Cardiomyocytes+with+Increased+Yield+of+C-Kit++Cardiac+Progenitor+Cells.">Improved Method for Isolation of Neonatal Rat Cardiomyocytes with Increased Yield of C-Kit+ Cardiac Progenitor Cells.</a> Journal of Stem Cell Research and Therapy. 5, 1-8 (2015).</li><li>Saravanakumar, M., Devaraj, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+homing+pattern+of+c-kit++Sca-1++CXCR4++resident+cardiac+stem+cells+in+neonatal,+postnatal,+and+adult+mouse+heart.">Distribution and homing pattern of c-kit+ Sca-1+ CXCR4+ resident cardiac stem cells in neonatal, postnatal, and adult mouse heart.</a> Cardiovascular Pathology. 22, 257-263 (2013).</li><li>Monsanto, M. 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=Concurrent+Isolation+of+3+Distinct+Cardiac+Stem+Cell+Populations+From+a+Single+Human+Heart+Biopsy.">Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy.</a> Circulation Research. 121, 113-124 (2017).</li><li>Vidyasekar, P., Shyamsunder, P., Santhakumar, R., Arun, R., Verma, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+protocol+for+the+isolation+and+culture+of+cardiomyocytes+and+progenitor+cells+from+neonatal+mouse+ventricles.">A simplified protocol for the isolation and culture of cardiomyocytes and progenitor cells from neonatal mouse ventricles.</a> European Journal of Cell Biology. 94, 444-452 (2015).</li><li>Dergilev, K. V., 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=Comparison+of+cardiac+stem+cell+sheets+detached+by+Versene+solution+and+from+thermoresponsive+dishes+reveals+similar+properties+of+constructs.">Comparison of cardiac stem cell sheets detached by Versene solution and from thermoresponsive dishes reveals similar properties of constructs.</a> Tissue Cell. 49, 64-71 (2017).</li><li>Zaruba, M. M., Soonpaa, M., Reuter, S., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyogenic+potential+of+C-kit(+)-expressing+cells+derived+from+neonatal+and+adult+mouse+hearts.">Cardiomyogenic potential of C-kit(+)-expressing cells derived from neonatal and adult mouse hearts.</a> Circulation. 121, 1992-2000 (1992).</li><li>Wang, 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=Isolation+and+characterization+of+a+Sca-1+/CD31-+progenitor+cell+lineage+derived+from+mouse+heart+tissue.">Isolation and characterization of a Sca-1+/CD31- progenitor cell lineage derived from mouse heart tissue.</a> BMC Biotechnology. 14, 75 (2014).</li><li>Smits, A. 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此 csc 隔离协议的关键步骤如下所示。1) 从小鼠身上提取心脏时, 必须保持灭菌状态。心脏提取过程中的任何污染都可能损害 csc 的质量. 2) 在切碎心脏之前, 必须完全切除血液, 这是通过用 hbss 溶液对整个心脏和心脏碎片进行几次清洗来完成的。3) 心脏碎片必须完全裂解成单细胞悬浮液与胶原酶溶液。4) 用于分离细胞的多蔗糖和二甲酸钠梯度溶液必须预热。5) csc 培养的介质必须预热。6) 在培养皿中...

缺血性心脏病, 包括心肌梗死 (mi), 是全世界死亡的主要原因1。干细胞治疗再生死亡心肌仍然是改善心肌梗死心脏 2,3,4, 5 的主要途径。不同类型的干细胞被用来补充死亡心肌和改善心肌梗死的心功能。它们大致可分为胚胎干细胞6和成人干细胞.在成人干细胞中, 使用了各种类型的干细胞, 如骨髓源的单个核细胞7,8, 骨髓间充质干细胞 9,10, 脂肪组织11、12和脐带13和 csc14、15。干细胞可以通过内分泌和/或辅助作用促进心脏再生16,17,18, 19,20.然而, 干细胞治疗的一个主要局限性是获得足够数量的干细胞, 这些干细胞能够增殖和/或分化到特定的心脏谱系21,22。干细胞自体同种异体移植是干细胞治疗中的一个重要挑战。csc 可能是心脏再生的更好方法, 因为它们来自心脏, 它们比非心脏干细胞更容易分化为心脏谱系。因此, 它降低了畸胎瘤的风险。此外, csc 的内分泌和副分泌效应, 如来自 csc 的外显子和 mirna, 可能比其他类型的干细胞更有效。因此, csc 仍然是心脏再生23,24 的更好选择。
尽管 csc 由于其心脏源, 是心肌心脏再生的较好候选, 但 csc 的一个主要限制是由于缺乏有效的隔离方法而导致的产量较低。另一个限制因素可能是 csc 向心肌细胞谱系 2、25、26、27 的分化受损。为了避免这些限制, 重要的是要为 csc 的分离、表征和向心脏谱系的分化制定一个有效的协议。csc 没有单一的可接受标记, 基于细胞表面标记的 csc 隔离可产生较少的 csc。在这里, 我们标准化了一种简单的梯度离心方法, 将 csc 从鼠标心脏中分离出来, 该方法具有成本效益, 并提高了 csc 的产量。这些孤立的 csc 可以选择特定的细胞表面标记通过荧光激活细胞短路。除了 csc 分离外, 我们还提供了 csc 培养、表征和向心肌细胞谱系分化的协议。因此, 我们提出了一种很好的方法来隔离、表征、培养和区分 csc 和成人鼠标心脏 (图 5)。

<table>
	<tbody>
		<tr>
			<td>Mice</td>
			<td>The Jackson laboratory, USA</td>
			<td>Stock no. 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OCT4-</td>
			<td>Abcam</td>
			<td>ab18976 (rabbit polyclonal)</td>
			<td>OCT4-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>SOX2</td>
			<td>Abcam</td>
			<td>ab97959 (rabbit polyclonal)</td>
			<td>SOX2-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Nanog</td>
			<td>Abcam</td>
			<td>ab80892 (rabbit polyclonal)</td>
			<td>Nanog-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Ki67</td>
			<td>Abcam</td>
			<td>ab16667 (rabbit polyclonal)</td>
			<td>Ki67-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Sca I</td>
			<td>Millipore</td>
			<td>AB4336 (rabbit polyclonal)</td>
			<td>Sca I Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>NKX2-5</td>
			<td>Santa Cruz</td>
			<td>sc-8697 (goat polyclonal)</td>
			<td>NKX2-5-Primary antibody- 1:50 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>GATA4</td>
			<td>Abcam</td>
			<td>ab84593 (rabbit polyclonal)</td>
			<td>GATA4-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>MEF2C</td>
			<td>Santa Cruz</td>
			<td>sc-13268 (goat polyclonal)</td>
			<td>MEF2C-Primary antibody- 1:50 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Troponin I</td>
			<td>Millipore</td>
			<td>MAB1691 (mouse monoclonal)</td>
			<td>Troponin I-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Actinin</td>
			<td>Millipore</td>
			<td>MAB1682 (mouse monoclonal)</td>
			<td>Actinin-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>ANP</td>
			<td>Millipore</td>
			<td>AB5490 (mouse polyclonal)</td>
			<td>ANP-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution</td>
		</tr>
		<tr>
			<td>Alex Fluor-488 checken anti-rabbit</td>
			<td>Life technology</td>
			<td>Ref no. A21441</td>
			<td></td>
		</tr>
		<tr>
			<td>Alex Fluor-594 goat anti-rabbit</td>
			<td>Life technology</td>
			<td>Ref no. A11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Alex Fluor-594 rabbit anti-goat</td>
			<td>Life technology</td>
			<td>Ref no. A11078</td>
			<td></td>
		</tr>
		<tr>
			<td>Alex Fluor-488 checken anti-mouse</td>
			<td>Life technology</td>
			<td>Ref no. A21200</td>
			<td></td>
		</tr>
		<tr>
			<td>Alex Fluor-594 checken anti-goat</td>
			<td>Life technology</td>
			<td>Ref no. A21468</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture medium: </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CSC maintenance medium</td>
			<td>Millipore</td>
			<td>SCM101</td>
			<td>Note: For CSC culture, PBS or incomplete DMEM medium was used for washing the cells</td>
		</tr>
		<tr>
			<td>cardiomyocytes differentiation medium</td>
			<td>Millipore</td>
			<td>SCM102</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D5546</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell Isolation buffer:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polysucrose and sodium diatrizoate solution (Histopaque1077)</td>
			<td>Sigma</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>2018-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase solution</td>
			<td>STEMCELL Technologies</td>
			<td>7913</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>LONZA</td>
			<td>S1226</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Thermoscientific</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Other reagents:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal checken serum</td>
			<td>Vector laboratory</td>
			<td>S3000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Applichem</td>
			<td>A100,0010</td>
			<td>Dapi, working concentration-1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Biorad</td>
			<td>145-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ACROS</td>
			<td>Cas No. 900-293-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Sceintific</td>
			<td>Lot No. 160170</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tissue culture materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 flask</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 flask</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell stainer</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m cell stainer</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Fisher Scientific</td>
			<td>09-719C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syring</td>
			<td>BD</td>
			<td>Ref no. 309604</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L, 200 &#181;L, 1000 &#181;L pipette tips</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL, 10mL, 25 mL disposible plastic pipette</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrufuge machine</td>
			<td>Thermo Scientific</td>
			<td>LEGEND X1R centrifuge</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS microscope</td>
			<td>Life technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting slide</td>
			<td>Biorad</td>
			<td>145-0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippte aid</td>
			<td>Thermo Scientific</td>
			<td>S1 pipet filler</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgical Instruments:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Scientific Tool</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine surgical scissors</td>
			<td>Fine Scientific Tool</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curve shank forceps</td>
			<td>Fine Scientific Tool</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Fine Scientific Tool</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, characterization, and differentiation of cardiac stem cells from the adult mouse heart

心肌梗塞 (mi) 是全球发病率和死亡率的主要原因。再生医学的一个主要目标是补充心肌梗死后的死亡心肌。虽然有几种策略已被用于再生心肌, 干细胞治疗仍然是补充心肌梗死心脏的死亡心肌的主要方法。越来越多的证据表明, 成人心脏中存在常驻心脏干细胞 (csc), 其内分泌和 (或旁分泌) 对心脏再生有影响。然而, csc 分离及其对心肌细胞, 特别是心肌细胞的表征和分化, 仍然是一个技术挑战。在本研究中, 我们提供了一个简单的方法, 分离, 表征和分化 csc 从成年小鼠心脏。在这里, 我们描述了一种密度梯度方法隔离 csc, 其中心脏被消化0.2% 胶原酶 ii 溶液。为了表征分离的 csc, 我们评估了 cscs1、nkx2-5 和 gat4 和多能/干细胞的表达。我们还通过在培养培养培养皿和评估扩散标记 ki-67 的表达来确定孤立的 csc 的增殖潜力。为了评估 csc 的分化潜力, 我们选择了七到十天培养的 csc。我们把它们转移到一个有心肌细胞分化培养基的新盘子里。它们在细胞培养孵化器中孵育 12天, 而分化培养基每三天改变一次。分化的 csc 表达心肌细胞特异性标记: 放线素和肌钙蛋白 i。因此, 使用该协议分离的 csc 具有干细胞和心脏标记物, 它们有向心肌细胞谱系增殖和分化的潜力。

在本研究中, 我们分离 csc 从10至12周的 c57bl/6j 雄性小鼠心脏。在这里, 我们提出了一个简单的方法, csc 隔离和表征使用标记的多能性。我们还提出了一个优雅的方法 csc 分化和描述的 csc 分化为心肌细胞谱系。我们在相对照显微镜下观察到2至3天培养的 csc 的纺锤形形态 (图 1 a 和1 b)。我们发现在维持培养基中培养7天的时候, csc 的形态发生?...

Watch this Scientific Journal Video about Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart at JoVE.com

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

本文的总体目标是规范心脏干细胞 (csc) 与成年小鼠心脏分离、表征和分化的协议。在这里, 我们描述了一种密度梯度离心方法, 以分离小鼠 csc 和详细的方法, csc 培养, 增殖, 并分化为心肌细胞。

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

本文的总体目标是标准化心脏干细胞、CCS与成人小鼠心脏的分离、表征和分化协议。这种方法可以帮助回答心血管疾病和区域治疗领域（如心脏干细胞治疗和心力衰竭）中关键问题。这种技术的主要优点是具有成本效益，在小时间内具有高助用性。开始本实验，如协议所述，在灭菌条件下准备所有必要的材料、仪器和隔离缓冲液。从安乐死小鼠腹部取出皮肤后，切开无菌罩内的肋骨以打开胸腔。暴露心脏，然后用一毫升注射器去除心脏附近的血液。要去除心脏附近的任何血液，请用冰冷的PBS清洗周围区域。使用手术剪刀和钳子解剖心脏，将心脏放在含有10毫升冰冷PBS的100毫米培养皿中。使用弯曲的刀柄钳子来心绞痛，并去除心脏内的残余血液。将四到五颗孤立的心转移到含有 10 毫升冰冷火腿平衡盐溶液的 100 毫升培养皿中。再次洗动心脏，洗掉心脏内残留的血液。为确保完全去除血液，每次洗涤后更换HSS溶液。用一对钳子握住每颗心脏，用手术刀片将每颗心脏切成两到四毫米，放入一个100毫米的培养皿中，其中含有5至7毫升的HPSS。经常更换溶液，以确保完全脱血。最后，在哈佛商学院解决方案中切碎心脏。将切碎的心在50毫微锥形管中以500倍G，在4摄氏度下放置5分钟，然后丢弃上流水线。加入5至6毫升，0.2%的胶原酶II溶液到颗粒中消化组织。在 37 摄氏度的摇床上摇动管，在 37 摄氏度下，45 至 60 分钟，彻底悬浮颗粒。每20至30分钟，用手大力摇动管子，增强解解。使用五毫升血清移液器和一毫升移液器尖端将糖化组织混合物分离成单细胞悬浮液。要停止组织的酶解，添加两到三倍的商用CSC维持介质，以体积的解酶组织。通过放置在50毫升锥形管上的100微米细胞过滤器将消化的细胞悬浮液传递，以去除任何未消化的组织碎片。要将细胞分离为密度梯度离心，在50毫升锥形管中加入37摄氏度、预热、多糖和三酸钠溶液。然后轻轻地缓慢地在溶液上添加等量的滤液，避免两种溶液的任何混合。在聚糖和三聚氰酸钠溶液上缓慢添加滤液，而不通过将管子倾斜在45度并倾斜在管壁上，这一点至关重要。不要过度添加聚糖和二三酸钠溶液。将管子非常缓慢地放在回转铲斗离心机中，确保不打扰溶液。在室温下以较低的加速和减速速度离心500倍G，20分钟，以避免混合两种溶液，并正确分离心脏干细胞。在离心机中非常小心地将梯度溶液置于无干扰状态至关重要。将离心机的加速和减速必须处于最低速度，如 1 或零。使用一毫米移液器将含有 CCS 的薄薄涂层以及额外的溶液从上层转移到 15 毫升消毒管。加入等量的CSC维护介质，并正确混合以中和残留的多糖和三酸钠溶液。在 4 摄氏度下，在 500 次 G 下将这种悬浮液离心五分钟。丢弃上一杯。使用不完整的 DMEM 解决方案重复洗涤 CCS 至少两次。在4摄氏度下以500倍G离心5分钟，去除残留的多糖和三酸钠溶液。去除上清液后，将含有纯化CSC的颗粒重新用一毫升CSC维护介质中，然后计算细胞数。将CSC播种在涂有0.02%明胶溶液的六井培养板中，该溶液含有0.5%纤维素，并加入两毫升完整的维护介质。在5%CO2中生长在37摄氏度的细胞。当CSC培养达到汇合时，将细胞转移到新的明胶和纤维素涂层板，如文本协议中所述。培养这些转移的P0CSC在37摄氏度在5%CO2培养箱，直到汇合的完整CSC维护介质。将细胞转移到新板上，以制造可用于进一步实验的P1 CCS。为了在初始阶段保持 CSC 区域性，请进一步播种单元格，如文本协议中所述。要描述培养细胞的特性，请将包含CSC的板放在荧光显微镜的目标上。使用十倍的放大率对细胞进行聚焦。要观察细胞，请使用相位对比度光圈，通过更改目标镜头将放大倍数提高至 20 倍，然后对 CCS 进行图像。进行免疫控制后，将培养板置于荧光显微镜下。聚焦不同放大倍率的单元格，观察相位对比度下的细胞和不同的激发滤镜，并保存图像。对于细胞分化，在六井板中生长CSC，其温度为37摄氏度，预热CSC维护介质。当细胞达到80至95%汇合时，用两毫升37摄氏度的预热心肌细胞分化介质取代维持介质。在37摄氏度下孵育，在5%CO2中孵育长达两到三周。每两到三天更换一次分化介质。在介质变化期间，在显微镜下观察细胞形态，以确保良好的培养条件。12 天后，按照标准免疫控制协议对细胞进行分化标记图像。在相对比显微镜下观察到两到三天的培养性 CSC 会显示主轴状形态。经过七天的文化，他们表现出形态的变化变得拉长。在对多能标记进行免疫污染后，CSC 将显示 OCT4、SOX2 和 Nanog 的表达。此外，CSC在文化媒介中扩散，并表达扩散标记Ki67。为了描述CSC的心脏起源，检查了心脏标记的表达，并且细胞对心脏标记Sca-1、NKX2.5和GATA4呈阳性。在心肌细胞分化介质中培养12天后，分化的CSC显示心肌细胞标记ACTININ和TROPONIN-I的表达。在尝试此过程时，必须记住不允许混合梯度溶液和滤液。将加速和减速设置为最低速度也很重要。无论谁污染它们，建议在生物安全柜内执行所有细胞隔离过程。按照这个程序，其他方法，如磁珠为基础的分离或流细胞为基础的细胞排序方法，可以执行，以回答其他问题，如，心脏干细胞的均质种群有多清晰？心脏干细胞的裂纹半径是大标记，与心肌细胞有差异。开发后，这项技术将为心血管疾病领域的研究人员铺平道路，他们往往从小鼠心脏中高产心脏干细胞，这对缺血性心力衰竭的再生治疗可能很重要。不要忘记，使用手术刀片、漂白剂和 DMSO 可能会很危险，在执行本程序时应始终采取预防措施，例如使用人员防护设备 PPE。

Research

JoVE Journal

Biology

Isolation of Retinal Stem Cells from the Mouse Eye

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

科研

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

成人从人体皮肤的上皮干细胞的分离和培养

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

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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

NaÃƒÆ’Ã‚Â¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

天真成人干细胞的分离从初级人类成纤维细胞培养

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

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

分离，培养和成年小鼠Cardiomyoctyes的功能分析

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

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

从人类Lipoaspirates脂肪来源干细胞的手动隔离

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

成年小鼠黏膜下层和固有层肠道胶质细胞的分离

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...