Name: induction of endothelial differentiation in cardiac progenitor cells under low serum conditions
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions at JoVE.com

作者感谢 vera lorenz 在实验期间提供的有益支持, 并感谢来自巴塞尔大学和大学医院的流式细胞术设施的工作人员。这项工作得到了巴塞尔大学 (到 michika mochizuki) 的 "固定轨道" 项目的支持。gabriela m. kuster 得到瑞士国家科学基金会赠款的支持 (赠款编号 310030 _ 156953)。

将小鼠用于细胞隔离, 符合《实验动物护理和使用指南》和《瑞士动物保护法》, 并得到瑞士州当局的批准。
注: 从鼠标心脏中分离 sca-1+/ cd31-sp-cpc 基本上是按照前面所述的34进行的, 但做了一些修改。有关所使用的材料和试剂, 请参阅材料表。在所有实验中, 从小鼠身上分离出的心脏 sp-cpc 都被扩增、传代, 并以类似细胞系的方式?...

<ol>
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本协议的优点:
该方案提供了一种 cpc 的内皮分化技术。我们发现, 低血清浓度和低细胞密度可以提高内皮分化的效率, 从而在这些条件下, ln 被证明是比 fn 更合适的底物。我们使用了两种不同类型的 cpc: 以类似细胞的方式使用的大鼠 cpc 和被隔离和扩展的小鼠 sp-cpc。值得注意的是, 该协议适用于这两种 cpc, 目前的技术使科学家能够从转基因小鼠和临床前疾病模型以及人类<sup class=...

最近的研究支持在成年哺乳动物心脏1,2,3 和 cpc 中存在的常驻心脏祖细胞 (cpc) 可以成为心脏损伤4后细胞治疗的有用来源.5. 此外, 扩大的 cpc 可为药物筛查和调查疾病机制提供一个富有成效的模式, 如果与罕见的心肌病患者或各自的疾病模型6,7分离。
从成人心脏中分离出的 cpc 具有干细胞细胞1、2、3、8, 因为它们是多能、克隆性的, 具有自我更新的能力。然而, 有许多不同的 (子) cpc 群表现出不同的表面标记特征, 例如, 包括 c-kit、sca-1 等, 或通过不同的隔离技术检索 (表 1)。已经制定了若干文化和差异化协议 1,2,8, 9,10,11,12, 13,14,15,16,17,18。这些协议在生长因子和血清含量方面的差异主要不同, 这些因素根据培养的目的进行调整, 并可能导致结果和结果的差异, 包括分化效率。
基于标记的隔离技术:
cpc 可以根据特定的表面标记表达式1、2、8、9、10、11、12 、13进行隔离,14,15,16,17,18。先前的研究表明, c-kit 和 sca-1 可能是分离居民cpc 1,11,14,19,20的最佳标记。因为这些标记都不是 cpc 的特定标记, 所以通常会应用不同标记的组合。例如, cpc表达的 c-kit 21 含量较低, 而 c-kit 也被其他细胞类型表达, 包括肥大细胞22、内皮细胞23和造血干细胞/祖细胞24。另一个问题是, 并非所有标记都在所有物种中表达。sca-1 的情况就是如此, 它在老鼠身上表达, 但在人类25中不表达。因此, 在临床试验和使用人体样本的研究中, 使用独立于隔离标记的协议可能是有利的。
与标记无关的隔离技术:
cpc 隔离有几种主要技术, 它们主要独立于曲面标记表达式, 但可以根据需要通过连续选择特定标记正亚分来细化 (另见表 1)。(1) 侧群 (sp) 技术最初的特征是原始的造血干细胞群体, 其基础是通过 atp 结合盒 (abc) 载体27排出 dna 染料 hoechst 3334226 的能力.心脏 sp 细胞已被不同的组分离, 并报告表达了各种标记与一些小差异的报告2,8,13, 14。(2) 成集细胞成纤维细胞 (cfu-f) 最初是根据间充质间质细胞 (msc) 样表型定义的。分离的骨髓间充质干细胞在菜肴上培养, 以诱导菌落的形成。这种形成细胞分裂的 msc 样 cfu-f 可以从成人心脏中分离, 并能够分化为心脏谱系15。(3) 心细胞衍生细胞 (cdc) 是由组织活检或外植体 28、29、30、31产生的细胞簇衍生的单个细胞。最近发现, cd105+/cd90/ct-kit-细胞部分表现出心肌和再生电位32.
在这里, 我们使用从小鼠身上分离出的 sp-cpc, 根据之前对大鼠 cpc 和小鼠 sp-cpc33的研究, 提供了一种有效诱导内皮谱系的协议。该协议在细胞密度、培养基血清含量和底物方面对培养和扩张技术进行了具体调整。它不仅适用于小鼠 sp-cpc, 也适用于不同类型的 cpc, 目的是诱导命运从放大到内皮承诺的 cpc, 无论是为了移植这些细胞还是用于机械体外研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Culture medium</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>ThermoFisher</td>
			<td>#12440</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)/Nutrient Mixture F12 Ham</td>
			<td>Merck</td>
			<td>#D8437</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin (P/S)</td>
			<td >ThermoFisher</td>
			<td >#15140122</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>#SH30071</td>
			<td>3.5% (0.1% for lineage induction)</td>
		</tr>
		<tr >
			<td >L-Glutamine&#160;</td>
			<td >ThermoFisher</td>
			<td>#25030</td>
			<td>Final concentration 2 mM</td>
		</tr>
		<tr >
			<td >Glutathione</td>
			<td>Merck</td>
			<td>#G6013</td>
			<td></td>
		</tr>
		<tr >
			<td >Recombinant Human Epidermal Growth Factor (EGF)</td>
			<td>Peprotech</td>
			<td>#AF-100-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Recombinant Basic Fibroblast Growth Factor (FGF)</td>
			<td>Peprotech</td>
			<td>#AF-100-18B</td>
			<td></td>
		</tr>
		<tr >
			<td >B27 Supplement</td>
			<td>ThermoFisher</td>
			<td>#17504044</td>
			<td></td>
		</tr>
		<tr >
			<td >Cardiotrophin 1&#160;</td>
			<td>Peprotech</td>
			<td>#250-25</td>
			<td></td>
		</tr>
		<tr >
			<td >Thrombin</td>
			<td>Diagontech AG, Switzerland</td>
			<td>#100-125</td>
			<td></td>
		</tr>
		<tr >
			<td >Hanks&#39; Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-)</td>
			<td >ThermoFisher</td>
			<td >#14170</td>
			<td></td>
		</tr>
		<tr >
			<td >0.05 % Trypsin-EDTA</td>
			<td>ThermoFisher</td>
			<td>#25300</td>
			<td></td>
		</tr>
		<tr >
			<td >T75 Flask</td>
			<td>Sarstedt</td>
			<td>#83.3911</td>
			<td></td>
		</tr>
		<tr >
			<td >Endothelial differentiation&#160;&#160;</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Endothelial Cell Growth Medium (EGM)-2 BulletKit</td>
			<td>Lonza</td>
			<td>#CC-3162</td>
			<td></td>
		</tr>
		<tr >
			<td >Ham&#39;s F-12K (Kaighn&#39;s) Medium</td>
			<td>ThermoFisher</td>
			<td>#21127</td>
			<td></td>
		</tr>
		<tr >
			<td >Laminin&#160;</td>
			<td>Merck</td>
			<td>#L2020</td>
			<td></td>
		</tr>
		<tr >
			<td >Fibronectin&#160;</td>
			<td>Merck</td>
			<td>#F4759</td>
			<td>Dilute in ddH2O</td>
		</tr>
		<tr >
			<td >6 Well Plate</td>
			<td>Falcon</td>
			<td>#353046</td>
			<td></td>
		</tr>
		<tr >
			<td >Formaldehyde Solution</td>
			<td>Merck</td>
			<td>#F1635</td>
			<td>Diluite 1:10 in PBS (3.7% final concentration)</td>
		</tr>
		<tr >
			<td >Triton X-100</td>
			<td>Merck</td>
			<td>#93420</td>
			<td>0.1% in ddH2O</td>
		</tr>
		<tr >
			<td >Normal Goat Serum (10%)</td>
			<td>ThermoFisher</td>
			<td>#50062Z</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-von Willebrand Factor antibody</td>
			<td>Abcam</td>
			<td>#ab6994</td>
			<td>1:100 in 10% goat serum</td>
		</tr>
		<tr >
			<td >Goat anti-Rabbit IgG, Alexa Fluor 546</td>
			<td>ThermoFisher</td>
			<td>#A11010</td>
			<td>1:500 in 10% goat serum</td>
		</tr>
		<tr >
			<td >4&#39;,6-diamidino-2-phenylindole, dihydrochloride (DAPI)</td>
			<td>ThermoFisher</td>
			<td>#62247</td>
			<td>1:500 in ddH2O&#160;</td>
		</tr>
		<tr >
			<td >SlowFade Antifade Kit</td>
			<td>ThermoFisher</td>
			<td>#S2828</td>
			<td></td>
		</tr>
		<tr >
			<td >BX63 widefield microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Tube formation</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >96 Well Plate</td>
			<td>Falcon</td>
			<td>#353072</td>
			<td></td>
		</tr>
		<tr >
			<td >5 ml Round Bottom Tube with Strainer Cap</td>
			<td >Falcon</td>
			<td >#352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Matrigel Growth Factor Reduced</td>
			<td >Corning</td>
			<td >#354230</td>
			<td></td>
		</tr>
		<tr >
			<td >IX50 widefield microscope</td>
			<td >Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Sca-1+/CD31- cardiac side population isolation34&#160;</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Reagents</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Pentobarbital Natrium 50 mg/ml ad usum vet.</td>
			<td >in house hospital pharmacy</td>
			<td >#9077862</td>
			<td>Working solution: 200 mg/kg</td>
		</tr>
		<tr >
			<td >Phosphate Buffered Saline (PBS) CaCl2(-), MgCl2(-)</td>
			<td >ThermoFisher</td>
			<td >#20012</td>
			<td></td>
		</tr>
		<tr >
			<td >Hanks&#39; Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-), phenol red (-)</td>
			<td >ThermoFisher</td>
			<td >#14175</td>
			<td>Prepare HBSS 500 mL + 2% FBS&#160; for quenching Collagenase B activity&#160;</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) 1g/L of D-Glucose, L-Glutamine, Pyruvate</td>
			<td >ThermoFisher</td>
			<td >#331885</td>
			<td>Prepare DMEM + 10% FBS&#160; + 25 mM HEPES+ P/S for Hoechst stanining</td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin&#160; (P/S)</td>
			<td>ThermoFisher</td>
			<td>#15140122</td>
			<td></td>
		</tr>
		<tr >
			<td >HEPES 1 M</td>
			<td >ThermoFisher</td>
			<td >#15630080</td>
			<td>Final concentration 25 mM</td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum (FBS)</td>
			<td >Hyclone</td>
			<td >#SH30071</td>
			<td></td>
		</tr>
		<tr >
			<td >RBC LysisBuffer (10X)</td>
			<td >BioLegend</td>
			<td >#420301/100mL</td>
			<td>Dilute to 1X in ddH2O and filter through a 0.2 &#181;m filter</td>
		</tr>
		<tr >
			<td >Collagenase B</td>
			<td>Merck</td>
			<td >#11088807001</td>
			<td>Final concentration 1 mg/mL in HBSS, filtered through a 0.2 &#181;m filter</td>
		</tr>
		<tr >
			<td >bisBenzimide H33342 Trihydrochloride (Hoechst)</td>
			<td>Merck</td>
			<td >#B2261</td>
			<td>Prepare 1 mg/mL in ddH2O</td>
		</tr>
		<tr >
			<td >Verapamil-hydrochloride&#160;</td>
			<td>Merck</td>
			<td >#V4629</td>
			<td>Final concentration 83.3 &#181;M&#160;</td>
		</tr>
		<tr >
			<td >APC Rat Anti-Mouse CD31</td>
			<td>BD Biosciences</td>
			<td>#551262</td>
			<td>0.25 &#181;g/107cells</td>
		</tr>
		<tr >
			<td >FITC Rat Anti-Mouse Ly-6A/E (Sca-1)</td>
			<td>BD Biosciences</td>
			<td>#557405</td>
			<td>0.6 &#181;g/107cells</td>
		</tr>
		<tr >
			<td >7-Aminoactinomycin D (7-ADD)</td>
			<td>ThermoFisher</td>
			<td>#A1310</td>
			<td>0.15 &#181;g/106cells</td>
		</tr>
		<tr >
			<td >APC rat IgG2a k Isotype Control</td>
			<td>BD Biosciences</td>
			<td>#553932</td>
			<td>0.25 &#181;g/107cells</td>
		</tr>
		<tr >
			<td >FITC Rat IgG2a k Isotype Control</td>
			<td>BD Biosciences</td>
			<td>#554688</td>
			<td>0.6 &#181;g/107cells</td>
		</tr>
		<tr >
			<td >Material</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Needles 27G</td>
			<td>Terumo</td>
			<td>#NN-2719R</td>
			<td></td>
		</tr>
		<tr >
			<td >Needles 18G</td>
			<td>Terumo</td>
			<td>#NN-1838S</td>
			<td></td>
		</tr>
		<tr >
			<td >Single Use Syringes 1 mL sterile</td>
			<td>CODAN</td>
			<td>#62.1640</td>
			<td></td>
		</tr>
		<tr >
			<td >Transferpipette 3.5 mL</td>
			<td>Sarstedt</td>
			<td>#86.1171.001</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell Strainer 40 &#181;m blue</td>
			<td>BD Biosciences&#160;</td>
			<td>#352340</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell Strainer 100 &#181;m yellow</td>
			<td>BD Biosciences</td>
			<td>#352360</td>
			<td></td>
		</tr>
		<tr >
			<td >Lumox Dish 50</td>
			<td>Sarstedt</td>
			<td>#94.6077.305</td>
			<td></td>
		</tr>
		<tr >
			<td >Culture Dishes P100</td>
			<td>Corning</td>
			<td >#353003</td>
			<td></td>
		</tr>
		<tr >
			<td >Culture Dishes P60</td>
			<td>Corning</td>
			<td >#353004</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Line</td>
			<td>Age</td>
			<td>Breeding</td>
			<td></td>
		</tr>
		<tr >
			<td >C57BL/6NRj / male</td>
			<td>12 weeks</td>
			<td>in house</td>
			<td></td>
		</tr>
		<tr >
			<td >Product Name</td>
			<td>Company</td>
			<td>Catalogue No.</td>
			<td></td>
		</tr>
		<tr >
			<td >Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>ThermoFisher</td>
			<td>#12440</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)/Nutrient Mixture F12 Ham</td>
			<td>Merck</td>
			<td>#D8437</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin&#160; (P/S)</td>
			<td>ThermoFisher</td>
			<td>#15140122</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>#SH30071</td>
			<td></td>
		</tr>
		<tr >
			<td >L-Glutamine&#160;</td>
			<td >ThermoFisher</td>
			<td>#25030</td>
			<td></td>
		</tr>
		<tr >
			<td >Glutathione</td>
			<td>Merck</td>
			<td>#G6013</td>
			<td></td>
		</tr>
		<tr >
			<td >B27 Supplement</td>
			<td>ThermoFisher</td>
			<td>#17504044</td>
			<td></td>
		</tr>
		<tr >
			<td >Recombinant Human Epidermal Growth Factor (EGF)</td>
			<td>Peprotech</td>
			<td>#AF-100-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Recombinant Basic Fibroblast Growth Factor (FGF)</td>
			<td>Peprotech</td>
			<td>#AF-100-18B</td>
			<td></td>
		</tr>
		<tr >
			<td >Cardiotrophin 1&#160;</td>
			<td>Peprotech</td>
			<td>#250-25</td>
			<td></td>
		</tr>
		<tr >
			<td >Thrombin</td>
			<td>Diagontech AG, Switzerland&#160;</td>
			<td>#100-125</td>
			<td></td>
		</tr>
		<tr >
			<td >Endothelial Cell Growth Medium (EGM)-2 BulletKit</td>
			<td>Lonza</td>
			<td>#CC-3162</td>
			<td></td>
		</tr>
		<tr >
			<td colspan="4" >Overview of medium compositions. Some of this infomation is identical with the one provided above, but sorted according to the composition of Media 1-3.&#160;</td>
		</tr>
		<tr >
			<td >Product Name</td>
			<td>Medium 18</td>
			<td>Medium 2</td>
			<td>Medium 3</td>
		</tr>
		<tr >
			<td >Reagents</td>
			<td>Culture</td>
			<td>Lineage induction</td>
			<td>Endothelial diff.</td>
		</tr>
		<tr >
			<td >Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>35%</td>
			<td>35%</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)/Nutrient Mixture F12 Ham</td>
			<td>65%</td>
			<td>65%</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin&#160; (P/S)</td>
			<td>1%</td>
			<td>1%</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum (FBS)</td>
			<td>3.5%</td>
			<td>&#8804;0.1%</td>
			<td></td>
		</tr>
		<tr >
			<td >L-Glutamine&#160;</td>
			<td>2 mM</td>
			<td>2 mM</td>
			<td></td>
		</tr>
		<tr >
			<td >Glutathione</td>
			<td>0.2 nM</td>
			<td>0.2 nM</td>
			<td></td>
		</tr>
		<tr >
			<td >B27 Supplement</td>
			<td>1.3%</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Recombinant Human Epidermal Growth Factor (EGF)</td>
			<td>6.5 ng/mL</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Recombinant Basic Fibroblast Growth Factor (FGF)</td>
			<td>13 ng/mL</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Cardiotrophin 1&#160;</td>
			<td>0.65 ng/mL</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of endothelial differentiation in cardiac progenitor cells under low serum conditions

心脏祖细胞 (cpc) 可能具有损伤后心脏再生的治疗潜力。在成年哺乳动物的心脏, 内在的 cpc 是极其稀缺的, 但扩大的 cpc 可能是有用的细胞治疗。它们使用的一个先决条件是它们能够使用定义和有效的协议以可控的方式区分到各种心脏谱系中。此外, 在体外扩张后, 从患者或临床前疾病模型中分离出的 cpc 可为疾病机制的调查提供富有成效的研究工具。
目前的研究使用不同的标记来识别 cpc。然而, 并不是所有的研究都是在人类身上表达的, 这限制了一些临床前研究的转化影响。无论隔离技术和标记表达如何, 都适用的差异化协议将允许为细胞治疗目的对 cpc 进行标准化扩展和启动。本文描述了在低胎牛血清 (fbs) 浓度和低细胞密度条件下对 cpc 的启动有利于 cpc 的内皮分化. 利用小鼠和大鼠 cpc 的两个不同亚群, 我们表明, 层压蛋白是一种更重要的根据以下协议, 为此目的, 适合基材比纤维连接蛋白: 在培养基中培养 2-3天, 包括维持多效能力的补充剂和3.5% 的 fbs 后, 在层压蛋白上播种 lt;60% 的融合, 并培养在内皮分化培养基分化前20-24小时内, 低浓度 fbs (0.1%) 的无补充培养基。由于 cpc 是异质性人群, 因此可能需要根据各自 cpc 亚群的特性调整血清浓度和培养时间。考虑到这一点, 该技术也可以应用于其他类型的 cpc, 并提供了一个有用的方法来调查的潜力和机制的分化, 以及他们如何受到疾病的影响时, 使用从各自的疾病模型隔离的 cpc。

鼠标 sp-pc 隔离:
在这项研究中, 我们使用了根据 sp 表型分离的小鼠 cpc, 而大鼠 cpc 的结果被修改和添加从以前的报告与许可 (图 8)33。
高、低细胞密度和不同血清浓度下的细胞增殖:
我们先前的研究表?...

Watch this Scientific Journal Video about Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions at JoVE.com

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

该方案描述了一种用于心脏祖细胞的内皮分化技术。它特别关注血清浓度和细胞播种密度如何影响内皮分化潜力。

低血清条件下心脏祖细胞内皮分化的诱导作用

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

在扩增时，心脏祖细胞（或CPC）的分化需要从增殖阶段到承诺阶段的相位转换，并且该协议在CPC中使用有效的内皮血统。该技术的一个主要优点是，它适用于基于多种技术和不同物种的分离的CPC。该协议可用于增强CPC在细胞治疗中的治疗潜力。它还可用于研究分化的潜力和机制，以及这些差异如何受到心脏病的影响。CPC 是细胞的不均匀总体，其形状、大小和效力可能不同。因此，协议必须根据使用的CPC的特定子组进行调整。麻醉鼠标后，用70%乙醇擦拭胸部。用剪刀切开皮肤和胸壁，露出胸腔。用钳子抬起心脏，然后用剪刀在底座上剪。将心脏转移到含有五毫升冷 PBS 的 P60 培养皿中，然后用钳子泵送心脏，将血液从腔中喷出。将心脏放在含有五毫升冷 PBS 的 P60 盘中并清洗。用小剪刀取出心，把心脏切成两个纵向碎片，用五毫升的冰冷PBS洗净。用小剪刀把心脏碎片切成小块。加入一滴 Hank 的平衡盐溶液，含有胶原酶 B，浓度为每毫升 1 毫克，并使用无菌剃刀刀片彻底切碎小心脏碎片。接下来，在菜中加入2.5毫升的胶原酶B溶液。将菜以30度角放在37摄氏度的培养箱中。让碎块孵育最多30分钟，同时在孵育过程中每10分钟反复通过巴斯德移液器。首先，在切碎和均质的心脏部分添加五毫升的冷HSS，并辅以2%的FBS。使用 100 微米过滤器，过滤心脏碎片以去除任何未消化的组织。在 470 倍 G 下离心，在室温下离心 5 分钟。丢弃上流剂，将颗粒重新悬浮在五毫升红细胞解液缓冲液中。在冰上孵育五分钟，偶尔摇晃。在此之后，加入10毫升PBS，并通过40微米过滤器过滤样品，以排除更大的细胞，包括残余心肌细胞。在 470 倍 G 下离心，在室温下离心 5 分钟，不休息。然后，重新悬浮DMEM中的细胞，辅以10%FBS，目标是最终细胞浓度为每毫升100万个细胞。将细胞分成两个管子进行染色和分拣，将1.5毫升的细胞溶液加入一个管中，将3.5毫升的细胞溶液添加到第二管中。加入 Hoechst 溶液，在 37 摄氏度下孵育 90 分钟，偶尔翻转，以确保染料的均匀分布，防止细胞凝血。侧人口出现在霍赫斯特左下角的主要人口之外。在 FACS 图中，可以根据阴值控制来识别，其中 verapamil 会提示侧总体消失。预热介质1至37摄氏度，将离心机冷却至4摄氏度。将分拣管在470倍G和4摄氏度下离心6分钟。然后，重新挂起介质中的单元格。将细胞转移到含有四毫升中培养的气体渗透P60盘中。每三天更换一次培养，直到细胞达到70至80%培养SP-CPC之间的汇合，在T75烧瓶中用8毫升的培养剂在每瓶瓶中产生2至3天。接下来，小心吸气介质，用五毫升的暖HSS轻轻冲洗烧瓶。用5毫升的尝试性EDTA治疗细胞，在37摄氏度下用5%的二氧化碳治疗5分钟。然后，加入五毫升的介质一，以停止三辛活性，并将细胞悬浮液转移到15毫升管。在室温下以 470 倍 G 离心五分钟。接下来，在一中或中二中重新挂起单元格。将P60盘上的细胞镀上，每盘电镀25万个SP-CPC，用三毫升的介质含有不同的血清浓度。经过两天的培养，从每道菜中收集培养的培养器放入15毫升的管子中，收集死细胞。如先前所述，将粘附细胞进行尝试，将细胞悬浮液收集到包含收集的培养的15毫升管中。在室温下以840倍G离心5分钟，然后吸升清液，并加入一毫升的介质一或中2用于细胞计数。用锥盘蓝色染色SP-CPC，并计算可行细胞和死细胞。首先，从准备好的细胞中吸出介质，然后用五毫升温暖的HSS轻轻冲洗。在细胞培养箱中用五毫升的尝试性EDTA治疗细胞五分钟。然后，添加五毫升的介质一，以停止尝试性活动。将电池悬浮液转移到15毫升管中，在室温下以470倍G离心，5分钟。吸上清液，并添加介质二进行细胞计数。接下来，将8万个细胞种子放入预涂的六井培养板的每一个井中，用三毫升的中二。将板在37摄氏度下保持20至24小时，然后将每井的介质更改为三毫升的中三。培养板21天，每三天更换一次介质。在此之后，用内皮标记染色细胞，并进行荧光显微镜，以验证分化细胞的内皮性质。首先，从准备好的细胞中吸出介质，然后用五毫升温暖的HSS轻轻冲洗。在细胞培养箱中用两毫升的尝试性EDTA治疗细胞五分钟，然后加入三毫升的中三，以停止尝试性活性。将电池悬浮液转移到15毫升管中，在室温下以470倍G离心，5分钟。吸上流液，并加入一毫升的中三，轻轻移液混合。在100微升的中三层中，将细胞和种子数到2000到4000个细胞之间，每个基质都涂有96井板的井。在37摄氏度下孵育板16小时。在此之后，使用亮场显微镜在两倍的放大率拍摄细胞的照片。本研究探讨了促进心脏祖细胞内皮血统承诺的适宜条件。当细胞汇合在或低于60%时，使用3.5%FBS处理的样品中无显著差异。当这种汇合的细胞被0.1%FBS治疗时，它们显示与纤维素相比，层压素上的细胞增殖减少，但细胞死亡没有增加。相比之下，高汇合的细胞在两种条件下的细胞增殖或细胞死亡方面没有显著差异。细胞形状的变化似乎是一个指标，表明在适当的培养条件下，内皮分化将取得成功。在内皮分化培养基的7至14天内，成功的培养物被认为含有具有不同形态的较大细胞。有趣的是，这些细胞在分化阶段结束时消失，并且往往以较低的数量出现，并在高密度培养物的后期出现。管的形成在低密度镀层和层压素上分化的细胞中始终是成功的，而在高密度镀层细胞中，管的形成大多失败。虽然管的形成在纤维素培养的细胞中大多不成功，而不管细胞密度如何，低密度分化的细胞有时形成初级管。血清浓度和细胞密度诱导内皮血统至关重要，必须调整它们，以确保增殖的减缓，同时保持生存能力。在血统诱导时，来自人类的CPC或临床前疾病模型可用于细胞移植研究，以评估其体内分化潜力。该协议可以帮助研究或建立新的心脏再生工具，它以前曾用于研究早期血统承诺的分子机制。

Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

脂肪干细胞的软骨细胞分化诱导离心重力

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

人脐血内皮祖细胞的分离

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

小鼠心脏场特定心脏祖细胞的体外生成

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

人脐带血衍生CD34的泛骨髓分化–造血干细胞和祖细胞

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

在无馈体和化学定义的培养条件下，使用人类多能干细胞的后多能感感神经元的有效分化

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...