Name: in vitro generation of heart field-specific cardiac progenitor cells
Uploaded: 2019-07-03T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells at JoVE.com

E.T.得到了《重要魔法》和《AHA》的支持。C. K. 得到 NICHD/NIH (R01HD086026)、AHA 和 MSCRF 的赠款的支持。

注:在体外生成心脏场特异性小鼠心脏祖细胞(图1)。
1. 鼠标 ESC 的维护
<ol><li>生长 mESC (mESCTbx1-Cre;罗莎-RFP;HCN4-GFP, mESCIsl1-RFP)25上 0.1% (w/v) 明胶涂层 T25 烧瓶在 2i 培养基 (870 mL 的格拉斯考最低必需介质 (GMEM), 100 mL 的胎儿牛血清 (FBS), 10 mL 的 GlutaMAX, 10 mL 的非必需氨基酸, 10 mL 钠丙酸酯,3 μL β-甲苯?...

<ol>
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在我们的协议中,我们描述了一种生成心脏球体和隔离心脏场特定Pc的方法。这些可用于研究CPC规范的机制及其特性,以及心脏室特定疾病建模。以前发表的一项工作使用mESC线与两个荧光报告器(Mef2c/Nkx2.5)在体外研究心形生成,然而,这两个标记在胚胎日9.5-10时,心肌细胞已经形成26。据我们所知,目前没有在体外分离心脏领域特定CP的方法。更重要的是,我们的协议也可以应用于人类干细...

多能干细胞(PSCs)的使用彻底改变了心脏再生领域和个性化医学领域,患者特异性肌细胞用于疾病建模和药物治疗1、2、3、4.最近,用于生成心房与心室以及心脏起搏器样 PSC 衍生心肌细胞的体外方案已经开发出来5、6。然而,心脏生成是否可以在体外重建,以研究心脏发育,并随后产生心室特异性心脏细胞仍不清楚。
在早期胚胎发育过程中,在BMP4、Wnts和ActivinA等分泌形态素的影响下,中皮细胞形成原始条纹7。以Mesp1的表达为标志的心脏心皮细胞,在前和后迁移形成心脏新月,然后原始心脏管7,8。这个迁移组细胞包括两个非常不同的心脏祖细胞(CPCs)群体,即第一和第二心脏场(FHF和SHF)9,10。来自SHF的细胞具有高度增殖性和迁移性,主要负责心脏管的伸长和循环。此外,SHF细胞分化到心肌细胞,成纤维细胞,平滑肌肉和内皮细胞,因为他们进入心脏管形成右心室,右心室流出道和大部分两个atria7,10。相反,FHF细胞的增殖性和迁移性较少,主要分化为心肌细胞,因为它们产生左心室和11室的较小部分。此外,SHF祖细胞的表达有Tbx1、FGF8、FGF10和Six2的表达,而FHF细胞表示Hcn4和Tbx511、12、13、14、15。
PSC可以分化到所有三个生殖层,然后分化到体内的任何细胞类型4,16。因此,它们为理解心脏发育和模拟导致先天性心脏病的特定发育缺陷提供了巨大的潜力,而先天性心脏病是导致先天缺陷的最常见原因17。先天性心脏病的一大亚群包括室特异性心脏异常18、19。然而,目前还不清楚这些是否源于异常的心场发展。此外,鉴于心肌细胞在出生后无法增殖,人们已为心脏再生1、7、20创建心脏组织进行了广泛的努力。考虑到心脏室之间的生理和形态差异,使用PSC生成腔部特定心脏组织非常重要。虽然发展性心脏病学的最新进展导致PSC产生强大的心脏细胞,但尚不清楚这两个心脏场是否可以诱导在PSC系统中。
为了在体外重述心脏发生,并研究CPC的规格和性质,我们以前使用一种基于PSC衍生心脏球体的系统21、22、23、24。最近,我们分别在FHF基因Hcn4和SHF基因Tbx1(mESCs Tbx1-Cre)的控制下,用GFP和RFP报告机生成了小鼠胚胎干细胞(mESCsTbx1-Cre;罗莎-RFP;HCN4-GFP) 25.体外分化的meESCs形成心脏球体,其中GFP+和RFP+细胞从两个不同的间皮细胞区域出现,以互补的方式模式化。由此产生的GFP+和RFP+细胞分别表现出FHF和SHF特性,由RNA测序和克隆分析确定。重要的是,使用携带Isl1-RFP报告器(mESCIsl1-RFP)的mESCs,我们发现SHF细胞忠实地被细胞表面蛋白CXCR4标记,这可以实现无转基因心脏场特异性细胞的分离。本议定书将描述心脏领域特定PC的产生和分离,从mESCs,这可能是研究腔部特定心脏病的宝贵工具。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">&#946;-mercaptoethanol</td>
			<td style="width:245px;">Sigma</td>
			<td style="width:344px;">M6250</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.1% (w/v) Gelatin</td>
			<td>EMD Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100mM Sodium Pyruvate</td>
			<td>Gibco</td>
			<td align="right">11360</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100X Pen/Strep</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1X PBS w/o Calcium and Magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">20% Paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>50-980-493</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:420px;">5 ml Polystyrene round-bottom tube with a 40&#956;m cell strainer</td>
			<td>BD Falcon</td>
			<td align="right">35223</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Activin A</td>
			<td>R &#38; D Systems</td>
			<td>338-AC-010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A-4544</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B27 minus vitamin A (50x)</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">12587010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BMP4</td>
			<td>R &#38; D Systems</td>
			<td>314-BP</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:420px;">Cell sorter</td>
			<td style="width:245px;">Sony</td>
			<td style="width:344px;">SH800</td>
			<td style="width:241px;">Sony or any other fluorescence-activated cell sorter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:420px;">Cell strainer 70&#956;m</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-771-2</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Centrifuge Sorvall Legend XT</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">75004508</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CHIR99021</td>
			<td>Selleck chemicals</td>
			<td>S2924</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:420px;">CO2 Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">51030285</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Corning Ultra Low Attachment T75 flask</td>
			<td>Corning</td>
			<td>07-200-875</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Countless II FL automated cell counter</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Donkey anti-mouse IgG secondary antibody, Alexa Fluor 647 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-31571, Lot #1757130</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s Modified Eagle&#39;s Medium high glucose (DMEM)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ESGRO (LIF)</td>
			<td>Millipore</td>
			<td>ESG1106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EVOS FL microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMF4300</td>
			<td style="width:241px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glasgow&#8217;s MEM (GMEM)</td>
			<td>Gibco</td>
			<td align="right">11710035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX (100 x)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ham&#8217;s F12</td>
			<td>Gibco</td>
			<td>10-080-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IMDM</td>
			<td>Gibco</td>
			<td align="right">12440053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monothioglycero (MTG)</td>
			<td>Sigma</td>
			<td>M-6145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Troponin T antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>MS-295-P1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2-SUPPLEMENT</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-essential amino acid solution (NEAA</td>
			<td>Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD0325901</td>
			<td>Selleckchem</td>
			<td>S1036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP-efluor 710 conjugated anti-Cxcr4 antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>46-9991-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suspension culture dish 150 mm x 25mm</td>
			<td>Corning</td>
			<td align="right">430597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T25 flasks</td>
			<td>Corning</td>
			<td align="right">353109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypLE (Trypsin)</td>
			<td>Gibco</td>
			<td align="right">12604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632 (ROCK inhibitor)</td>
			<td>Stem cell technologies</td>
			<td align="right">72304</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro generation of heart field-specific cardiac progenitor cells

多能干细胞为理解心脏发育和疾病以及再生医学提供了巨大的潜力。虽然发展性心脏病学的最新进展导致从多能干细胞中产生心脏细胞,但不清楚这两个心脏领域——第一和第二心脏场(FHF和SHF)——是否诱导多能干细胞系统。为了解决这个问题,我们生成了一个方案,用于体外规范和分离心脏领域特定的心脏祖细胞。我们使用携带Hcn4-GFP和Tbx1-Cre的胚胎干细胞系;罗莎-RFP记者分别从FHF和SHF,和活细胞免疫染色的细胞膜蛋白Cxcr4,一个SHF标记。通过这种方法,我们生成了祖细胞,重述了它们体内对应物的功能特性和转录组。我们的协议可用于研究两个心脏场的早期规范和分离,并生成用于心脏病建模的腔室特定心脏细胞。由于这是一个体外器官系统,它可能无法提供精确的解剖信息。然而,该系统克服了胃化阶段胚胎的不良可及性,可以针对高通量屏幕进行升级。

经过大约132小时的分化后,可以使用荧光显微镜检测Tbx1-RFP和Hcn4-GFPCP(图2)。通常,GFP 和 RFP 细胞大约同时出现。聚居会的两种人口继续以接近和通常互补的模式扩大。调整Activin A和BMP4的浓度将改变FHF与SHFCPC的百分比(图3)。体外CPC规格主要由BMP4浓度决定。因此,我们的心脏球形系统可用于研究CPC规范。
同样,使用 Isl1-RFP 报告器 mESC 线路,...

Watch this Scientific Journal Video about In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells at JoVE.com

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

该方法的目的是在体外生成心脏场特异性心脏祖细胞,以研究祖细胞的规范和功能特性,并生成用于心脏病建模的腔内特定心脏细胞。

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 ...

我们的方法旨在从小鼠胚胎干细胞中生成心脏场特异性心脏祖细胞。这个心脏场记者干细胞系允许对先天性心脏病背后的机制进行高通量研究，提供一个适合遗传和药理操作的系统。有几个腔腔特异性先天性心脏病。通过回顾体外心电生成，该协议允许研究疾病机制和未来再生疗法的发展。该协议可以提供洞察如何指定不同腔室的心脏祖细胞在心脏发育期间。首先在0.1%的明胶涂层T25烧瓶中生长转基因小鼠胚胎干细胞。在2i介质中。当细胞达到70至80%的汇合时，用PBS冲洗培养物，并每瓶加入一毫升的三辛，在37摄氏度下将培养物分离成单细胞3分钟。当细胞分离时，用四毫升10 FBS在DMEM中中和中和反应，用适当的方法计算细胞。将细胞稀释到每新鲜介质浓度约三倍十至五个细胞，通过离心收集细胞。然后将颗粒重新用五毫升新鲜 2i 介质重新装到新的 0.1% 明胶涂层 T25 烧瓶上。对于来自心脏球状物的CPC生成，通过离心收集2.5倍10至6个分离的转基因小鼠胚胎干细胞样本，并在25毫升SFD培养的细胞中重新补充细胞。将细胞悬浮板板放入一个150至25毫米的无菌板中，在37摄氏度和5%的二氧化碳下孵育48小时。在孵育结束时，将心脏球状体收集到锥形管中。通过离心沉淀球体，以方便球体的选择性分离，并避免单个细胞。在25毫升新鲜SFD介质中重新补充球体，每毫升活性素A1毫微克和骨形态遗传蛋白4的1.5毫微克。将球体重新板回同一培养板，在细胞培养箱中24小时孵化。第二天，通过离心回忆所有的心脏球状体。将分化胚胎体重新用 25 毫升新鲜 SFD 培养基，在细胞培养培养箱中电镀在超低附着、75 厘米方形的烧瓶中，48 小时。为了使用荧光报告器分离心脏场特异性CPC，通过离心收集胚胎体，并在37摄氏度下用一毫升的三丁蛋白分离3分钟。在孵化结束时，通过移液混合，分离细胞，并在DMEM中用四毫升10%FBS中和中和反应。将混合物通过70微米滤株，去除任何非分离胚胎体，并通过离心沉淀过滤细胞。要通过荧光处理器表达式对CPC进行排序，在500微升法发剂ACCS溶液中重新暂停颗粒，并通过40微米细胞滤株将细胞过滤成冰上5毫升聚苯乙烯圆底管。然后对细胞进行排序，通过 FACS 分离 RFP 和 GFP 表达细胞，在 FBS 的一毫升中收集细胞。为了根据表面蛋白Cxcr4表达分离第一和第二心脏场CPC，在PBS中300微升10%FBS中重新增殖单小鼠胚胎干细胞RFP表达心脏祖细胞，并辅以荧光结合抗Cxcr4抗体。在室温下五分钟后，每次洗涤一到两毫升新鲜、冷的 PBS 中，将细胞洗涤三次。上次洗涤后，将 500 微升的 FACS 溶液中的细胞重新暂停，并通过 40 微米滤株将细胞过滤成 5 毫升的圆底管。然后按其Cxcr4表达对细胞进行排序，将Cxcr4阳性和负量收集到每个管中含有一毫升FSP的单个管中。要重新培养FACS分离、心脏场特异性心脏祖细胞，通过离心收集分的细胞，并在SFD介质中重新补充颗粒。然后在0.1%的明胶涂层、384井板中播种约3倍至4个细胞。如果在分拣后注意到细胞死亡增加，则在每个井中加入10微摩尔 ROCK抑制剂。经过两天的文化，应观察自发的殴打。为了分析镀CPC分化心肌细胞的能力，如演示，在分化的第12天收集细胞，如所证明的，分离单个心肌细胞，并在4%的半成甲醛中重新暂停细胞。在室温下30分钟后，通过离心收集固定细胞，并在PBS中清洗颗粒，以去除多余的固定剂。接下来，在PBS中重新暂停10%FBS中的细胞，用小鼠抗肌酸T抗体孵育一半的细胞样本，并使用样本的另一半作为阴性对照。在室温下30分钟后，用新鲜PBS清洗细胞两次，并在10%FBS加PBS中重新暂停样品，并辅以适当的二次抗体。在室温下再孵育30分钟后，每次洗涤用新鲜PBS清洗细胞两次，每管200微升PBS中重新给细胞，用于流式细胞仪分析。经过约132小时的分化，Tbx1-RFP和Hcn4-GFP心脏祖细胞可以通过荧光显微镜检测。一般来说，GFP和RFP细胞大约在同一时间出现，而前细胞的两个种群继续近距离扩展，通常以互补模式生长。调整活性素 A 和骨形态遗传蛋白 4 的浓度将改变第一和第二心脏场心脏祖细胞的百分比。同样，使用RFP记者小鼠胚胎干细胞系，经过132小时的分化，RFP阳性心脏祖细胞出现。在对Cxcr4进行免疫抑制后，可以分离RFP阳性、Cxcr4阳性和RFP阳性。分化第12天心脏肌蛋白T的免疫抑制证实，第一个心脏场细胞主要分化成肌细胞。同样，与Cxcr4单阳性心脏祖细胞相比，来自RFP阳性、Cxcr4阴性心脏祖细胞的细胞产生心肌细胞的比例要高得多。有时，小鼠胚胎干细胞不能有效地区分，形成非常低的心肌特异性心脏祖细胞。细胞因子添加的时间、浓度和一致性以及细胞数量对于成功生成腔体特异性心脏祖细胞至关重要。按照这个程序，研究人员可以分析不同人群的心脏祖细胞的生理特性，以进一步深入了解其规范和功能。

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

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

基于ES-Cell的编程组合MYH6启动子，选择小鼠心脏起搏器细胞聚集一代

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

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 ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

定量造血干和祖细胞 (HSPCs) 在斑马鱼胚胎发育中的体外试验研究

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

人多潜能干细胞第一心场样心脏祖细胞和心室样细胞的生成

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

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

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 ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...