小鼠心脏场特定心脏祖细胞的体外生成
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| β-mercaptoethanol | Sigma | M6250 | |
| 0.1% (w/v) Gelatin | EMD Millipore | ES-006-B | |
| 100mM Sodium Pyruvate | Gibco | 11360 | |
| 100X Pen/Strep | Gibco | 15070-063 | |
| 1X PBS w/o Calcium and Magnesium | Thermo Fisher Scientific | 21-040-CV | |
| 20% Paraformaldehyde | Thermo Fisher Scientific | 50-980-493 | |
| 5 ml Polystyrene round-bottom tube with a 40μm cell strainer | BD Falcon | 35223 | |
| Activin A | R & D Systems | 338-AC-010 | |
| Ascorbic Acid | Sigma | A-4544 | |
| B27 minus vitamin A (50x) | Thermo Fisher Scientific | 12587010 | |
| BMP4 | R & D Systems | 314-BP | |
| Bovine Serum Albumin | Sigma | A2153 | |
| Cell sorter | Sony | SH800 | Sony or any other fluorescence-activated cell sorter |
| Cell strainer 70μm | Thermo Fisher Scientific | 08-771-2 | |
| Centrifuge Sorvall Legend XT | Thermo Fisher Scientific | 75004508 | |
| CHIR99021 | Selleck chemicals | S2924 | |
| CO2 Incubator | Thermo Fisher Scientific | 51030285 | |
| Corning Ultra Low Attachment T75 flask | Corning | 07-200-875 | |
| Countless II FL automated cell counter | Thermo Fisher Scientific | ||
| Donkey anti-mouse IgG secondary antibody, Alexa Fluor 647 conjugate | Thermo Fisher Scientific | A-31571, Lot #1757130 | |
| Dulbecco's Modified Eagle's Medium high glucose (DMEM) | Gibco | 11965-092 | |
| EDTA | Sigma | E6758 | |
| ESGRO (LIF) | Millipore | ESG1106 | |
| EVOS FL microscope | Thermo Fisher Scientific | AMF4300 | |
| Fetal Bovine Serum | Invitrogen | SH30071.03 | |
| Glasgow’s MEM (GMEM) | Gibco | 11710035 | |
| GlutaMAX (100 x) | Gibco | 35050-061 | |
| Ham’s F12 | Gibco | 10-080-CV | |
| HEPES | Sigma | H3375 | |
| IMDM | Gibco | 12440053 | |
| Monothioglycero (MTG) | Sigma | M-6145 | |
| Mouse anti-Troponin T antibody | Thermo Fisher Scientific | MS-295-P1 | |
| N2-SUPPLEMENT | Gibco | 17502-048 | |
| Non-essential amino acid solution (NEAA | Invitrogen | 11140-050 | |
| PD0325901 | Selleckchem | S1036 | |
| PerCP-efluor 710 conjugated anti-Cxcr4 antibody | Thermo Fisher Scientific | 46-9991-82 | |
| Suspension culture dish 150 mm x 25mm | Corning | 430597 | |
| T25 flasks | Corning | 353109 | |
| TrypLE (Trypsin) | Gibco | 12604 | |
| Y-27632 (ROCK inhibitor) | Stem cell technologies | 72304 |
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