低血清条件下心脏祖细胞内皮分化的诱导作用
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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?…
Representative Results
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?…
Discussion
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=…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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)。
Materials
| Culture medium | |||
| Iscove's Modified Dulbecco's Medium (IMDM) | ThermoFisher | #12440 | |
| Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F12 Ham | Merck | #D8437 | |
| Penicillin-Streptomycin (P/S) | ThermoFisher | #15140122 | |
| Fetal Bovine Serum (FBS) | Hyclone | #SH30071 | 3.5% (0.1% for lineage induction) |
| L-Glutamine | ThermoFisher | #25030 | Final concentration 2 mM |
| Glutathione | Merck | #G6013 | |
| Recombinant Human Epidermal Growth Factor (EGF) | Peprotech | #AF-100-15 | |
| Recombinant Basic Fibroblast Growth Factor (FGF) | Peprotech | #AF-100-18B | |
| B27 Supplement | ThermoFisher | #17504044 | |
| Cardiotrophin 1 | Peprotech | #250-25 | |
| Thrombin | Diagontech AG, Switzerland | #100-125 | |
| Hanks' Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-) | ThermoFisher | #14170 | |
| 0.05 % Trypsin-EDTA | ThermoFisher | #25300 | |
| T75 Flask | Sarstedt | #83.3911 | |
| Endothelial differentiation | |||
| Endothelial Cell Growth Medium (EGM)-2 BulletKit | Lonza | #CC-3162 | |
| Ham's F-12K (Kaighn's) Medium | ThermoFisher | #21127 | |
| Laminin | Merck | #L2020 | |
| Fibronectin | Merck | #F4759 | Dilute in ddH2O |
| 6 Well Plate | Falcon | #353046 | |
| Formaldehyde Solution | Merck | #F1635 | Diluite 1:10 in PBS (3.7% final concentration) |
| Triton X-100 | Merck | #93420 | 0.1% in ddH2O |
| Normal Goat Serum (10%) | ThermoFisher | #50062Z | |
| Anti-von Willebrand Factor antibody | Abcam | #ab6994 | 1:100 in 10% goat serum |
| Goat anti-Rabbit IgG, Alexa Fluor 546 | ThermoFisher | #A11010 | 1:500 in 10% goat serum |
| 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) | ThermoFisher | #62247 | 1:500 in ddH2O |
| SlowFade Antifade Kit | ThermoFisher | #S2828 | |
| BX63 widefield microscope | Olympus | ||
| Tube formation | |||
| 96 Well Plate | Falcon | #353072 | |
| 5 ml Round Bottom Tube with Strainer Cap | Falcon | #352235 | |
| Matrigel Growth Factor Reduced | Corning | #354230 | |
| IX50 widefield microscope | Olympus | ||
| Sca-1+/CD31- cardiac side population isolation34 | |||
| Reagents | |||
| Pentobarbital Natrium 50 mg/ml ad usum vet. | in house hospital pharmacy | #9077862 | Working solution: 200 mg/kg |
| Phosphate Buffered Saline (PBS) CaCl2(-), MgCl2(-) | ThermoFisher | #20012 | |
| Hanks' Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-), phenol red (-) | ThermoFisher | #14175 | Prepare HBSS 500 mL + 2% FBS for quenching Collagenase B activity |
| Dulbecco's Modified Eagle's Medium (DMEM) 1g/L of D-Glucose, L-Glutamine, Pyruvate | ThermoFisher | #331885 | Prepare DMEM + 10% FBS + 25 mM HEPES+ P/S for Hoechst stanining |
| Penicillin-Streptomycin (P/S) | ThermoFisher | #15140122 | |
| HEPES 1 M | ThermoFisher | #15630080 | Final concentration 25 mM |
| Fetal Bovine Serum (FBS) | Hyclone | #SH30071 | |
| RBC LysisBuffer (10X) | BioLegend | #420301/100mL | Dilute to 1X in ddH2O and filter through a 0.2 µm filter |
| Collagenase B | Merck | #11088807001 | Final concentration 1 mg/mL in HBSS, filtered through a 0.2 µm filter |
| bisBenzimide H33342 Trihydrochloride (Hoechst) | Merck | #B2261 | Prepare 1 mg/mL in ddH2O |
| Verapamil-hydrochloride | Merck | #V4629 | Final concentration 83.3 µM |
| APC Rat Anti-Mouse CD31 | BD Biosciences | #551262 | 0.25 µg/107cells |
| FITC Rat Anti-Mouse Ly-6A/E (Sca-1) | BD Biosciences | #557405 | 0.6 µg/107cells |
| 7-Aminoactinomycin D (7-ADD) | ThermoFisher | #A1310 | 0.15 µg/106cells |
| APC rat IgG2a k Isotype Control | BD Biosciences | #553932 | 0.25 µg/107cells |
| FITC Rat IgG2a k Isotype Control | BD Biosciences | #554688 | 0.6 µg/107cells |
| Material | |||
| Needles 27G | Terumo | #NN-2719R | |
| Needles 18G | Terumo | #NN-1838S | |
| Single Use Syringes 1 mL sterile | CODAN | #62.1640 | |
| Transferpipette 3.5 mL | Sarstedt | #86.1171.001 | |
| Cell Strainer 40 µm blue | BD Biosciences | #352340 | |
| Cell Strainer 100 µm yellow | BD Biosciences | #352360 | |
| Lumox Dish 50 | Sarstedt | #94.6077.305 | |
| Culture Dishes P100 | Corning | #353003 | |
| Culture Dishes P60 | Corning | #353004 | |
| Mouse | |||
| Line | Age | Breeding | |
| C57BL/6NRj / male | 12 weeks | in house | |
| Product Name | Company | Catalogue No. | |
| Reagents | |||
| Iscove's Modified Dulbecco's Medium (IMDM) | ThermoFisher | #12440 | |
| Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F12 Ham | Merck | #D8437 | |
| Penicillin-Streptomycin (P/S) | ThermoFisher | #15140122 | |
| Fetal Bovine Serum (FBS) | Hyclone | #SH30071 | |
| L-Glutamine | ThermoFisher | #25030 | |
| Glutathione | Merck | #G6013 | |
| B27 Supplement | ThermoFisher | #17504044 | |
| Recombinant Human Epidermal Growth Factor (EGF) | Peprotech | #AF-100-15 | |
| Recombinant Basic Fibroblast Growth Factor (FGF) | Peprotech | #AF-100-18B | |
| Cardiotrophin 1 | Peprotech | #250-25 | |
| Thrombin | Diagontech AG, Switzerland | #100-125 | |
| Endothelial Cell Growth Medium (EGM)-2 BulletKit | Lonza | #CC-3162 | |
| 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. | |||
| Product Name | Medium 18 | Medium 2 | Medium 3 |
| Reagents | Culture | Lineage induction | Endothelial diff. |
| Iscove's Modified Dulbecco's Medium (IMDM) | 35% | 35% | |
| Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F12 Ham | 65% | 65% | |
| Penicillin-Streptomycin (P/S) | 1% | 1% | |
| Fetal Bovine Serum (FBS) | 3.5% | ≤0.1% | |
| L-Glutamine | 2 mM | 2 mM | |
| Glutathione | 0.2 nM | 0.2 nM | |
| B27 Supplement | 1.3% | ||
| Recombinant Human Epidermal Growth Factor (EGF) | 6.5 ng/mL | ||
| Recombinant Basic Fibroblast Growth Factor (FGF) | 13 ng/mL | ||
| Cardiotrophin 1 | 0.65 ng/mL | ||
References
- Beltrami, A. P., et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 114 (6), 763-776 (2003).
- Hierlihy, A. M., Seale, P., Lobe, C. G., Rudnicki, M. A., Megeney, L. A. The post-natal heart contains a myocardial stem cell population. FEBS Letters. 530 (1-3), 239-243 (2002).
- Sandstedt, J., et al. Left atrium of the human adult heart contains a population of side population cells. Basic Research in Cardiology. 107 (2), 255 (2012).
- Bolli, R., et al. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. Lancet. 378 (9806), 1847-1857 (2011).
- Makkar, R. R., et al. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet. 379 (9819), 895-904 (2012).
- Wu, S. M., Chien, K. R., Mummery, C. Origins and fates of cardiovascular progenitor cells. Cell. 132 (4), 537-543 (2008).
- Smits, A. M., et al. Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology. Nature Protocols. 4 (2), 232-243 (2009).
- Noseda, M., et al. PDGFRalpha demarcates the cardiogenic clonogenic Sca1+ stem/progenitor cell in adult murine myocardium. Nature Communications. 6, 6930 (2015).
- Linke, A., et al. Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function. Proceeding of the National Acadademy of Sciences of the United States of America. 102 (25), 8966-8971 (2005).
- El-Mounayri, O., et al. Serum-free differentiation of functional human coronary-like vascular smooth muscle cells from embryonic stem cells. Cardiovascular Research. 98 (1), 125-135 (2013).
- Ellison, G. M., et al. Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair. Cell. 154 (4), 827-842 (2013).
- Oh, H., et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proceeding of the National Acadademy of Sciences of the United States of America. 100 (21), 12313-12318 (2003).
- Martin, C. M., et al. Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart. Developmental Biology. 265 (1), 262-275 (2004).
- Pfister, O., et al. CD31- but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circulation Research. 97 (1), 52-61 (2005).
- Pelekanos, R. A., et al. Comprehensive transcriptome and immunophenotype analysis of renal and cardiac MSC-like populations supports strong congruence with bone marrow MSC despite maintenance of distinct identities. Stem Cell Research. 8 (1), 58-73 (2012).
- Laugwitz, K. L., et al. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature. 433 (7026), 647-653 (2005).
- Bu, L., et al. Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature. 460 (7251), 113-117 (2009).
- Smith, A. J., et al. Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart. Nature Protocols. 9 (7), 1662-1681 (2014).
- Matsuura, K., et al. Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes. Journal of Biological Chemistry. 279 (12), 11384-11391 (2004).
- Liang, S. X., Tan, T. Y., Gaudry, L., Chong, B. Differentiation and migration of Sca1+/CD31- cardiac side population cells in a murine myocardial ischemic model. International Journal of Cardiology. 138 (1), 40-49 (2010).
- Vicinanza, C., et al. Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification. Cell Death and Differentiation. 24 (12), 2101-2116 (2017).
- Galli, S. J., Tsai, M., Wershil, B. K. The c-kit receptor, stem cell factor, and mast cells. What each is teaching us about the others. American Journal of Pathology. 142 (4), 965-974 (1993).
- Konig, A., Corbacioglu, S., Ballmaier, M., Welte, K. Downregulation of c-kit expression in human endothelial cells by inflammatory stimuli. Blood. 90 (1), 148-155 (1997).
- Ogawa, M., et al. Expression and function of c-kit in hemopoietic progenitor cells. Journal of Experimental Medicine. 174 (1), 63-71 (1991).
- Bradfute, S. B., Graubert, T. A., Goodell, M. A. Roles of Sca-1 in hematopoietic stem/progenitor cell function. Experimental Hematology. 33 (7), 836-843 (2005).
- Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. Journal of Experimental Medicine. 183 (4), 1797-1806 (1996).
- Pfister, O., et al. Role of the ATP-binding cassette transporter Abcg2 in the phenotype and function of cardiac side population cells. Circulation Research. 103 (8), 825-835 (2008).
- Messina, E., et al. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circulation Research. 95 (9), 911-921 (2004).
- Davis, D. R., et al. Validation of the cardiosphere method to culture cardiac progenitor cells from myocardial tissue. PLoS One. 4 (9), e7195 (2009).
- Carr, C. A., et al. Cardiosphere-derived cells improve function in the infarcted rat heart for at least 16 weeks–an MRI study. PLoS One. 6 (10), e25669 (2011).
- Martens, A., et al. Rhesus monkey cardiosphere-derived cells for myocardial restoration. Cytotherapy. 13 (7), 864-872 (2011).
- Cheng, K., et al. Relative roles of CD90 and c-kit to the regenerative efficacy of cardiosphere-derived cells in humans and in a mouse model of myocardial infarction. Journal of the American Heart Association. 3 (5), e001260 (2014).
- Mochizuki, M., et al. Polo-Like Kinase 2 is Dynamically Regulated to Coordinate Proliferation and Early Lineage Specification Downstream of Yes-Associated Protein 1 in Cardiac Progenitor Cells. Journal of the American Heart Association. 6 (10), (2017).
- Pfister, O., Oikonomopoulos, A., Sereti, K. I., Liao, R. Isolation of resident cardiac progenitor cells by Hoechst 33342 staining. Methods in Molecular Biology. 660, 53-63 (2010).
- Discher, D. E., Mooney, D. J., Zandstra, P. W. Growth factors, matrices, and forces combine and control stem cells. Science. 324 (5935), 1673-1677 (2009).
- Plaisance, I., et al. Cardiomyocyte Lineage Specification in Adult Human Cardiac Precursor Cells Via Modulation of Enhancer-Associated Long Noncoding RNA Expression. JACC: Basic to Translational Science. 1 (6), 472-493 (2016).
Cite This Article
Mochizuki, M., Della Verde, G., Soliman, H., Pfister, O., Kuster, G. M. Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions. J. Vis. Exp. (143), e58370, doi:10.3791/58370 (2019).