人脐血内皮祖细胞的分离
Summary
该协议的目的是分离脐血内皮祖细胞。一些应用包括利用这些细胞作为生物标志物来识别心血管风险患者, 治疗缺血性疾病, 并创建组织工程的血管和心脏瓣膜结构。
Abstract
Ashara 和同事们首次报告了外周血中内皮祖细胞 (内皮) 的存在及其在血管中的参与情况1。后来, 其他人记录了来自骨髓的类似类型的内皮的存在2,3。最近, 奥约德和英格拉姆表明, 内皮从脐血获得了较高的增殖潜能与孤立的成人外周血4,5,6。除了参与产后血管, 内皮也显示了承诺作为一个细胞源创建组织工程血管和心脏瓣膜构建7,8。存在各种隔离协议, 其中一些涉及在内皮和造血标志物的帮助下从先前提到的来源获得的单个核细胞的细胞分类, 或培养这些具有特殊内皮生长的跨国公司中, 或这些技术的组合9。在这里, 我们提出了一个内皮的分离和培养的协议使用专门的内皮培养基补充生长因子, 不使用 immunosorting, 其次是对孤立细胞的表征使用西部印迹和免疫.
Introduction
一些调查人员研究了人类内皮的特征和潜能5,10,11,12,13。内皮可以被描述为在缺氧、缺血、损伤或肿瘤形成的部位有能力黏附于内皮组织的循环细胞, 并有助于形成新的血管结构4,14。他们观察到的参与新生血管, 以产后血管的形式, 导致了对这些细胞的病理生理学的理解和他们的用途在治疗应用4,15, 16. 一个人的内皮的数量已被证明与心血管病病理学相关9、15、161718 ,20。其他研究也将内皮分化成成纤维细胞样表型, 并建议这些细胞可用于组织工程心脏瓣膜7,21。
由于调查4之间的差异, 没有明确确定分离内皮所需的特定细胞表面分子。跨国公司对某一矩阵的附着力, 暴露于各种培养条件下, 已由几个组1、17、22、23进行, 建议假定内皮可能显示不同的表型属性。这些性质包括缺乏吞噬能力, 管形成在基质, 和摄取 Dil-乙酰低密度脂蛋白。高克隆和增殖电位是内皮可以排列5的两个属性。内皮也可以形成体外小管当共与人胎肺成纤维细胞的4。这些细胞是已知的表达内皮细胞表面标记和分享一些造血标记13,24,25。积极表达的标记, 被广泛接受的分内皮是 CD31, CD34, 血管内皮生长因子受体 2 (VEGFR2), 冯血友病因子 (vWF), CD133, c-试剂盒, 血管内皮钙黏蛋白 (VE 钙黏附素)4,18. co-express CD90、CD45、CD14、CD115 或α-平滑肌肌动蛋白 (α-SMA) 的细胞不被认为是内皮, 因为它们的增殖潜能有限, 吞噬细菌的能力, 以及无法形成de 从头人类容器在体内4,7。本文概述了一个改进的协议, 分离内皮祖细胞从人脐血不需要任何细胞排序协议。本文以 CD31、CD34 和 VEGFR2 为阳性标记, 用α-SMA 作为负性指标。
本文提出了一种利用生长因子 (华) 对脐血内皮祖细胞进行无细胞分类的分离培养方法。该华包含血管内皮生长因子 (VEGF) 和成纤维细胞生长因子 (增殖素), 它能增强内皮细胞膜的存活、增生和迁移26。它还包括抗坏血酸, 它负责维持细胞的鹅卵石形态;胰岛素样生长 factor-1 (IGF-1), 提供血管生成和迁移功能;和肝素, 在培养基26中导致生长因子的长期稳定性改善。其他生长因子添加到内皮细胞培养基包括补充表皮生长因子 (egf), 这有助于刺激细胞增殖和分化, 和氢化可的松, 其中使细胞的 egf26.我们表明, 这一特定生长培养基的使用产生的内皮比内皮基培养基 (循证医学) 或 Dulbecco 氏改良鹰培养基 (DMEM) 的数量更高。
Protocol
这项研究是在阿肯色大学机构审查委员会的批准下进行的 (批准编号 16-04-722)。在阿肯色州脐血库的柠檬酸盐磷酸葡萄糖 (方案) 溶液中收集脐血单位, 而不符合储存要求的单位则捐给研究。在环境温度下, 脐带血单位在收集24小时内快递到实验室. 1. 从脐血中分离内皮祖细胞 试剂的制备。 通过将内皮基底培养基 (循证医学) 添加到10% 胎牛血清 (FBS), 并?…
Representative Results
内皮祖细胞的分离和扩展:提供了一个示意图 (图 1) 描述了整个协议。采用密度梯度介质对人脐血密度梯度离心法进行了不同的血成分层观察。在将跨国公司播种到胶原处理的盘子上时, 首先观察到5天和 7 (图 2A) 之间的菌落的生长。这些殖民地继续生长并且有纺锤形的细胞形态学 (图 2…
Discussion
如前所述, 附着内皮具有鹅卵石形态。我们的孤立的跨国公司从一个纺锤形的细胞殖民地 (图 2A-2D) 发展到一个鹅卵石殖民地 (图 2E-2F), 在文化中的十天内。内皮的标记不同的研究组, 即晚期内皮祖细胞10, 血管内皮细胞的形成,5, 或内皮祖细胞12。应该指出的是, 这些细胞在功?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这种材料是根据国家科学基金会资助的 No. 的工作。CMMI-1452943 和阿肯色大学荣誉学院。我们还要感谢阿肯色州脐带血库为我们提供脐血单位。
Materials
| A) For isolation and culturing | |||
| EGM-2 BulletKit | Lonza | CC-3162 | This product comes with all the growth factors needed to make the Endothelial Growth Medium |
| Fetal Bovine Serum | Thermofisher Scientific | 26140079 | |
| Pencillin-Streptomycin-Glutamine (100X) | Thermofisher Scientific | 10378016 | |
| Ficoll-Paque | GE Heatlhcare | 17-1440-02 | |
| Hank's Balanced Salt Solution | Thermofisher Scientific | 14170-112 | |
| Ammonium Chloride | Stem Cell Technologies | 7850 | |
| 1X Phosphate Buffer Saline | Thermofisher Scientific | 14190250 | |
| Rat Tail I Collagen | Corning | 354236 | |
| Glacial Acetic Acid | Amresco | 0714-500ML | |
| 0.05% Trypsin-EDTA | Thermofisher Scientific | 25300054 | |
| HEPES buffer | Thermofisher Scientific | 15630080 | |
| Dulbecco's Modified Eagle's Medium | Thermofisher Scientific | 10566-016 | |
| B) Antibodies and cell lysates | |||
| CD31 | Abcam | ab28364 | 1:250 dilution for Western blotting |
| CD34 | Santa Cruz Biotechnology | sc-7045 | 1:100 dilution for Western blotting |
| α-SMA | abcam | ab5694 | 1:100 dilution for Western blotting |
| α-tubulin | abcam | ab7291 | 1:2500 dilution for Western blotting |
| VEGFR2 | abcam | sc504 | 1:100 dilution for Western blotting |
| Human umbilical vein endothelial cell lysate | Santa Cruz Biotechnology | sc24709 | |
| Valve interstitial cell lysate | Primary cell line cultured from own lab and lysed with RIPA buffer | ||
| C) Western blotting and immunostaining | |||
| 10X Tris/Glycine/SDS buffer | Biorad | 161-0772 | Used as running buffer |
| 10X Tris/Glycine buffer | Biorad | 161-0771 | Used as transfer buffer |
| Immobilon-FL transfer membrane | Merck Millipore | IPFL0010 | This is a PVDF transfer membrane that has 45 µm pore size and is mentioned in the protocol as western blot membrane |
| 4X Laemmli sample buffer | Biorad | 161-0747 | |
| 2-mercaptoethanol | Biorad | 161-0710 | |
| 10% Criterion TGX precast gel | Biorad | 5671033 | |
| Prolong Gold antifade | Thermofisher Scientific | P36930 | Used for mounting immunostained coverslips for long term storage |
| Methanol | VWR Analytical | BDH1135-4LP |
Riferimenti
- Asahara, T., et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 275 (5302), 964-967 (1997).
- Lin, Y., Weisdorf, D. J., Solovey, A., Hebbel, R. P. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest. 105 (1), 71-77 (2000).
- Shi, Q., et al. Evidence for circulating bone marrow-derived endothelial cells. Blood. 92 (2), 362-367 (1998).
- Hirschi, K. K., Ingram, D. A., Yoder, M. C. Assessing identity, phenotype, and fate of endothelial progenitor cells. Arterioscler Thromb Vasc Biol. 28 (9), 1584-1595 (2008).
- Ingram, D. A., et al. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood. 104 (9), 2752-2760 (2004).
- Yoder, M. C., et al. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood. 109 (5), 1801-1809 (2007).
- Sales, V. L., et al. Transforming growth factor-beta1 modulates extracellular matrix production, proliferation, and apoptosis of endothelial progenitor cells in tissue-engineering scaffolds. Circulation. 114, 193-199 (2006).
- Sales, V. L., et al. Endothelial Progenitor Cells as a Sole Source for Ex Vivo Seeding of Tissue-Engineered Heart Valves. Tissue Eng Pt A. 16 (1), 257-267 (2010).
- Liew, A., Barry, F., O’Brien, T. Endothelial progenitor cells: diagnostic and therapeutic considerations. Bioessays. 28 (3), 261-270 (2006).
- Hur, J., et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol. 24 (2), 288-293 (2004).
- Ingram, D. A., Caplice, N. M., Yoder, M. C. Unresolved questions, changing definitions, and novel paradigms for defining endothelial progenitor cells. Blood. 106 (5), 1525-1531 (2005).
- Melero-Martin, J. M., et al. In vivo vasculogenic potential of human blood-derived endothelial progenitor cells. Blood. 109 (11), 4761-4768 (2007).
- Melero-Martin, J. M., Bischoff, J. Chapter 13. An in vivo experimental model for postnatal vasculogenesis. Methods Enzymol. 445, 303-329 (2008).
- Yoder, M. C. Human endothelial progenitor cells. Cold Spring Harb Perspect Med. 2 (7), 006692 (2012).
- Siddique, A., Shantsila, E., Lip, G. Y. H., Varma, C. Endothelial progenitor cells: what use for the cardiologist. J Angiogenes Res. 2 (6), (2010).
- Camci-Unal, G., et al. Surface-modified hyaluronic acid hydrogels to capture endothelial progenitor cells. Soft Matter. 6 (20), 5120-5126 (2010).
- Hill, J. M., et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 348 (7), 593-600 (2003).
- Young, P. P., Vaughan, D. E., Hatzopoulos, A. K. Biologic properties of endothelial progenitor cells and their potential for cell therapy. Prog Cardiovasc Dis. 49 (6), 421-429 (2007).
- Mehta, J. L., Szwedo, J. Circulating endothelial progenitor cells, microparticles and vascular disease. J Hypertens. 28 (8), 1611-1613 (2010).
- Nevskaya, T., et al. Circulating endothelial progenitor cells in systemic sclerosis are related to impaired angiogenesis and vascular disease manifestations. Ann Rheum Dis. 66, 67-67 (2007).
- Cebotari, S., et al. Clinical application of tissue engineered human heart valves using autologous progenitor cells. Circulation. 114, 132-137 (2006).
- Ito, H., et al. Endothelial progenitor cells as putative targets for angiostatin. Cancer Res. 59 (23), 5875-5877 (1999).
- Vasa, M., et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation. 103 (24), 2885-2890 (2001).
- Wu, X., et al. Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells. Am J Physiol Heart Circ Physiol. 287 (2), 480-487 (2004).
- Boyer, M., et al. Isolation of endothelial cells and their progenitor cells from human peripheral blood. J Vasc Surg. 31 (1), 181-189 (2000).
- Huber, B., Czaja, A. M., Kluger, P. J. Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering. Cell Biol Int. 40 (5), 569-578 (2016).
- Sturdivant, N. M., Smith, S. G., Ali, S. F., Wolchok, J. C., Balachandran, K. Acetazolamide Mitigates Astrocyte Cellular Edema Following Mild Traumatic Brain Injury. Sci Rep. 6, 33330 (2016).
- Lam, N. T., Muldoon, T. J., Quinn, K. P., Rajaram, N., Balachandran, K. Valve interstitial cell contractile strength and metabolic state are dependent on its shape. Integr Biol (Camb). 8 (10), 1079-1089 (2016).
- Tandon, I., et al. Valve interstitial cell shape modulates cell contractility independent of cell phenotype. J Biomech. 49 (14), 3289-3297 (2016).
- Cockshell, M. P., Bonder, C. S. Isolation and Culture of Human CD133+ Non-adherent Endothelial Forming Cells. Bio-Protocol. 6 (7), (2016).
Citazione di questo articolo
Ravishankar, P., Zeballos, M. A., Balachandran, K. Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood. J. Vis. Exp. (127), e56021, doi:10.3791/56021 (2017).