从人类心脏组织血管周围多能前体细胞群的分离
Summary
人体心脏组织窝藏多能血管周围的前体细胞群可能适合于心肌再生。这里描述的技术允许与天然血管， 即 CD146 + CD34相关联的两个多能基质干细胞群的同时分离和纯化–周细胞和CD34 + CD146 –外膜细胞，从人的心肌。
Abstract
Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34– pericytes and vascular CD34+CD146– adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.
Introduction
该心脏一直被认为是一个有丝分裂后的器官。然而，最近的研究已经证明有限心肌周转的成年人的心脏1的存在。原生干/祖细胞，心肌细胞分化的潜能也被认定为成人啮齿动物和人的心，其中的Sca-1 +，C-KIT +，cardiosphere形成心肌内，以及最近的血管周围前体细胞2,3。这些细胞代表了旨在通过细胞移植或原位增殖的刺激增强心脏修复/再生疗法具有吸引力的候选者。
间充质干细胞/基质细胞（MSC）已经从几乎每个人组织4,5的MSC的治疗应用的临床试验已进行了多种病理条件下分离如心血管修补6，移植物抗宿主病7 </sup>和肝硬化8。有益的作用已被归因于MSCs向的能力：家里炎症9的部位;分化成不同细胞类型10;分泌促修复分子11;和调节宿主的免疫反应12。干细胞的分离历来依靠其优惠的坚持塑料基板。然而，所得到的细胞群是典型地显着异质13。通过使用荧光激活细胞分选（FACS）用的关键血管周围细胞标记物的结合，我们已经能够分离和纯化多能的MSC样前体群体（CD146 + / CD31 – / CD34 – / CD45 – / CD56 – ）从多种人体组织包括成人骨骼肌和白色脂肪14。
在各种非心脏组织的血管周围细胞群已被显示出具有干/祖细胞特性的第二正在研究在心血管设置临床使用。周细胞，最知名的血管周围细胞亚群之一，是一个异质人口玩几个病理生理作用，包括在新船15的发展，血压16和血管完整性17,18的维护管理。由于在多种组织所示，周细胞的特定子集本身表达MSC抗原和FACS纯化后14维持在原代培养的MSC样表型。此外，这些细胞稳定地维持培养于他们的长期的表型，并表现出多谱系分化潜能，类似的MSCs 19,20。这些结果表明，周细胞是难以捉摸的MSC 14的起源地之一。周细胞的治疗潜力已经证明在心肌疤痕的减小和增强移植后的心脏功能到缺血性损伤心21。最近，我们成功地从人类心肌纯化周细胞和无骨骼肌肉发生3展示了他们MSC样表型和多能（脂肪形成，软骨和骨）。此外，心肌当周细胞与其他器官纯化的同行相比表现差心肌潜力和血管生成能力。
多能血管周围干/祖细胞，外膜细胞的第二群体，已经从人隐静脉分离阳性CD34的表达22的基础上的。静脉外膜细胞已被证明具有克隆的潜力，中胚层分化能力和体外促血管生成潜力。这些细胞注入小鼠的缺血性损伤的心移植导致间质纤维化的减少，增加血管生成和心肌血流量，减少心室稀通货膨胀，而增加心脏射血分数23。有趣的是，脂肪外膜细胞已被证明失去CD34的表达和上调响应于血管生成素II治疗中培养CD146表达，提示通过一个周细胞表型与刺激24。内的心脏，然而，外膜细胞群体尚未前瞻性通过FACS和/或良好表征纯化。利用在以下各节中描述的细胞分离方法，我们目前正在表征心肌外膜细胞和调查其对再生的应用潜力。
这里我们描述了分离和从人胎儿或成年心肌纯化血管周围干/祖细胞的两个亚群的方法。本前瞻性细胞分离方法将使研究人员能够从人的心脏活检的比较研究和furthe获得等基因血管周围干/祖细胞亚群- [R探讨其在各种心脏病理状态的治疗潜力。
Protocol
Representative Results
Discussion
越来越多的证据支持成人心脏损伤后有限的再生能力。负责受伤的心如再生反应本土前体细胞的识别和鉴定是相关的机制和信号通路双方的理解和制定办法来治疗利用这些细胞的关键。
先前协议已经从人骨骼肌25中所述的血管周围的前体细胞亚群的分离。然而，这些技术对心脏组织的直接应用经常导致非常差的细胞产量。因此，我们以丰富血管周围的前体细胞，即周?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
The authors wish to thank Shonna Johnston, Claire Cryer, Fiona Rossi and Will Ramsay at the University of Edinburgh and Alison Logar and Megan Blanchard at the University of Pittsburgh for their expert assistance with flow cytometry. We also wish to thank Anne Saunderson and Lindsay Mock for their help with obtaining human tissues. Human adult and fetal heart tissue samples were procured with full ethics permission of the NHS Scotland Tayside Committee on Medical Research Ethics and the NHS Lothian Research Ethics Committee (REC08/S1101/1) respectively. This work was supported by grants from the Medical Research Council (BP), British Heart Foundation (BP), Commonwealth of Pennsylvania (BP), Children’s Hospital of Pittsburgh (BP), National Institute of Health R01AR49684 (JH) and R21HL083057 (BP), and the Henry J. Mankin Endowed Chair at University of Pittsburgh (JH). JEB was supported by a British Heart Foundation Centre of Research Excellence doctoral training award (RE/08/001/23904). WC was supported in part by an American Heart Association predoctoral fellowship (11PRE7490001).
Materials
| AbC Anti-mouse Bead Kit | Molecular Probes | A-10344 | |
| Collagenase I | Gibco | 17100-017 | Reconstitute powder as required and filter sterilise |
| Collagenase II | Gibco | 17101-015 | |
| Collagenase IV | Gibco | 17104-019 | |
| anti-human CD34-PE | BD Pharmingen | 555822 | Keep sterile |
| anti-human CD45-APC-Cy7 | BD Pharmingen | 557833 | Keep sterile |
| anti-human CD56-PE-Cy7 | BD Pharmingen | 557747 | Keep sterile |
| anti-human CD144-PerCP-Cy5.5 | BD Pharmingen | 561566 | Keep sterile |
| anti-human CD146-AF647 | AbD Serotec | MCA2141A647 | Keep sterile |
| EGM2-BulletKit | Lonza | CC-3162 | For collection of cells and culture until adhered |
| DMEM, high glucose, GlutaMAX without sodium pyruvate | ThermoFischer Scientific | 10566-016 | |
| Fetal Bovine Serum | ThermoFischer Scientific | 10500-064 | Freeze in aliquots and keep sterile |
| Gelatin | Sigma Aldrich | G1393 | Dilute with sterile water |
| IgG1k-PE | BD Pharmingen | 559320 | Keep sterile |
| IgG1k-APC-Cy7 | BD Pharmingen | 557873 | Keep sterile |
| IgG1k-PE-Cy7 | BD Pharmingen | 557872 | Keep sterile |
| IgG1k-PerCP-Cy5.5 | BD Pharmingen | 561566 | Keep sterile |
| IgG1k-647 | AbD Serotec | MCA1209A647 | Keep sterile |
| Mouse serum | Sigma Aldrich | M5905 | Keep sterile |
| Paraffin Film – Parafilm M | Sigma Aldrich | P7793 | |
| Penicillin-Streptomycin | Gibco | 15979-063 | Freeze in aliquots and keep sterile |
| Phosphate buffered saline pH 7.4 | ThermoFischer Scientific | 10010-023 | Keep sterile |
| Red Blood Cell Lysing Buffer Hybri-Max | Sigma Aldrich | R7757 | Keep sterile |
| Trypan Blue Solution | Sigma Aldrich | T8154 | |
| Trypsin-EDTA 0.5%(10X) | Invitrogen | 15400-054 | |
| FACSARIA FUSION | BD Pharmingen | Fluorescence Activated Cell Sorter |
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