来自人类脐带血内皮细胞集落形成细胞的表型和功能特性
Summary
具有强大的克隆增殖潜能内皮细胞集落形成细胞（ECFCs）循环内皮细胞，显示内在<em>在体内</em>血管形成能力。会CB衍生内皮细胞生长的表型和功能特性，是重要的识别和分离<em>善意</em> ECFCs修复受损的组织，为潜在的临床应用。
Abstract
新的血液通过血管生成，血管，小动脉血管的形成长期的看法，最近已检讨1。循环内皮祖细胞（EPCs）的存在，首次发现麻原等。成人外周血中，在1997年2将注入新的假说和血管再生和修复策略。内皮祖细胞是罕见的，但正常的血液循环的组成部分，血管形成或血管重塑的网站，并在很大程度上促进通过旁分泌刺激现有的血管壁细胞中的3任产后的血管生成，血管生成，或小动脉。没有特定的标记，以确定一个EPC已经确定，目前该领域的国家是理解，许多类型的细胞，包括血管形成的造血干细胞和祖细胞，循环血管细胞，Tie2的+单核细胞 ，髓系祖CELLS，肿瘤相关巨噬细胞和M2激活巨噬细胞在多种临床前动物模型系统和人类受试者在许多疾病状态4，5，刺激血管生成过程的参与。内皮细胞集落形成细胞（ECFCs）是可行的罕见循环内皮细胞，特点是强大的克隆增殖潜能，二级和三级集落形成能力后replating，为6-8免疫缺陷小鼠在移植后体内血管形成的内在能力。虽然ECFCs已成功地从外周血中分离的健康成人受试者，健康新生儿脐带血（CB）的，和血管壁无数人的动脉和静脉血管6-9，CB拥有ECFCs 7的频率最高，显示最强大的克隆增殖潜能和耐用，功能血管的形式在体内 8，10-13。而推导从成人外周血ECFC已提交14，15，在这里，我们描述的推导方法，克隆，扩展，并在体外以及在从人脐CB ECFCs 体内表征。
Protocol
试剂和溶液肌电图-2媒体（龙沙，猫。编号：CC-3162包含的EBM-2培养基和临时股东大会-2 SingleQuot的套件补充，生长因子） EBM-2（龙沙，货号CC-3156），辅以整个SingleQuot套件补充和生长因子（龙沙，猫。CC-4176号），胎牛血清（FBS）和10％（V / V） 1％（V / V），青霉素（1万U /毫升）/链霉素（10,000微克/毫升）/两性霉素（25微克/毫升）。在4°C存储长达1个月建议的临?…
Discussion
假定内皮祖细胞的表型和功能特性，是重要的，以确定是否真正是能够克隆，并连续在文化再镀的的善意 ECFCs和耐用和功能， 在体内植入血管引起。脐血丰富与ECFCs这些循环细胞老化或疾病的10下降浓度。最近的研究表明，ECFC可能在血管修复或再生血管损伤，心肌梗死，或视网膜病变17-19的情况下发挥重要作用。
在这里，我们描述了简单?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
尤德博士是EndGenitor科技公司的顾问和Rimedion科技公司的董事会成员
Materials
| Name of the reagent | Company | Catalogue number |
| Heparin Sodium Injection, USP | APP Pharmaceuticals | 504031 |
| Ficoll-Pague | Amersham Biosciences | 17-1440-03 |
| Mixing cannula | Maersk Medical | 500.11.012 |
| EGM-2 | Lonza | CC-3162 |
| Defined FBS | Hyclone | SH30070.03 |
| TrypLE express | Gibco | 12605 |
| Rat type I collagen | BD Biosciences | 354236 |
| Matrigel | BD Biosciences | 356234 |
| FcR Block | Miltenyi Biotech | 130-059-901 |
| hCD31, FITC conjugated | BD Pharmingen | 555445 |
| hCD45, FITC conjugated | BD Pharmingen | 555482 |
| hCD14, FITC conjugated | BD Pharmingen | 555397 |
| hCD144, PE conjugated | eBioscience | 12-1449-80 |
| hCD146, PE conjugated | BD Pharmingen | 550315 |
| hCD105, PE conjugated | Invitrogen | MHCD10504 |
| Ms IgG1,k antibody, FITC conjugated | BD Pharmingen | 555748 |
| Ms IgG1,k antibody, PE conjugated | BD Pharmingen | 559320 |
| Ms IgG2a,k antibody, FITC conjugated | BD Pharmingen | 555573 |
| Anti-human CD31 | Dako | clone JC70/A |
| Anti-mouse CD31 | BD Pharmingen | 553370 |
| 0.22-μm vacuum filtration system | Millipore | SCGPU05RE |
| Glacial acetic acid, 17.4N | Fisher | A38-500 |
| Antibiotic-Antimycotic | Invitrogen | 15240-062 |
| Fetal bovine serum (FBS) | Hyclone | SH30070.03 |
| IHC Zinc Fixative | BD Biosciences | 550523 |
| Sytox green reagent | Invitrogen | S33025 |
| Cloning cylinders, sterile | Fisher Scientific | 07-907-10 |
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Tags
Phenotypic CharacterizationFunctional CharacterizationEndothelial Colony Forming CellsHuman Umbilical Cord BloodAngiogenesisVasculogenesisArteriogenesisCirculating Endothelial Progenitor CellsVascular RegenerationVascular RepairParacrine StimulationEPC MarkerProangiogenic Hematopoietic Stem CellsCirculating Angiogenic CellsTie2+ MonocytesMyeloid Progenitor CellsTumor Associated MacrophagesM2 Activated Macrophages
Cite This Article
Prasain, N., Meador, J. L., Yoder, M. C. Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood. J. Vis. Exp. (62), e3872, doi:10.3791/3872 (2012).