Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria; Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria.
Cytotherapy. 2013 Nov;15(11):1426-35. doi: 10.1016/j.jcyt.2013.06.018.
Human endothelial progenitor cells (EPC) play an important role in regenerative medicine and contribute to neovascularization on vessel injury. They are usually enriched from peripheral blood, cord blood and bone marrow. In human fat tissue, EPC are rare and their isolation remains a challenge.
Fat tissue was prepared by collagenase digestion, and the expression of specific marker proteins was evaluated by flow cytometry in the stromal vascular fraction (SVF). For enrichment, magnetic cell sorting was performed with the use of CD133 microbeads and EPC were cultured until colonies appeared. A second purification was performed with CD34; additional isolation steps were performed with the use of a combination of CD34 and CD31 microbeads. Enriched cells were investigated by flow cytometry for the expression of endothelial specific markers, by Matrigel assay and by the uptake of acetylated low-density lipoprotein.
The expression pattern confirmed the heterogeneous nature of the SVF, with rare numbers of CD133+ detectable. EPC gained from the SVF by magnetic enrichment showed cobblestone morphology of outgrowth endothelial cells and expressed the specific markers CD31, CD144, vascular endothelial growth factor (VEGF)R2, CD146, CD73 and CD105. Functional integrity was confirmed by uptake of acetylated low-density lipoprotein and the formation of tube-like structures on Matrigel.
Rare EPC can be enriched from human fat tissue by magnetic cell sorting with the use of a combination of microbeads directed against CD133, an early EPC marker, CD34, a stem cell marker, and CD31, a typical marker for endothelial cells. In culture, they differentiate into EC and hence could have the potential to contribute to neovascularization in regenerative medicine.
人类内皮祖细胞（EPC）在再生医学中发挥重要作用，并有助于血管损伤后的血管新生。它们通常从外周血、脐带血和骨髓中富集。在人类脂肪组织中，EPC 很少，其分离仍然是一个挑战。
通过胶原酶消化制备脂肪组织，并通过流式细胞术评估基质血管部分（SVF）中特定标记蛋白的表达。为了富集，使用 CD133 微珠进行磁性细胞分选，然后培养 EPC 直到出现集落。用 CD34 进行第二次纯化；用 CD34 和 CD31 微珠的组合进行额外的分离步骤。通过流式细胞术检测内皮特异性标记物的表达、Matrigel 测定和乙酰化低密度脂蛋白的摄取来研究富集细胞。
表达模式证实了 SVF 的异质性，可检测到少数 CD133+。通过磁性富集从 SVF 中获得的 EPC 显示出鹅卵石状的细胞形态，并表达特异性标记物 CD31、CD144、血管内皮生长因子（VEGF）R2、CD146、CD73 和 CD105。通过摄取乙酰化低密度脂蛋白和在 Matrigel 上形成管状结构来证实功能完整性。
通过使用针对早期 EPC 标记物 CD133、干细胞标记物 CD34 和内皮细胞典型标记物 CD31 的微珠组合进行磁性细胞分选，可以从人类脂肪组织中富集稀有 EPC。在培养中，它们分化为 EC，因此有可能为再生医学中的血管新生做出贡献。
