Lu Yang, Gong Yiyi, Lian Jie, Wang Ling, Kretlow James D, Zhou Guangdong, Cao Yilin, Liu Wei, Zhang Wen Jie
Department of Plastic and Reconstructive Surgery, Shanghai 9th People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Tissue Engineering, National Tissue Engineering Center of China, Shanghai, China.
Division of Plastic Surgery, Baylor College of Medicine, Houston, Texas, United States of America.
PLoS One. 2014 Sep 25;9(9):e107127. doi: 10.1371/journal.pone.0107127. eCollection 2014.
In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2 × 10(5) cells/cm(2)) by seeding total 9 × 10(5) cells into six high density dots or cultured in regular density (1.6 × 10(4) cells/cm(2)) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells.
内皮祖细胞（EPCs）的体外扩增在干细胞研究及其应用中仍然是一个挑战。我们假设高密度培养能够通过模拟骨髓微环境中的细胞间相互作用来扩增骨髓中的EPCs。为了验证这一假设，将大鼠骨髓细胞以两种方式培养：一种是高密度培养（2×10⁵个细胞/cm²），即将总共9×10⁵个细胞接种到六个高密度点中；另一种是常规密度培养（1.6×10⁴个细胞/cm²），细胞总数相同。对培养15天的细胞进行流式细胞术分析表明，与常规密度培养的细胞相比，高密度培养的细胞体积更小，与EPCs相关的标志物表达水平更高。在功能上，这些细胞表现出强大的血管生成潜力，在体外具有更好的成管能力，并且在体内能够有效地挽救小鼠缺血肢体，它们可整合到新生毛细血管结构中。全局基因芯片和酶联免疫吸附测定分析显示，高密度培养的细胞中黏附分子的基因表达上调，促血管生成生长因子的蛋白释放增加。总之，高密度细胞培养促进了骨髓中EPCs的扩增，这些EPCs能够通过旁分泌生长因子和直接分化为内皮细胞来增强组织血管生成。
