Supplementary Material for: Osteogenic Differentiation of Late-Outgrowth CD45-Negative Endothelial Progenitor Cells

<b><i>Background/Aims:</i></b> Late-outgrowth CD45-negative endothelial colony-forming cells are implicated to be circulating endothelial progenitor cells (EPCs), as they express endothelial cell markers and can directly form blood vessels. As these cells share characteristics of other progenitor cell phenotypes, late-outgrowth EPCs were assayed for both multilineage differentiation capability and for markers of pluripotency. <b><i>Methods:</i></b> Clonal single-colony late-outgrowth EPCs were derived from human cord blood and assayed both for multilineage differentiation capability in vitro and for markers of pluripotency by qPCR. <b><i>Results:</i></b> Under osteogenic growth conditions, these EPCs expressed the osteogenic markers RUNX2, COL1A1, ALPL, and osteocalcin and demonstrated calcium deposition and bone mineralization. Endothelial colony-forming cells expressed markers associated with induced pluripotent stem cells, including SOX2, POU5F1, c-MYC, and KLF4. <b><i>Conclusions:</i></b> Late-outgrowth EPCs express markers associated with pluripotency and can directly express an osteogenic phenotype under bone differentiation conditions in vitro.

**<i>背景与目的：</i>** 迟发性增殖CD45阴性内皮集落形成细胞（late-outgrowth CD45-negative endothelial colony-forming cells）被认为是循环内皮祖细胞（endothelial progenitor cells, EPCs），因其可表达内皮细胞标志物并能直接形成血管。由于这类细胞与其他祖细胞表型具有共同特征，本研究对迟发性增殖EPCs进行了多向分化能力及多能性标志物检测。
**<i>方法：</i>** 本研究从人脐带血中分离得到单克隆单集落迟发性增殖EPCs，通过体外实验检测其多向分化能力，并采用定量聚合酶链反应（qPCR）检测其多能性标志物表达水平。
**<i>结果：</i>** 在成骨诱导培养条件下，此类EPCs可表达成骨标志物RUNX2、COL1A1、ALPL及骨钙素，并出现钙沉积与骨矿化现象。此类内皮集落形成细胞还可表达与诱导多能干细胞相关的标志物，包括SOX2、POU5F1、c-MYC及KLF4。
**<i>结论：</i>** 迟发性增殖EPCs可表达多能性相关标志物，并可在体外骨分化培养条件下直接呈现成骨表型。