Hosoda Chihiro, Mitani Seiji, Sakata Asuka, Kasuda Shogo, Onodera Yu, Takabayashi Yoko, Shima Midori, Tatsumi Kohei
Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan.
Medicinal Biology of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan.
Regen Ther. 2024 Aug 29;26:654-662. doi: 10.1016/j.reth.2024.08.009. eCollection 2024 Jun.
CD34-positive endothelial progenitor cells (EPCs) promote angiogenesis and are a promising tool for regenerative cell therapy of ischemic diseases. However, the number and quality of CD34-positive cells decrease owing to various external and internal factors; thus, an efficient method is needed to establish CD34-positive EPCs. The generation of functional cells by reprogramming, that is, manipulating cell fate via gene transfer and/or treatment with chemical compounds, has recently been reported. Therefore, we aimed to generate CD34-positive cells by the reprogramming of endothelial cells (ECs).
Based on previous reports, seven candidate chemical compounds were selected to reprogram human umbilical vein ECs (HUVECs) to CD34-positive cells. Following stimulation with the chemical compounds, the expression of CD34 was evaluated using quantitative PCR, flow cytometry, and immunocytochemistry.
HUVECs treated with the compounds exhibited increased CD34 expression. We cultured cells in alternate media lacking one of the seven compounds and found no CD34 expression in cells treated with PD0325901-free media, suggesting that PD0325901-a MEK inhibitor-mainly contributed to the increase in CD34 expression. We found that 98% of cells were CD34-positive after PD0325901 treatment alone for 7 d. Western blotting revealed that the phosphorylation of ERK was suppressed in PD0325901-treated cells. No upregulation of CD34 was observed in fibroblast cell lines, even after PD0325901 treatment. These results suggested that PD0325901 induces CD34-positive cells by inhibiting ERK phosphorylation in ECs.
CD34 expression was strongly induced in ECs by treatment with the MEK inhibitor PD0325901 in vitro. Our study provides a useful reference for the establishment of CD34-positive EPCs and will contribute to the development of regenerative therapies, especially for ischemic diseases.
CD34 阳性内皮祖细胞（EPCs）可促进血管生成，是缺血性疾病再生细胞治疗的一种有前景的工具。然而，由于各种外部和内部因素，CD34 阳性细胞的数量和质量会下降；因此，需要一种有效的方法来建立 CD34 阳性 EPCs。最近有报道称，通过重编程，即通过基因转移和/或用化学化合物处理来操纵细胞命运，可产生功能性细胞。因此，我们旨在通过内皮细胞（ECs）的重编程来产生 CD34 阳性细胞。
根据先前的报道，选择七种候选化学化合物将人脐静脉 ECs（HUVECs）重编程为 CD34 阳性细胞。在用化学化合物刺激后，使用定量 PCR、流式细胞术和免疫细胞化学评估 CD34 的表达。
用这些化合物处理的 HUVECs 显示 CD34 表达增加。我们在缺乏七种化合物之一的交替培养基中培养细胞，发现在不含 PD0325901 的培养基处理的细胞中没有 CD34 表达，这表明 MEK 抑制剂 PD0325901 主要促成了 CD34 表达的增加。我们发现单独用 PD0325901 处理 7 天后，98%的细胞为 CD34 阳性。蛋白质印迹显示，在 PD0325901 处理的细胞中 ERK 的磷酸化受到抑制。即使在 PD0325901 处理后，在成纤维细胞系中也未观察到 CD34 的上调。这些结果表明，PD0325901 通过抑制 ECs 中的 ERK 磷酸化诱导 CD34 阳性细胞。
在体外，用 MEK 抑制剂 PD0325901 处理可强烈诱导 ECs 中 CD34 的表达。我们的研究为建立 CD34 阳性 EPCs 提供了有用的参考，并将有助于再生疗法的发展，特别是针对缺血性疾病的疗法。
