Introduction: circulating CD34-positive endothelial progenitor cells (EPCs) promote angiogenesis. Recent decades, the clinical efficacy of autologous transplantation of CD34-positive cells has been demonstrated in ischemic diseases. However, it is known that the quality and number of circulating EPCs reduce with aging, smoking, diabetes, and so on. Then, we aimed to generate CD34-positive cells from endothelial cells (ECs) by chemical reprogramming.

Methods: based on previous reports about reprogramming, seven candidate chemical compounds were selected. Human umbilical venous endothelial cells (HUVECs) were used as ECs and the following chemical compounds are added to medium: CHIR-99021, A83-01, LDN193189, Y-27632, valproic acid, forskolin, PD0325901. The expression of CD34 was evaluated by quantitative PCR, flowcytometry, and immunocytochemistry. Western blotting was used to assess the state of intracellular signaling.

Results: after exposure to seven chemical compounds, the expression of CD34 increased significantly. Their combinations were investigated and PD0325901, an inhibitor of the MEK pathway, contributed to significant upregulation of CD34 expression. To confirm the effect of PD0325901 to MEK/ERK pathway, western blotting was performed, and it is revealed that the phosphorylated ERK1/2 levels were substantially reduced in PD0325901-treated cells. These results suggest that PD0325901 upregulate CD34 expression via inhibition of MEK/ERK pathway. This pathway is known to be closely associated with cell proliferation. In our studies, the proliferative ability of PD0325901-treated cells reduced, and the number of harvested cells was almost equal to the number of cells seeded.

Conclusion: we found the MEK inhibitor PD0325901 increase the expression of CD34 in ECs. However, the proliferative ability decreased by inhibition of MEK/ERK pathway. To apply our findings to regenerative therapy, further functional analysis and investigations into the mechanisms of CD34 upregulation are required.