Background: Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are considered a new and favorable source of stem cells for cell replacement-based therapy. Some microRNAs (miRNAs) have been reported to participate in the regulation of immune responses. Our aim was to investigate the effects of miR-21 on the biological characteristics, immunoregulatory properties, and potential mechanisms of hAM-MSCs.
Methods: hAM-MSCs were isolated from the placental amnion membrane of a newborn. Cell proliferation, cell cycle, apoptosis, and expressions of cell surface markers were measured by CCK-8 and flow cytometric assays in hAM-MSCs. The expression of mesenchymal-specific antigens vimentin and stage-specific embryonic antigen-4 (SSEA-4) were identified by immunofluorescence staining. Tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin-10 (IL-10) expressions in the cocultured supernatant of hAM-MSCs and peripheral blood mononuclear cells (PBMC) were detected via enzyme-linked immunosorbent assays (ELISA).
Results: Flow cytometric analyses revealed that the positive expression rates of the cell surface markers CD29, CD44, CD73, and CD90 in hAM-MSCs were 97.3%, 96.3%, 97.8%, and 98.2%, respectively, while the rates of CD34 and CD45 expression were only 0.6% and 0.84%, respectively. The immunofluorescent staining results showed that vimentin and SSEA-4 were positive in hAM-MSCs. CCK-8 assays revealed that miR-21 overexpression significantly promoted hAM-MSC proliferation. Cell cycle analyses revealed that the number of hAM-MSCs-miR-21+ cells during the synthesis phase (S phase) was significantly increased. miR-21 overexpression also significantly inhibited apoptosis in hAM-MSCs. The ELISA analyses revealed that miR-21 overexpression enhanced the inhibitory effect of hAM-MSCs on the secretion of TNF-α and MCP-1 as well as the promotive effect on the secretion of IL-10 in PBMC cocultured with miR-21-hAM-MSCs. In addition, miR-21 downregulation reduced the inhibitory effect of hAM-MSCs on the secretion of TNF-α, MCP-1, and the promotive effect on the secretion of IL-10 in PBMC cocultured with anti-miR-21-hAM-MSCs.
Conclusions: Our data showed that miR-21 promoted hAM-MSCs proliferation, inhibited apoptosis, and was involved in controlling the immunoregulatory capacity of hAM-MSCs.
Keywords: cell growth; hAM-MSCs; immunoregulation; miR-21.