Stemness maintenance of stem cells derived from human exfoliated deciduous teeth (SHED) in 3D spheroid formation through the TGF-β/Smad signaling pathway

Mesenchymal stem cells (MSCs) have shown great potential as important therapeutic tools for dental pulp tissue engineering, with the maintenance and enhancement of their stemness being crucial for successful therapeutic application in vivo and threedimensional (3D) spheroid formation considered a reliable technique for enhancing their pluripotency. Human exfoliated deciduous tooth stem cells (SHED) were cultured in a low attachment plate to form aggregates for five days. Then, the resulting spheroids were analyzed for pluripotent marker expression, paracrine secretory function, proliferation, signaling pathways involved, and distribution of key proteins within the spheroids. The results indicated that 3D spheroid formation significantly increased the activation of the transforming growth factor beta (TGF-f3)/Smad signaling pathway and upregulated the secretion and mRNA expression levels of TGF-f3, which in turn enhanced the expression of pluripotency markers in SHED spheroids. The activation of the TGFf3/Smad signaling pathway through 3D spheroid formation was found to preserve the stemness properties of SHED. Thus, understanding the mechanisms behind pluripotency maintenance of SHED culture through 3D spheroid formation could have implications for the therapeutic application of MSCs in regenerative medicine and tissue engineering.