Stem cells from human exfoliated deciduous teeth (SHEDs) are ideal seed cells in bone tissue engineering. As a first‐line antidiabetic drug, metformin has recently been found to promote bone formation. The purpose of this study was to investigate the effect of metformin on the osteogenic differentiation of SHEDs and its underlying mechanism. SHEDs were isolated from the dental pulp of deciduous teeth from healthy children aged 6 to 12, and their surface antigen markers of stem cells were detected by flow cytometry. The effect of metformin (10–200 μM) treatment on SHEDs cell viability, proliferation, and osteogenic differentiation was analyzed. The activation of adenosine 5′‐monophosphate‐activated protein kinase (AMPK) phosphorylation Thr172 (p‐AMPK) was determined by western blot assay. SHEDs were confirmed as mesenchymal stem cells (MSCs) on the basis of the expression of characteristic surface antigens. Metformin (10–200 μM) did not affect the viability and proliferation of SHEDs but significantly increased the expression of osteogenic genes, alkaline phosphatase activity, matrix mineralization, and p‐AMPK level expression in SHEDs. Compound C, a specific inhibitor of the AMPK pathway, abolished metformin‐induced osteogenic differentiation of SHEDs. Moreover, metformin treatment enhanced the expression of proangiogenic/osteogenic growth factors BMP2 and VEGF but reduced the osteoclastogenic factor RANKL/OPG expression in SHEDs. In conclusion, metformin could induce the osteogenic differentiation of SHEDs by activating the AMPK pathway and regulates the expression of proangiogenic/osteogenic growth factors and osteoclastogenic factors in SHEDs. Therefore, metformin‐pretreated SHEDs could be a potential source of seed cells during stem cell‐based bone tissue engineering.

Stem cells from human exfoliated deciduous teeth (SHEDs) are ideal seed cells in bone tissue engineering. As a first-line anti-diabetic drug, metformin has recently been found to promote bone formation. The purpose of this study was to investigate the effect of metformin on osteogenic differentiation of SHEDs and its underlying mechanism. SHEDs were isolated from the dental pulp of deciduous teeth from healthy children aged from 6 to 12, and their surface antigen markers of stem cells were detected by flow cytometry. The effect of metformin (10 - 200 μM) treatment on SHEDs cell viability, proliferation, and osteogenic differentiation was analyzed. The activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) was determined by western blot assay for the AMPK phosphorylated at Thr172 (p-AMPK). SHEDs were confirmed as mesenchymal stem cells (MSCs) based on the expression of characteristic surface antigens. Metformin (10-200 μM) did not affect the viability and proliferation of SHEDs, but significantly increased the expression of osteogenic genes, the activity of alkaline phosphatase, matrix mineralization, and p-AMPK level in SHEDs. Compound C, a specific inhibitor of AMPK pathway, abolished metformin-induced osteogenic differentiation of SHEDs. Moreover, metformin treatment enhanced pro-angiogenic/osteogenic growth factors BMP2 and VEGF but reduced the osteoclastogenic factor RANKL/OPG expression in SHEDs. In conclusion, metformin could induce the osteogenic differentiation of SHEDs by activating the AMPK pathway and regulates the expression of pro-angiogenic/osteogenic growth factors and osteoclastogenic factors in SHEDs. Therefore, SHEDs, combined with metformin possesses therapeutic potential for bone regeneration and bone defect repair.

The stem cells of human exfoliated deciduous teeth (SHEDs) are considered to be one of the main sources of seed cells in stem cell therapy. The aim of this study was to examine the effect of ciliary neurotrophic factor (CNTF) on neurogenic differentiation of SHEDs. With the consent of parents, SHEDs from 6 to 8 year old children were isolated and cultured. The mesenchymal stemness and the potential of multidirectional (adipogenic and osteogenic) differentiation for the isolated SHEDs were firstly determined. The effect of CNTF on specific neurogenic differentiation of SHEDs was then examined by detecting the expression of marker genes and proteins via RT-PCR, immunoblotting, and immunofluorescence microscopy. The isolated SHEDs expressed specific surface markers of mesenchymal stem cells, and their potential of osteogenic and adipogenic differentiation were confirmed. CNTF promoted the differentiation of SHEDs into neuron-like cells with a high expression of acetylcholine transferase (CHAT), a marker of cholinergic neurons. The expression of other neuron markers including nestin, microtubule-associated protein 2 (MAP 2), and β-tublin III was also detected. Interestingly, the expression of neurogenic markers was maintained at a high level after neurogenic induction. SHEDs can be induced by CNTF to differentiate into cholinergic neuron-like cells under appropriate culture conditions. Our findings have laid a foundation for future use of SHEDs to treat neurological diseases.