miR-21 negatively regulates stemness-related properties of miR-21 knock-in neonatal dermal cells via a reactive oxygen species dependent pathway. N-acetyl cysteine (NAC; 5 mM) was added into culture medium and adipogenic and osteogenic induced medium of miR-21 knock-in neonatal dermal cells to scavenge intracellular reactive oxygen species (ROS), and stemness related-properties of miR-21 knock-in neonatal dermal cells with and without NAC were detected. (A) Proliferation was measured every 2 days after seeding. Colony formation assay including (B) colony numbers, (C) average colony sizes, and (D) colony size distribution was present. (E) Representative migration photographs at indicated time points. (F) Relative migration rate presented as mean ± standard deviation (n = 6 per time point for each group). (G) Quantification and representative photographs of the Oil-Red O staining of the adipogenic differentiation. (H) Quantification and representative photographs of the Alizarin Red staining of the osteogenic differentiation. CFU, colony-forming units; d, days; h, hours; miR-21 cells, miR-21 knock in neonatal dermal cells; OD, optical density. **P <0.01. Scale bar = 500 μm.