The Effect of Liposomal DMU-212 on the Differentiation of Human Ovarian Granulosa Cells in a Primary 3D Culture Model

<b>Background/Objectives:</b> Human ovarian granulosa cells (hGCs) are crucial to ovarian follicle development and function, exhibiting multipotency and the ability to differentiate into neuronal cells, chondrocytes, and osteoblasts in vitro. 3,4,5,4'-tetramethoxystilbene (DMU-212) is a methylated derivative of resveratrol, a natural polyphenol found in grapes and berries, with a wide spectrum of biological activities, including notable anticancer properties. Interestingly, DMU-212 exhibits cytotoxic effects predominantly on cancer cells while sparing non-cancerous ones, and evidence suggests that similar to resveratrol, it may also promote hGC differentiation. This study aimed to investigate the effects of the liposomal formulation of this methylated resveratrol analog-lipDMU-212-on the osteogenic differentiation ability of hGCs in a primary three-dimensional cell culture model. <b>Methods:</b> lipDMU-212 was formulated using the thin-film hydration method. GC spheroids' viability was evaluated after exposure to lipDMU-212, an osteoinductive medium, or both. Osteogenic differentiation was confirmed using Alizarin Red staining and quantified by measuring Alkaline Phosphatase (ALP) activity on days 1, 7, and 15. RNA sequencing (RNA-seq) was performed to explore molecular mechanisms underlying lipDMU-212-induced differentiation. <b>Results:</b> lipDMU-212 promoted osteogenic differentiation of hGCs in the 3D cell culture model, as evidenced by increased mineralization and a ~4-fold increase in ALP activity compared with the control. RNA-seq revealed up-regulation of genes related to cell differentiation and cellular identity. Furthermore, <i>JUN</i> (+2.82, <i>p</i> = 0.003), <i>LRP1</i> (+2.06, <i>p</i> = 0.05), <i>AXIN1</i> (+3.02, <i>p</i> = 0.03), and <i>FYN</i> (+3.30, <i>p</i> = 0.01) were up-regulated, indicating modulation of the Wnt/β-catenin signaling pathway, a key regulator of osteoblast differentiation. <b>Conclusions:</b> The ability of GCs to differentiate into diverse tissue-specific cell types underscores their potential in regenerative medicine. This study contributes to the understanding of lipDMU-212's role in osteogenic differentiation and highlights its potential in developing future therapies for degenerative bone diseases.