Background/Objectives: 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. Methods: 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. Results: 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, JUN (+2.82, p = 0.003), LRP1 (+2.06, p = 0.05), AXIN1 (+3.02, p = 0.03), and FYN (+3.30, p = 0.01) were up-regulated, indicating modulation of the Wnt/β-catenin signaling pathway, a key regulator of osteoblast differentiation. Conclusions: 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.

Center for Gynecology Obstetrics and Infertility Treatment Pastelova 60 198 Poznan Poland

Collegium Medicum University of Zielona Gora 65 046 Zielona Gora Poland

Department of Basic and Preclinical Sciences Institute of Veterinary Medicine Nicolaus Copernicus University in Torun 87 100 Torun Poland

Department of Computational Biology Institute of Molecular Biology Faculty of Biology Adam Mickiewicz University 61 614 Poznan Poland

Department of Inorganic and Analytical Chemistry Poznan University of Medical Sciences 60 806 Poznan Poland

Department of Molecular Neurooncology Institute of Bioorganic Chemistry Polish Academy of Sciences 61 704 Poznan Poland

Department of Obstetrics and Gynecology University Hospital and Masaryk University 62500 Brno Czech Republic

Department of Stem Cells and Regenerative Medicine Institute of Natural Fibres and Medicinal Plants 62 064 Plewiska Poland

Department of Toxicology Poznan University of Medical Sciences 60 806 Poznan Poland

Department of Veterinary Surgery Institute of Veterinary Medicine Nicolaus Copernicus University in Torun 87 100 Torun Poland

Division of Anatomy Department of Human Morphology and Embryology Wroclaw Medical University 50 368 Wroclaw Poland

Doctoral School Poznan University of Medical Sciences 60 812 Poznan Poland

Physiology Graduate Faculty North Carolina State University Raleigh NC 27613 USA

Prestage Department of Poultry Science North Carolina State University Raleigh NC 27695 USA

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