Human Decellularized Endometrial Scaffold Seeded by Myometrial Smooth Muscle Cells Supports the Differentiation of Human Endometrial Mesenchymal Cells to Epithelial and Stromal Cells ​

Abstract

Background: This study designed to evaluate the co-culturing of human mesenchymal endometrial cells (EMCs) and myometrial smooth muscle cells (MSMCs) in decellularized scaffold as a natural bioscaffold to formation of the endometrial-like structure. Methods: After decellularization of the human endometrium, cell seeding was performed by centrifugation of human EMCs with different speeds and times in 15 experimental subgroups. Analysis of residual cell count in suspension was done in all subgroups and the method with the lower amount of suspended cells was selected for subsequent study. Then, the human EMCs and the MSMCs were seeded on the decellularized tissue and cultured for one week then, differentiation of the seeded cells was assessed by morphological and gene expression analysis. Results: The cell seeding method by centrifuging at 7000 rpm for 2 minutes showed the highest number of seeded cells and the lowest number of residual cells in suspension. The endometrial-like structure formed and the epithelial-like cells had some protrusions on their surface and the stromal cells had spindle and polyhedral morphology. The MSMCs almost were homed at the periphery of mesenchymal cells similar to their arrangement in the native uterus. The expression of endometrial-related genes (SPP1, MMP2, ZO-1, LAMA2 and COL4A1) confirmed these observations. The low level of expression of the OCT4 gene as a pluripotency marker in seeded cells confirmed the differentiation of these cells. Conclusion: This study showed that the co-culturing of human EMCs with the MSMCs on the decellularized scaffold could improve the formation of endometrial-like structures.