Bone Marrow- and Umbilical Cord-Derived Mesenchymal Stem Cell Secretome Alters Gene Expression and Upregulates Motility of Human Endometrial Stromal Cells
Authors: Zhao Qingshi , Larios Karla , Naaldijk Yahaira , Sherman Lauren , Chemerinski Anat , Okereke Kennisha , Rameshwar Pranela , Lemenze Alexander , Douglas Nataki C. , Morelli Sara S. 
Published: November 2022

ABSTRACTIntroductionCyclic regeneration of the endometrium, and its repair after parturition or injury, are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) and umbilical cord (UC-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, the mechanisms remain unclear. This study tested the hypothesis that the secretome of MSCs from human BM and UC upregulates human endometrial stromal cell (HESC) proliferation, migration and invasion, and activates pathways to increase HESC motility.MethodsMSCs were purchased from ATCC (BM-MSC-1) and cultured from the BM aspirate of three healthy female donors (BM-MSC-2-4), and from umbilical cords of two healthy male term infants (UC-MSC-1-2). Indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system studied the effect of the BM-MSC and UC-MSC secretome on HESC proliferation, migration, and invasion. To study the effect of the MSC secretome on HESC gene expression, HESCs were exposed to the BM-MSC secretome via indirect co-culture for 24 h. Total RNA was extracted from HESCs for RNA sequencing (RNA-Seq). Differentially expressed genes (DEG) and significantly altered pathways were identified. MSigDB was used to identify the top 15 enriched biological pathways (padj < 0.05). RT-qPCR was performed to validate changes in mRNA expression of DEG common to both BM-MSC exposures. Given robust upregulation ofCCL2mRNA expression in HESCs exposed to the BM- and UC-MSC secretomes, transwell migration and invasion assays were performed to determine the effect of recombinant CCL2 on HESC motility. Statistical significance was defined asp<0.05.ResultsIndirect co-culture of HESCs with BM- or UC-MSCs resulted in significant increase in HESC migration and invasion regardless of the source of MSCs. However, effects on cellular proliferation varied among the MSC donors. Exposure of HESCs to the secretome of BM-MSCs changed the expression of 10,139 genes with FDR < 0.05. There was overlap among 4350 genes between HESCs exposed to BM-MSC-1 and BM-MSC-2. Within four biological pathways enriched in HESCs, 4 genes (CCL2, HGF, PLAU, andBDKRB2) were differentially expressed in HESCs that had been cocultured with BM-MSC-1 and BM-MSC-2. qRT-PCR showed significantly increased mRNA expression ofCCL2in HESCs exposed to BM-MSC-1 (5-fold) and BM-MSC-2 (7.7-fold). In contrast, the increase inHGFexpression was significant after exposure to BM-MSC-2 (1.8-fold) but not BM-MSC-1. Exposure to the UC-MSC secretome had similar effects on HESC-derivedCCL2andHGFlevels.CCL2expression was significantly increased (6.5-fold) by UC-MSC-2 but not by UC-MSC-1;HGFexpression was significantly increased (1.6-fold) by UC-MSC-2 but not by UC-MSC-1. Validation studies indicated that exposure to recombinant CCL2 for 48 hours significantly increased HESC migration (1.2-fold) and invasion (1.4-fold). These data suggest that CCL2 is a key factor in mediating MSC-induced HESC motility.ConclusionIncreased HESC motility by the secretome of BM- and UC-MSC appears to be mediated by paracrine and autocrine mechanisms, in part by upregulatedCCL2expression in HESC. Together, our data support the potential for leveraging the MSC secretome as a novel cell-free therapy in the treatment of disorders of endometrial regeneration.