IntroductionCyclic 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.
Cyclic regeneration and repair of the endometrium 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, mechanisms remain unclear. This study tested the hypothesis that the BM-MSC and UC-MSC secretomes upregulate human endometrial stromal cell (HESC) proliferation, migration, and invasion, and activate pathways to increase HESC motility. BM-MSCs were purchased from ATCC and cultured from the BM aspirate of three healthy female donors. UC-MSCs were cultured from umbilical cords of two healthy male term infants. Using indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system, we demonstrated that co-culture of HESCs with BM-MSCs or UC-MSCs from all donors significantly increased HESC migration and invasion, whereas effects on HESC proliferation varied among BM-MSC and UC-MSC donors. Analysis of gene expression by mRNA sequencing and RT-qPCR showed that expression of CCL2 and HGF was upregulated in HESCs that had been cocultured with BM-MSCs or UC-MSCs. Validation studies revealed that exposure to recombinant CCL2 for 48 hours significantly increased HESC migration and invasion. Increased HESC motility by the BM-MSC and UC-MSC secretome appears to be mediated in part by upregulated HESC CCL2 expression. Our data support the potential for leveraging MSC secretome as a novel cell-free therapy to treat disorders of endometrial regeneration.
Introduction: Uterine leiomyosarcoma (uLMS) is a rare tumor with poor survival and few therapeutic options. The Notch pathway is evolutionarily conserved, is active in uterine processes, has oncogenic properties in many cancers, but has not been well studied in uLMS. Cancer stem cells (CSC) are subpopulations of cancer cells that are treatment resistant. In certain tumors, activation of canonical Notch signaling, via gamma secretase, supports CSC survival and the acquisition of chemoresistance. Our objective is to identify and characterize the subpopulation of uLMS cells that are viable after treatment with the half maximal inhibitory concentration (IC50) of MK-0752, a gamma secretase inhibitor (GSI) in phase I trials. We will determine Notch signaling activity and expression of markers associated with stem-like potential in GSI resistant uLMS cells. Methods: MTT assays were performed on SK-LMS-1 and SK-UT-1B, two uLMS cell lines, to identify the IC50 for MK-0752. Cells were exposed to MK-0752 at the IC50 or DMSO (control) for 24 hours and viable cells were collected. First, expression of stemness markers, CD133, cMYC and SOX2, in uLMS cells was compared to human uterine smooth muscle cells (hUT-SMC) at baseline. After treatment with MK-0752, the remaining subpopulations of resistant uLMS cells were evaluated for Notch signaling activity by expression of HES1. Stemness marker expression was also assessed. Gene expression was measured with qRT-PCR. Mean fold change (FC) gene expression was determined by FC experimental divided by FC control, relative to expression of 18S rRNA. P≤0.05 was considered significant. Results: In untreated uLMS cells, expression of CD133 and c-MYC was significantly higher in SK-LMS-1 (fibroblast morphology) vs hUT-SMC, while expression of CD133 and SOX2 was significantly higher in SK-UT-1B (epithelial morphology) vs hUT-SMC. After treatment with MK-0752 at IC50 (427.4 µM for SK-LMS-1 or 128.4µM for SK-UT-1B) expression of HES1 was decreased in SK-LMS-1 (0.35x, p≤0.05) and increased in SK-UT-1B (2.38x, p≤0.01) in treatment resistant, viable cells as compared to untreated cells. Expression of c-MYC was decreased (0.38x, p≤0.01) and CD133 was similar (1.64x, p>0.05) in GSI resistant SK-LMS-1 cells, while expression of CD133 and SOX2 was similar (1.43x and 1.30x, respectively, p>0.05) in GSI resistant SK-UT-1B cells compared to untreated cells. Conclusions: Cellular morphology, Notch signaling activity and expression of stemness markers differs in GSI resistant SK-LMS-1 compared to GSI resistant SK-UT-1B cells. The subpopulation of MK-0752 resistant SK-LMS-1 cells have reduced Notch activity and reduced expression of stemness marker, c-MYC, while the resistant SK-UT-1B cells have increased Notch activity. Further studies are required to identify additional factors associated with uLMS resistance to GSIs and the importance of this heterogeneity of uLMS in vivo. Citation Format: Yasmin Abedin, Emily Alpert, Erica Rego, Karla Larios, Emma Cheung, Sofia Gabrilovich, Qingshi Zhao, Mark H. Einstein, Nataki Douglas. Resistance to MK-0752 alters Notch activity and expression of stemness markers in uterine leiomyosarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6074.
Introduction: During the pandemic, every state was at liberty to determine if abortion services were an essential or non-essential service. This furthered disparities in resource availability based on location. Our study analyzes changes in abortion care practices as a response to COVID-19 on a regional basis.
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