An investigation of matrix metalloproteinase 3 (MMP3) derived from myometrial cells on endometrial mesenchymal stem-like cells (eMSCs) maintenance

Abstract

Endometrial mesenchymal stem-like cells (eMSCs) are responsible for the regeneration of the endometrium after each menstrual cycle during the reproductive years in women. The stem niche can mediate stem cell self-renewal and differentiation. Our group previously found the myometrium as a prospective niche for eMSCs and identified matrix metalloproteinase-3 (MMP3) as a potential stem cell regulator. MMP3 is important in tissue remodeling events and parturition. Cytokines and progesterone withdrawal can regulate MMP3. In this study, we investigated the factor(s) involved in regulating myometrial cells on stem cell maintenance. 1) We confirmed the effect of myometrial cells and the role of MMP3 on eMSCs and, 2) examined the factor(s) regulating MMP3 by the myometrial cells. EMSCs were co-cultured with myometrial cells at a ratio of 1:90 for 15 days. Clonogenicity and the phenotypic expression of eMSCs were determined by counting the colony forming units (CFUs) and analyzing the co-expression of CD140b and CD146 (eMSC markers) by flow cytometry or double immunofluorescence (IF), respectively. Large CFUs are generated from stem/progenitor cells while small CFUs are from differentiated transit amplifying (TA) cells. The relative total clonogenicity of eMSCs significantly increased after co-culture with myometrial cells to 1.67 ± 0.22 fold (n=4, P<0.05). The relative clonogenicity of large CFUs also significantly increased to 4.02 ± 2.61 fold (n=4, p<0.05). Coculture of myometrial cells can also increase the relative proportion of eMSCs to 1.93 ± 2.61 fold (flow, n=4, P<0.05). Addition of neutralizing MMP3 antibody reduced the relative total clonogenicity to 0.12 ± 0.07 fold (n=4, P<0.05) and proportion of eMSCs to 0.49 ± 0.25 fold (IF staining, n=3, P<0.05). Next, the role of cytokines in regulating myometrial cells to secrete MMP3 was assessed. Three cytokines: TNF α, IL1ß and IL6 at 10 and 100ng/ml were used to treat myometrial cells for 5 days in culture. The pro-inflammatory cytokines, TNF α and IL1ß showed significant increase in MMP3 secretion (ELISA, n=5, p<0.05) and protein expression (western blot, n=5, p<0.05). Using lower concentration of TNF α and IL1β (0.1, 1ng/ml), only IL1β significantly increase the MMP3 protein secretion (ELISA, n=4, p<0.05) and protein expression (western blot, n=3, p<0.05) but there was no difference in the mRNA expression (qPCR, n=3, p>0.05). The conditioned medium (CM) collected from myometrial cells treated with cytokines were then use to culture with eMSCs at different ratios of CM to growth medium (1:10, 1:20, 1:30). EMSCs cultured with CM from myometrial cells treated with TNF-α to GM at 1:20 showed significant increase in phenotypic expression to 1.4 ± 0.25 fold (flow, n=6, p<0.01). In conclusion, our findings indicate that myometrial cells can maintain eMSCs and is likely to be mediated by MMP3. During inflammatory events, cytokines such as IL-1β or TNF-α may regulate MMP3 in the myometrial cells to enable eMSCs for the regeneration of the human endometrium.