Development and validation of primary human myometrial cell culture models to study pregnancy and labour

Abstract

Background The development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal) segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated. Methods Myometrial cells isolated from paired upper segment (US) and lower segment (LS) biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; α smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2), oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL)-1β was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8), were measured. ANOVA followed by Bonferroni’s multiple comparisons test was performed. Results We demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10). Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1β, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1) through to p10. Conclusions Isolated primary myometrial cells maintain expression of smooth muscle and pregnancy-associated markers and retain their ability to respond to an inflammatory stimulus. These distinct myometrial cell models will provide a useful tool to investigate mechanisms underlying the process of human labour and the concept of functional regionalization of the pregnant uterus.

Methods and Model Systems Used to Study Pregnant Human Uterine Smooth Muscle

“…Furthermore, the comparison between primary and telomerase-immortalized cells revealed no significant differences in signaling pathways, such as epidermal growth factor (EGF)-stimulated phosphorylation of the EGF receptor, insulinstimulated Akt phosphorylation, oxytocin and lysophosphatidic acid-stimulated extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation, myosin light chain (MYL) phosphorylation, and interleukin-1 induction of IκBα degradation [25]. Although this study found no significant difference between immortalized and primary cell lines, it has been suggested that immortalization could cause fundamental changes in the cells [26], and as such many studies use primary cells only at low passages (passage five and lower) [27].…”

“…Mosher et al [26] established primary cultures of human myometrial cells isolated from paired upper and lower segment uterine biopsies for 10 passages and determined the expression of smooth muscle markers, fibroblast markers, contractile proteins or labor-associated proteins over time. It was found that both upper and lower segment human myometrial cells stably expressed smooth muscle markers (α-SMA, calponin, caldesmon, tropomyosin) and fibroblast markers (vimentin, 1B10) to at least 10 passages [26].…”

“…Mosher et al [26] established primary cultures of human myometrial cells isolated from paired upper and lower segment uterine biopsies for 10 passages and determined the expression of smooth muscle markers, fibroblast markers, contractile proteins or labor-associated proteins over time. It was found that both upper and lower segment human myometrial cells stably expressed smooth muscle markers (α-SMA, calponin, caldesmon, tropomyosin) and fibroblast markers (vimentin, 1B10) to at least 10 passages [26]. Interestingly, comparison of paired upper and lower segment myometrial cells revealed that mRNA levels for Connexin 43 (GJA1), Prostaglandin-endoperoxide synthase 2 (PTGS2) and vimentin (VIM) were significantly higher in lower segment cells compared to upper segment cells [26].…”

“…It was found that both upper and lower segment human myometrial cells stably expressed smooth muscle markers (α-SMA, calponin, caldesmon, tropomyosin) and fibroblast markers (vimentin, 1B10) to at least 10 passages [26]. Interestingly, comparison of paired upper and lower segment myometrial cells revealed that mRNA levels for Connexin 43 (GJA1), Prostaglandin-endoperoxide synthase 2 (PTGS2) and vimentin (VIM) were significantly higher in lower segment cells compared to upper segment cells [26]. These findings supported the concept of a functional regionalization of the upper and lower segment of the human uterus and indicated that both upper and lower segments should be examined in order to garner maximum insight into the mechanisms underlying human parturition.…”

“…These findings supported the concept of a functional regionalization of the upper and lower segment of the human uterus and indicated that both upper and lower segments should be examined in order to garner maximum insight into the mechanisms underlying human parturition. Furthermore, both cell populations retained their ability to respond to inflammatory stimuli from passage 1 through to passage 10, as demonstrated by increased expression of PTGS2 and release of the pro-inflammatory chemokine, CXCL8, following treatment with the interleukin-1β (IL-1β) [26]. Mosher et al [26] concluded that primary myometrial cells are viable and responsive for at least 10 passages, and therefore represent a useful tool for investigating human parturition.…”