Telocytes (TCs) have been described in various organs and species (www.telocytes.com) as cells with telopodes (Tps) -very long cellular extensions with an alternation of thin segments (podomers) and dilated portions (podoms). We examined TCs using electron microscopy (EM), immunohistochemistry (IHC), immunofluorescence (IF), time-lapse videomicroscopy and whole-cell patch voltage clamp. EM showed a three-dimensional network of dichotomous-branching Tps, a labyrinthine system with homocellular and heterocellular junctions. Tps release extracellular vesicles (mean diameter of 160.6 +/- 6.9 nm in non-pregnant myometrium and 171.6 +/- 4.6 nm in pregnant myometrium), sending macromolecular signals to neighbouring cells. Comparative measurements (non-pregnant and pregnant myometrium) of podomer thickness revealed values of 81.94 +/- 1.77 vs 75.53 +/- 1.81 nm, while the podoms'diameters were 268.6 +/- 8.27 vs 316.38 +/- 17.56 nm. IHC as well as IF revealed double c-kit and CD34 positive results. Time-lapse videomicroscopy of cell culture showed dynamic interactions between Tps and myocytes. In non-pregnant myometrium, patch-clamp recordings of TCs revealed a hyperpolarisation-activated chloride inward current with calcium dependence and the absence of L-type calcium channels. TCs seem to have no excitable properties similar to the surrounding smooth muscle cells (SMCs). In conclusion, this study shows the presence of TCs as a distinct cell type in human non-pregnant and pregnant myometrium and describes morphometric differences between the two physiological states. In addition, we provide a preliminary in vitro electrophysiological evaluation of the non-pregnant state, suggesting that TCs could influence timing of the contractile activity of SMCs.
Telocytes ( TC s), a novel type of interstitial cells, were recently described in the interstitial space of tissues (www.telocytes.com). Telocytes TC s have several very long, moniliform extensions, namely telopodes (Tps). However, the functional role(s) of TC s is not yet understood. Successive photomicrographs of ultrathin sections were concatenated to capture the entire length of Tps which usually measure tens to hundreds of micrometres. Besides the podoms (dilations) and podomers (thin segments), ultrastructural features of Tps include the dichotomous branching and establishing homo‐ and heterocellular contacts. Telopodes make a labyrinthine system by 3D convolution and overlapping, their number being roughly estimated at approximately 20 per 1000 μm 2 . Moreover, the presence of extracellular vesicles (shedding vesicles/exosomes) along the Tps suggests an active intercellular signalling (micro‐ and macromolecules), with possible significance in regulating uterine contractility.
Expression and function of the follicle-stimulating hormone receptor (FSHR) in females were long thought to be limited to the ovary. Here, however, we identify extragonadal FSHR in both the human female reproductive tract and the placenta, and test its physiological relevance in mice. We show that in nonpregnant women FSHR is present on: endothelial cells of blood vessels in the endometrium, myometrium, and cervix; endometrial glands of the proliferative and secretory endometrium; cervical glands and the cervical stroma; and (at low levels) stromal cells and muscle fibers of the myometrium. In pregnant women, placental FSHR was detected as early as 8-10 wk of gestation and continued through term. It was expressed on: endothelial cells in fetal portions of the placenta and the umbilical cord; epithelial cells of the amnion; decidualized cells surrounding the maternal arteries in the maternal decidua; and the stromal cells and muscle fibers of the myometrium, with particularly strong expression at term. These findings suggest that FSHR expression is upregulated during decidualization and upregulated in myometrium as a function of pregnancy. The presence of FSHR in the placental vasculature suggests a role in placental angiogenesis. Analysis of genetically modified mice in which Fshr is lacking in fetal portions of the placenta revealed adverse effects on fetoplacental development. Our data further demonstrate FSHB and CGA mRNAs in placenta and uterus, consistent with potential local sources of FSH. Collectively, our data suggest heretofore unappreciated roles of extragonadal FSHR in female reproductive physiology.
Over the course of pregnancy, the human uterus undergoes a 500- to 1,000-fold increase in volume and a 24-fold increase in weight. The uterine smooth muscle layer or myometrium is remodeled, and both cell hypertrophy and hyperplasia are evident. The origin of the new smooth muscle cells, however, is unclear. They may arise from existing smooth muscle cells, or they may be the product of stem cell differentiation. This study describes a subset of myometrial cells isolated from nonpregnant human myometrium that represents the myometrial stem cell population. This was characterized as side population of myometrial cells (myoSP) by a distinct Hoechst dye efflux pattern. In contrast to the main population of myometrial cells (myoMP), myoSP resided in quiescence, underexpressed or lacked myometrial cell markers, and could proliferate and eventually differentiate into mature myometrial cells in vitro only under low oxygen concentration. Although myoMP displayed mature myometrial phenotypes before and after in vitro cultivation, only myoSP, not myoMP, generated functional human myometrial tissues efficiently when transplanted into the uteri of severely immunodeficient mice. Finally, myoSP were multipotent and made to differentiate into osteocytes and adipocytes in vitro under the appropriate differentiation-inducing conditions. Thus, myoSP exhibited phenotypic and functional characteristics of myometrial stem cells. Study of myoSP will improve the understanding of myometrial physiology and the pathogenesis of myometrium-derived diseases such as leiomyoma. myoSP may also represent a novel source of biological material that could be used in the reconstruction of not only the human uterus but also other organs as well.
Tissue-specific (or somatic) stem cells constitute a subset of cells residing in normal adult tissues. By undergoing asymmetric division, they retain their ability to self-renew while producing daughter cells that go on to differentiate and play a role in tissue regeneration and repair. The human uterus consists primarily of endometrium and myometrium (the smooth muscle layer) that rapidly enlarges through its tremendous regenerative and remodeling capacity to accommodate the developing fetus. Such uterine enlargement and remodeling can take place repeatedly and cyclically over the course of a woman's reproductive life. These unique properties of the uterus suggest the existence of endometrial and myometrial stem cell systems. In addition, like somatic cells, tumor stem cells or tumor-initiating cells, a subset of cells within a tumor, retain the ability to reconstitute tumors. Uterine smooth muscle cells are thought to be the origin of leiomyomas that are the most common type of gynecologic tumor. Recent work has identified, isolated, and characterized putative stem/progenitor cells in the myometrium and in leiomyomas. Here, we review current studies of myometrial and leiomyoma stem/progenitor cells and provide a new paradigm for understanding myometrial physiology and pathology and how these cells might contribute to uterine remodeling during pregnancy and the formation of leiomyomas. The role of the WNT/CTNNB1 pathway in the pathogenesis of leiomyoma is also discussed.
Small‐conductance calcium‐activated potassium ( SK 3) channels have been detected in human myometrium and we have previously shown a functional role of SK channels in human myometrium in vitro . The aims of this study were to identify the precise localization of SK 3 channels and to quantify SK 3 m RNA expression in myometrium from pregnant and non‐pregnant women. Myometrial biopsies were obtained from pregnant ( n = 11) and non‐pregnant ( n = 11) women. The expression of SK 3 channels was assessed using immunohistochemistry and SK 3 m RNA was determined by q RT ‐ PCR . In non‐pregnant myometrium SK 3 immunoreactivity was observed in CD 34 positive ( CD 34 + ) interstitial Cajal‐like cells ( ICLC ), now called telocytes. Although CD 34 + cells were also present in pregnant myometrium, they lacked SK 3 immunoreactivity. Furthermore, the immunohistochemical results showed that SK 3 expression in vascular endothelium was similar between the two groups. CD 117 immunoreactivity was only detected in small round cells that resemble mast cells. Compared to non‐pregnant myometrium we found significantly less SK 3 m RNA in pregnant myometrium. We demonstrate that SK 3 channels are localized solely in CD 34 + cells and not in smooth muscle cells, and that the molecular expression of SK 3 channels is higher in non‐pregnant compared to pregnant myometrium. On the basis of our previous study and the present findings, we propose that SK 3 activators reduce contractility in human myometrium by modulating telocyte function. This is the first report to provide evidence for a possible role of SK 3 channels in human uterine telocytes.
Background: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Each leiomyoma is thought to be a benign monoclonal tumor arising from a single transformed myometrial smooth muscle cell; however, it is not known what leiomyoma cell type is responsible for tumor growth. Thus, we tested the hypothesis that a distinct stem/reservoir cell-enriched population, designated as the leiomyoma-derived side population (LMSP), is responsible for cell proliferation and tumor growth. Principal Findings: LMSP comprised approximately 1% of all leiomyoma and 2% of all myometrium-derived cells. All LMSP and leiomyoma-derived main population (LMMP) but none of the side or main population cells isolated from adjacent myometrium carried a mediator complex subunit 12 mutation, a genetic marker of neoplastic transformation. Messenger RNA levels for estrogen receptor-alpha, progesterone receptor and smooth muscle cell markers were barely detectable and significantly lower in the LMSP compared with the LMMP. LMSP alone did not attach or survive in monolayer culture in the presence or absence of estradiol and progestin, whereas LMMP readily grew under these conditions. LMSP did attach and survive when directly mixed with unsorted myometrial cells in monolayer culture. After resorting and reculturing, LMSP gained full potential of proliferation. Intriguingly, xenografts comprised of LMSP and unsorted myometrial smooth muscle cells grew into relatively large tumors (3.67 +/- 1.07 mm(3)), whereas xenografts comprised of LMMP and unsorted myometrial smooth muscle cells produced smaller tumors (0.54 +/- 0.20 mm(3), p<0.05, n = 10 paired patient samples). LMSP xenografts displayed significantly higher proliferative activity compared with LMMP xenografts (p<0.05). Conclusions: Our data suggest that LMSP, which have stem/reservoir cell characteristics, are necessary for in vivo growth of leiomyoma xenograft tumors. Lower estrogen and progesterone receptor levels in LMSP suggests an indirect paracrine effect of steroid hormones on stem cells via the mature neighboring cells.
Inflammatory mediators in the cervix, placenta and fetal membranes play a crucial role in human parturition. The aim of this study was to determine whether the upper and lower segments of the myometrium are infiltrated by inflammatory cells during pregnancy and parturition. Myometrial biopsies were obtained from non-pregnant women, and pregnant women at term before and after the onset of spontaneous labour. Subpopulations of inflammatory cells were identified using immunocytochemistry. The intercellular adhesion molecules, 1 and 2, platelet endothelial cell adhesion molecule, vascular cell adhesion molecule and E-selectin were immunolocalized to investigate their involvement in leukocyte accumulation. Histological analysis demonstrated that inflammatory cells, predominantly neutrophils and macrophages, infiltrate human myometrium during spontaneous labour at term. The infiltrate is predominant in the lower uterine segment but is also present in the upper segment. Increased expression of E-selectin was found on the vascular endothelium of biopsies obtained during labour, suggesting a role for this molecule in the accumulation of leukocytes. These results suggest that inflammatory cell infiltration is part of the physiological mechanisms that occur in the myometrium during parturition. Further understanding of this process may suggest new strategies aimed at preventing preterm delivery.
Previous reports describing Cajal‐like interstitial cells in human uterus are contradictory in terms of c‐kit immunoreactivity: either negative (but vimentin‐positive) in pregnant myometrium, or positive, presumably in the endometrium. The aim of this study was to verify the existence of human m yometrial C ajal‐ l ike i nterstitial c ells (m‐CLIC). Six different, complementary approaches were used: 1) methylene‐blue supravital staining of tissue samples (cryosections), 2) methylene blue and Janus green B vital staining (m‐CLIC and mitochondrial markers, respectively), and 3) extracellular single‐unit electrophysiological recordings in cell cultures , 4) non‐conventional light microscopy on glutaraldehyde/osmium fixed, Epon‐embedded semi‐thin sections (less than 1μm) stained with toluidine blue (TSM), 5) transmission electron microscopy (TEM), and 6) immunofluorescence (IF). We found m‐CLIC in myometrial cryosections and in cell cultures. In vitro , m‐CLIC represented ∼7% of the total cell number. m‐CLIC had 2–3 characteristic processes which were very long (∼ 60 μm), very thin (±0.5μm) and moniliform. The dilated portions of processes usually accomodated mitochondria. In vitro , m‐CLIC exhibited spontaneous electrical activity (62.4 ± 7.22 mV field potentials, short duration: 1.197 ± 0.04ms). Moreover, m‐CLIC fulfilled the usual TEM criteria, the so‐called ‘gold’ or ‘platinum’ standards ( e.g. the presence of discontinuos basal lamina, caveolae, endoplasmic reticulum, and close contacts between each other, with myocytes, nerve fibers and/or capillaries etc.). IF showed that m‐CLIC express CD117/c‐kit, sometimes associated with CD34 and with vimentin along their processes. In conclusion, we describe myometrial Cajal‐like interstitial cells that have affinity for methylene blue and Janus green B vital dyes, fulfill (all) TEM criteria, express CD117/c‐kit and have spontaneous electric activity.
Objective Secretory leukocyte protease inhibitor (SLPI) has been shown to have antimicrobial and antiinflammatory properties. The aim of this study was to verify its expression in human myometrium. Study Design Myometrium was obtained at time of cesarean delivery with (n = 9) or without (n = 11) labor. Expression of SLPI was detected using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. SLPI expression relative to nuclear factor-κB p65 subunit was compared between subjects. SLPI response to inflammatory mediators was studied in myometrial explants. Results SLPI was predominantly localized in the nuclei of myocytes and colocalized with CD68+ macrophages. The nuclear immunoreactivity of SLPI was increased after the onset of labor and was associated with increased nuclear translocation of nuclear factor-κB p65 subunit. Treatment with lipopolysaccharide, interleukin-1β, or tumor necrosis factor-α increased SLPI messenger RNA and protein concentrations slightly in myometrium explants. Conclusion SLPI was expressed in human myometrium and increased after the onset of labor.