Arrangement into layers and mechanobiology of multi-cell co-culture models of the uterine wall.

STUDY QUESTION: Can a co-culture of three cell types mimic the in vivo layers of the uterine wall? SUMMARY ANSWER: Three protocols tested for co-culture of endometrial epithelial cells (EEC), endometrial stromal cells (ESC), and myometrial smooth muscle cells (MSMC) led to formation of the distinct layers that are characteristic of the structure of the uterine wall in vivo. WHAT IS KNOWN ALREADY: We previously showed that a layer-by-layer co-culture of EEC and MSMC responded to peristaltic wall shear stresses (WSS) by increasing the polymerization of F-actin in both layers. Other studies showed that WSS induced significant cellular alterations in epithelial and endothelial cells. STUDY DESIGN, SIZE, DURATION: Human EEC and ESC cell lines and primary MSMC were co-cultured on a collagen-coated synthetic membrane in custom-designed wells. The co-culture model, created by seeding a mixture of all cells at once, was exposed to steady WSS of 0.5 dyne/cm2 for 10 and 30 min. PARTICIPANTS/MATERIALS, SETTING, METHODS: The co-culture of the three different cells was seeded either layer-by-layer or as a mixture of all cells at once. Validation of the models was by specific immunofluorescence staining and confocal microscopy. Alterations of the cytoskeletal F-actin in response to WSS were analyzed from the 2-dimensional confocal images through the Z-stacks following a previously published algorithm. MAIN RESULTS AND THE ROLE OF CHANCE: We generated three multi-cell in vitro models of the uterine wall with distinct layers of EEC, ESC, and MSMC that mimic the in vivo morphology. Exposure of the mixed seeding model to WSS induced increased polymerization of F-actin in all the three layers relative to the unexposed controls. Moreover, the increased polymerization of F-actin was higher (P-value < 0.05) when the length of exposure was increased from 10 to 30 min. Furthermore, the inner layers of ESC and MSMC, which are not in direct contact with the applied shearing fluid, also increased their F-actin polymerization. LARGE SCALE DATA: N/A. LIMITATIONS, RESONS FOR CAUTION: The mixed seeding co-culture model was exposed to steady WSS of one magnitude, whereas the uterus is a dynamic organ with intra-uterine peristaltic fluid motions that vary in vivo with different time-dependent magnitude. Further in vitro studies may explore the response to peristaltic WSS or other physical and/or hormonal perturbations that may mimic the spectrum of pathophysiological aspects. WIDER IMPLICATIONS OF THE FINDINGS: Numerous in vitro models were developed in order to mimic the human endometrium and endometrium-myometrium interface (EMI) region. The present co-culture models seem to be the first constructed from EEC, ESC, and MSMC on a collagen-coated synthetic membrane. These multi-cell in vitro models better represent the complex in vivo anatomy of the EMI region. The mixed seeding multi-cell in vitro model may easily be implemented in controlled studies of uterine function in reproduction and the pathogenesis of diseases. STUDY FINDING/COMPETING INTEREST(S): This study was supported in part by Tel Aviv University funds. All authors declare no conflict of interest.

Transmembrane Mucin Response in Conjunctival Epithelial Cells Exposed to Wall Shear Stresses.

Human conjunctival epithelium cells (HCEC) line the inner surface of the eyelid and cover the sclera and are continuously subjected to wall shear stresses (WSS). The effects of external forces on the conjunctival epithelium are not fully known. The conjunctival epithelium contains stratified squamous cells that synthesize the membrane-spanning mucins MUC1 and MUC16, which play important roles in protecting the ocular surface. Alterations in both gel-forming and membrane-tethered mucins occur in drying ocular surface diseases. The aim of this study was to explore the mechanobiological characteristics of transmembrane mucin secretion and cellular alterations of primary HCEC exposed to airflow-induced WSS perturbations. We exposed the HCEC to a steady WSS of 0.5 dyne/cm2 for durations of 15 and 30 min. Cytoskeletal alterations and MUC1 secretions were studied using immunohistochemically fluorescent staining with specific antibodies. We investigated for the first time an in vitro model of membrane-tethered mucin secretion by HCEC in response to WSS. The exposure of HCEC to WSS increased the polymerization of F-actin, altered the cytoskeletal shape and reduced the secretion of membrane-tethered MUC1.

Controlled amnioreduction for twin-to-twin transfusion syndrome.

Background: Twin-to-twin transfusion syndrome (TTTS) is a severe condition causing preterm delivery, fetal death, and neurodevelopmental disorders. This study presents a data-based controlled amnioreduction (AR) protocol composed of sequential amniodrainage in treatment of TTTS. Methods: A total of 18 procedures were performed in 11 TTTS pregnancies at 17 to 34 weeks of gestation. The amniotic pressure was measured along with sequential removal of the amniotic fluid, 500 mL each step. The umbilical artery systolic/diastolic (S/D) ratio for each twin was measured pre- and post-AR. Long-term neurodevelopmental outcomes of all TTTS survivors were evaluated from parental answers to a phone survey. Results: The amniotic pressure decreased exponentially with the increased volume of removed amniotic fluid until a plateau was obtained. Changes of the S/D ratio between pre- and post-AR procedure did not reveal a clear tendency. The survival rate was 86.4% although 91% of all twins were at Quintero stage III. Long-term neurodevelopment outcomes in the 19 surviving twins were 68.4% optimal, 26.3% suboptimal, and 5.3% abnormal. Conclusion: The controlled AR procedure resulted in a relatively high rate of twin survival with favorable long-term neurodevelopment outcomes.

In Utero Programming of Testicular Cancer.

It is well established that the intrauterine biological environment plays important roles in fetal development. In this review, we re-visit the hypothesis that testicular germ cell cancer (TGCC), especially in adolescents and young adults, has been programmed in utero. The origin for extreme in utero environments is mostly maternal driven and may be due to nutritional, physical and psychological stressful conditions that alter the optimal molecular and biophysical in utero environments. Moreover, precursors for TGCC may originate as early as during fertilization or implantation of the blastocyst. Further investigations of human developmental biology, both in vivo and in vitro, are needed in order to establish better understanding of in utero programming of future wellbeing or diseases.

Mechanobiology of conjunctival epithelial cells exposed to wall shear stresses.

The human conjunctival epithelial cells (HCEC) line the inner sides of the eyelids and the anterior part of the sclera. They include goblet cells that secret mucus into the tear film that protects the ocular surface. The conjunctival epithelium is subjected to mechano-physical stimuli due to eyelid movement during blinking, during wiping and rubbing the eyes, and when exposed to wind and air currents. We cultured primary HCEC under air-liquid interface (ALI) conditions in custom-designed wells that can be disassembled for installation of the in vitro model in a flow chamber. We exposed the HCEC after ALI culture of 8-10 days to steady and oscillatory airflows. The in vitro model of HCEC was exposed to steady wall shear stresses (sWSS) of 0.5 and 1.0 dyne/cm2 for lengths of 30 and 60 min and to oscillatory wall shear stresses (oWSS) of 0.5 and 0.77 dyne/cm2 amplitudes for a length of 10 min. Cytoskeletal alterations and MUC5AC mucin secretion in response to WSS were investigated using immunohistochemically fluorescent staining and enzyme-linked lectin assay (ELLA), respectively. The results revealed that both exposure times and sWSS values increased the polymerization of F-actin filaments while mucin secretion decreased. However, after a recovery of 24 h in the incubator we observed a decrease of F-actin fibers and mucin secretion only for exposure of 30 min. The length of exposure was more influential on cytoskeletal alterations than the level of sWSS. The very small effect of sWSS on mucin secretion is most likely related to the much smaller amount of goblet cell than in other mucus-secreting tissue. The results for both oWSS amplitudes revealed similar trends regarding F-actin and mucin secretion. Immediately post-exposure we observed an increase in polymerization of F-actin filaments while mucin secretion decreased. However, after 24-h recovery we observed that both F-actin and mucin secretion returned to the same values as for unexposed cultures. The results of this study suggest that WSS should be considered while exploring the physiological characteristics of HCEC.

Quantitative imaging of tongue kinematics during infant feeding and adult swallowing reveals highly conserved patterns.

Tongue motility is an essential physiological component of human feeding from infancy through adulthood. At present, it is a challenge to distinguish among the many pathologies of swallowing due to the absence of quantitative tools. We objectively quantified tongue kinematics from ultrasound imaging during infant and adult feeding. The functional advantage of this method is presented in several subjects with swallowing difficulties. We demonstrated for the first time the differences in tongue kinematics during breast- and bottle-feeding, showing the arrhythmic sucking pattern during bottle-feeding as compared with breastfeeding in the same infant with torticollis. The method clearly displayed the improvement of tongue motility after frenotomy in infants with either tongue-tie or restrictive labial frenulum. The analysis also revealed the absence of posterior tongue peristalsis required for safe swallowing in an infant with dysphagia. We also analyzed for the first time the tongue kinematics in an adult during water bolus swallowing demonstrating tongue peristaltic-like movements in both anterior and posterior segments. First, the anterior segment undulates to close off the oral cavity and the posterior segment held the bolus, and then, the posterior tongue propelled the bolus to the pharynx. The present methodology of quantitative imaging revealed highly conserved patterns of tongue kinematics that can differentiate between swallowing pathologies and evaluate treatment interventions. The method is novel and objective and has the potential to advance knowledge about the normal swallowing and management of feeding disorders.

Tissue-engineered arterial intima model exposed to steady wall shear stresses.

The arterial intima is continuously under pulsatile wall shear stresses (WSS) imposed by the circulating blood. The knowledge of the contribution of smooth muscle cells (SMC) to the response of endothelial cell (EC) to WSS is still incomplete. We developed a co-culture model of EC on top of SMC that mimics the inner in vivo structure of the arterial intima of large arteries. The co-cultured model, as well as a monolayer model of EC, were developed in custom-designed wells that allowed for mechanobiology experiments. Both the monolayer and co-culture models were exposed to steady flow induced WSS of up to 24 dyne/cm2 and for lengths of 60 min. Quantification of WSS induced alterations in the cytoskeletal actin filaments (F-actin) and vascular endothelial cadherin (VE-cadherin) junctions were utilized from confocal images and flow cytometry. High confluency of both models was observed even after exposure to the high WSS. The quantitive analysis revealed larger post WSS amounts of EC F-actin polymerization in the monolayer, which may be explained by the relative help of the SMC to resist the external load of WSS. The VE-cadherin demonstrated morphological alterations in the monolayer model, but without significant changes in their content. The SMC in the co-culture maintained their contractile phenotype post high WSS which is more physiological, but not post low WSS. Generally, the results of this work demonstrate the active role of SMC in the intima performance to resist flow induced WSS.

Matrix stiffness and shear stresses modulate hepatocyte functions in a fibrotic liver sinusoidal model.

Extracellular matrix (ECM) rigidity has important effects on cell behaviors and increases sharply in liver fibrosis and cirrhosis. Hepatic blood flow is essential in maintaining hepatocytes' (HCs) functions. However, it is still unclear how matrix stiffness and shear stresses orchestrate HC phenotype in concert. A fibrotic three-dimensional (3-D) liver sinusoidal model is constructed using a porous membrane sandwiched between two polydimethylsiloxane (PDMS) layers with respective flow channels. The HCs are cultured in collagen gels of various stiffnesses in the lower channel, whereas the upper channel is pre-seeded with liver sinusoidal endothelial cells (LSECs) and accessible to shear flow. The results reveal that HCs cultured within stiffer matrices exhibit reduced albumin production and cytochrome P450 (CYP450) reductase expression. Low shear stresses enhance synthetic and metabolic functions of HC, whereas high shear stresses lead to the loss of HC phenotype. Furthermore, both mechanical factors regulate HC functions by complementing each other. These observations are likely attributed to mechanically induced mass transport or key signaling molecule of hepatocyte nuclear factor 4α (HNF4α). The present study results provide an insight into understanding the mechanisms of HC dysfunction in liver fibrosis and cirrhosis, especially from the viewpoint of matrix stiffness and blood flow.NEW & NOTEWORTHY A fibrotic three-dimensional (3-D) liver sinusoidal model was constructed to mimic different stages of liver fibrosis in vivo and to explore the cooperative effects of matrix stiffness and shear stresses on hepatocyte (HC) functions. Mechanically induced alterations of mass transport mainly contributed to HC functions via typical mechanosensitive signaling.

Tissue engineered endometrial barrier exposed to peristaltic flow shear stresses.

Cyclic myometrial contractions of the non-pregnant uterus induce intra-uterine peristaltic flows, which have important roles in transport of sperm and embryos during early stages of reproduction. Hyperperistalsis in young females may lead to migration of endometrial cells and development of adenomyosis or endometriosis. We conducted an in vitro study of the biological response of a tissue engineered endometrial barrier exposed to peristaltic wall shear stresses (PWSSs). The endometrial barrier model was co-cultured of endometrial epithelial cells on top of myometrial smooth muscle cells (MSMCs) in custom-designed wells that can be disassembled for mechanobiology experiments. A new experimental setup was developed for exposing the uterine wall in vitro model to PWSSs that mimic the in vivo intra-uterine environment. Peristaltic flow was induced by moving a belt with bulges to deform the elastic cover of a fluid filled chamber that held the uterine wall model at the bottom. The in vitro biological model was exposed to peristaltic flows for 60 and 120 min and then stained for immunofluorescence studies of alternations in the cytoskeleton. Quantification of the F-actin mass in both layers revealed a significant increase with the length of exposure to PWSSs. Moreover, the inner layer of MSMCs that were not in direct contact with the fluid also responded with an increase in the F-actin mass. This new experimental approach can be expanded to in vitro studies of multiple structural changes and genetic expressions, while the tissue engineered uterine wall models are tested under conditions that mimic the in vivo physiological environment.

Biomechanics of Early Life in the Female Reproductive Tract.

Early human life that starts at the onset of fertilization and ends with implantation of the embryo in the uterine wall is the foundation for a successful pregnancy. The different stages during this period require biomechanical mechanisms, which are mostly unknown due to difficulties to conduct in vivo studies in humans.

Tissue-engineered multi-cellular models of the uterine wall.

The human uterus is composed of three layers: endometrium, myometrium and perimetrium. It remodels during the monthly menstrual cycle and more significantly during the complex stages of reproduction. In vivo studies of the human uterine wall are yet incomplete due to ethical and technical limitations. The objective of this study was to develop in vitro uterine wall models that mimic the in vivo structure in humans. We co-cultured multiple cellular models of endometrial epithelial cells, endometrial stromal cells and smooth muscle cells on a synthetic membrane mounted in multi-purpose custom-designed wells. Immunofluorescence staining and confocal imaging confirmed that the new model represents the in vivo anatomical architecture of the inner uterine wall. Hormonal treatment with progesterone and ß-estradiol demonstrated increased expression of progestogen-associated endometrial protein, which is associated with the in vivo receptive uterus. The new tissue-engineered in vitro models of the uterine wall will enable deeper investigation of molecular and biomechanical aspects of the blastocyst-uterus interaction during the window of implantation.

Analysis of in vivo uterine peristalsis in the non-pregnant female mouse.

Uterine peristalsis due to spontaneous contractions of the myometrial smooth muscles has important roles in pre-implantation processes of intra-uterine sperm transport to the fertilization site, and then embryo transport to the implantation sites. We developed a new objective methodology to study in vivo uterine peristalsis in female mice during the pro-oestrus phase. The acquisition procedure of the uterine organ is remote without interfering with the organ function. The uniqueness of the new approach is that video images of physiological pattern were converted using image processing and new algorithms to biological time-dependent signals that can be processed with existing algorithms for signal processing. Using this methodology we found that uterine peristalsis in the pro-oestrus mouse is in the range of 0.008-0.029 Hz, which is about one contraction per minute and with fairly symmetric contractions that occasionally propagate caudally. This rate of contractions is similar to that of human uterine peristalsis acquired in vivo, which is important information for a popular animal model.

In vitro exposure of nasal epithelial cells to atmospheric dust.

Dust storms are common phenomena in many parts of the world, and significantly increase the level of atmospheric particulate matter (PM). The soil-derived dust is a mixture of organic and inorganic particles and even remnants of pesticides from agricultural areas nearby. The risk of human exposure to atmospheric dust is well documented, but very little is known on the impact of inhaled PM on the biological lining of the nasal cavity, which is the natural filter between the external environment and the respiratory tract. We developed a new system and methodology for in vitro exposure of cultured nasal epithelial cells (NEC) to atmospheric soil-dust pollutants under realistic and controlled laboratory simulations that mimic nasal breathing. We exposed cultured NEC to clean and dust-polluted airflows that mimic physiological conditions. The results revealed that the secretion of mucin and IL-8 from the NEC exposed to clean and dust-polluted airflows was less than the secretion at static conditions under clean air. The secretion of IL-8 from NEC exposed to dust-polluted air was larger than that of clean air, but not larger than in the static case. The experiments with dust air pollution that also contained agricultural pesticides did not reveal differences in the secretion of mucin and IL-8 as compared to the same pollution without pesticides.

Objective Analysis of Vaginal Ultrasound Video Clips for Exploring Uterine Peristalsis Post Vaginal and Cesarean Section Deliveries.

The nonpregnant uterus is characterized by cyclic contractions that assist in sperm transport to the fallopian tube, embryo transport to implantation site, and expulsion of menstrual debris. The effect of post-Cesarean section (CS) scar on uterine peristalsis is unclear, while worldwide the prevalence of CS deliveries is increasing. In this study, we developed a new objective method for analysis of dynamic characteristics of the nonpregnant uterus from transvaginal ultrasound (TVUS) recordings when the uterine cavity is not clearly observed, as may be the case in post-CS uteri. The method of active contours was utilized to detect the contours of the endometrium-myometrium interface (EMI) from sagittal cross-section TVUS images of nonpregnant uteri. The contours were straightened along the uterus centerline and registered with respect to the fundal end in order to reduce the noise due to movements of the physician and the participant. A dynamic analysis was conducted on these time-dependent contours in order to explore the frequency and amplitude of the EMI motility. The analysis was conducted on TVUS video clips from 12 nonpregnant participants, 7 post-CS and 5 controls. The frequencies of the EMI motility was 0.010 to 0.064 Hz at days 8 to 17 in the control participants and 0.014 to 0.073 Hz at days 9 to 15 in post-CS participants. The maximal amplitude of motility was 0.67 to 2.00 mm and 0.48 to 2.58 mm for the control and post-CS participants, respectively. In this preliminary study, we have not observed significant difference between the EMI motility of healthy and post-CS uteri.

IFPA meeting 2017 workshop report: Clinical placentology, 3D structure-based modeling of placental function, placental bed, and treating placental dysfunction.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2017 there were four themed workshops, all of which are summarized in this report. These workshops discussed new knowledge and technological innovations in the following areas of research: 1) placental bed; 2) 3D structural modeling; 3) clinical placentology; 4) treatment of placental dysfunction.

Biomechanics of the human uterus.

The appropriate biomechanical function of the uterus is required for the execution of human reproduction. These functions range from aiding the transport of the embryo to the implantation site, to remodeling its tissue walls to host the placenta, to protecting the fetus during gestation, to contracting forcefully for a safe parturition and postpartum, to remodeling back to its nonpregnant condition to renew the cycle of menstruation. To serve these remarkably diverse functions, the uterus is optimally geared with evolving and contractile muscle and tissue layers that are cued by chemical, hormonal, electrical, and mechanical signals. The relationship between these highly active biological signaling mechanisms and uterine biomechanical function is not completely understood for normal reproductive processes and pathological conditions such as adenomyosis, endometriosis, infertility and preterm labor. Animal studies have illuminated the rich structural function of the uterus, particularly in pregnancy. In humans, medical imaging techniques in ultrasound and magnetic resonance have been combined with computational engineering techniques to characterize the uterus in vivo, and advanced experimental techniques have explored uterine function using ex vivo tissue samples. The collective evidence presented in this review gives an overall perspective on uterine biomechanics related to both its nonpregnant and pregnant function, highlighting open research topics in the field. Additionally, uterine disease and infertility are discussed in the context of tissue injury and repair processes and the role of computational modeling in uncovering etiologies of disease. WIREs Syst Biol Med 2017, 9:e1388. doi: 10.1002/wsbm.1388 For further resources related to this article, please visit the WIREs website.

Quercetin alters the DNA damage response in human hematopoietic stem and progenitor cells via TopoII- and PI3K-dependent mechanisms synergizing in leukemogenic rearrangements.

Quercetin (Que) is an abundant flavonoid in the human diet and high-concentration food supplement with reported pro- and anti-carcinogenic activities. Topoisomerase II (TopoII) inhibition and subsequent DNA damage induction by Que was implicated in the mixed lineage leukemia gene (MLL) rearrangements that can induce infant and adult leukemias. This notion raised concerns regarding possible genotoxicities of Que in hematopoietic stem and progenitor cells (HSPCs). However, molecular targets mediating Que effects on DNA repair relevant to MLL translocations have not been defined. In this study we describe novel and potentially genotoxic Que activities in suppressing non-homologous end joining and homologous recombination pathways downstream of MLL cleavage. Using pharmacological dissection of DNA-PK, ATM and PI3K signalling we defined PI3K inhibition by Que with a concomitant decrease in the abundance of key DNA repair genes to be responsible for DNA repair inhibition. Evidence for the downstream TopoII-independent mutagenic potential of Que was obtained by documenting further increased frequencies of MLL rearrangements in human HSPCs concomitantly treated with Etoposide and Que versus single treatments. Importantly, by engaging a tissue engineered placental barrier, we have established the extent of Que transplacental transfer and hence provided the evidence for Que reaching fetal HSPCs. Thus, Que exhibits genotoxic effects in human HSPCs via different mechanisms when applied continuously and at high concentrations. In light of the demonstrated Que transfer to the fetal compartment our findings are key to understanding the mechanisms underlying infant leukemia and provide molecular markers for the development of safety values.

The Driving Mechanism for Unidirectional Blood Flow in the Tubular Embryonic Heart.

The embryonic heart of vertebrate embryos, including humans, has a tubular thick-wall structure when it first starts to beat. The tubular embryonic heart (TEH) does not have valves, and yet, it produces an effective unidirectional blood flow. The actual pumping mechanism of the TEH is still controversial with pros and cons for either peristaltic pumping (PP) or impedance pumping (IP). On the other hand, observation of movies of the contractile TEH of the quail revealed a propagating wave from the venous end towards the arterial end that occludes the lumen behind the leading edge. This pattern of contraction represents a complex PP with a duty cycle, and was defined here as biological pumping (BP). In this work we developed a heart-like model that represents the main features of the chick TEH and allows for numerical analysis of all the three pumping mechanisms (i.e., IP, PP, and BP) as well as a comprehensive sensitivity evaluation of the structural, operating, and mechanical parameters. The physical model also included components representing the whole circulatory system of the TEH. The simulations results revealed that the BP mechanism yielded the level and time-dependent pattern of blood flow and blood pressure, as well as contractility that were observed in experiments.

Ex Vivo Human Placental Perfusion Model for Analysis of Fetal Circulation in the Chorionic Plate.

OBJECTIVES: The purpose of this study was to develop an ex vivo placental perfusion model to assess changes in the umbilical artery systolic-to-diastolic (S/D) ratio due to progressive occlusion of the placental arterial system. METHODS: Ex vivo human placentas were connected to a computerized pulse duplicator mimicking pulsatile flow from the fetal heart. Doppler sonographic measurements were conducted on the umbilical and chorionic arteries of 25 mature placentas. Simulation of placental occlusion was performed by progressive ligature of the chorionic arteries, including one umbilical artery. The correlation between the umbilical artery S/D ratio and the severity of simulated placental occlusion was analyzed. RESULTS: The normal mean S/D ratio ± SD decreased gradually along the chorionic plate from 2.66 ± 0.47 at the cord insertion to 1.90 ± 0.59 in generation IV of the chorionic vessels. The Doppler index initially increased slowly with simulated placental occlusion. Only when all 4 generations were occluded was the umbilical artery S/D ratio elevated. Complete occlusion of one umbilical artery resulted in a 39% increase in the umbilical artery S/D ratio. CONCLUSIONS: This unique model combining Doppler sonography with perfusion of an ex vivo placenta can be used for a better understudying of pathologic placental blood flow circulation.