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Anatomy of human placenta. A, Fetal side: the amnion, umbilical cord, and fetal vessels are visible. B, Maternal side: the cotyledons and their circulatory units are visible. Images by Chiara Mannelli.
Source publication
The identification of reproductive toxicants is a major scientific challenge for human health. Prenatal life is the most vulnerable and important time span of human development. For obvious ethical reasons, in vivo models cannot be used in human pregnancy, and animal models do not perfectly reflect human physiology. This review describes the in vit...
Citations
... Mouse models have been used for the study of endometrium, however, there are significant biologic differences between mouse and human endometrium. Namely, human endometrium is shed cyclically due to the hormonal milieu while mouse endometrium is only shed in settings of pregnancy or trauma (10)(11)(12). ...
Endometrial cancer is the most common gynecologic malignancy in the United States and is one of the few malignancies that had an increasing incidence and mortality rate over the last 10 years. Current research models fail to recapitulate actual characteristics of the tumor that are necessary for the proper understanding and treatment of this heterogenous disease. Patient-derived organoids provide a durable and versatile culture system that can capture patient-specific characteristics such as the mutational profile and response to therapy of the primary tumor. Here we describe the methods for establishing, expansion and banking of endometrial cancer organoids to develop a living biobank. Samples of both endometrial tumor tissue and matched normal endometrium were collected from 10 patients. The tissue was digested into single cells and then cultured in optimized media to establish matched patient endometrial cancer and normal endometrial tissue organoids. Organoids were created from all major endometrial cancer histologic subtypes. These organoids are passaged long term, banked and can be utilized for downstream histological and genomic characterization as well as functional assays such as assessing the response to therapeutic drugs.
... Notably, BeWo cells also show a spontaneous ability to form syncytial units mimicking the syncytiotrophoblast, whereas a marked self-renewal ability makes HTR-8/ SVneo cells a good model of cytotrophoblast cells (Weber et al. 2013). Although freshly isolated cytotrophoblast can differentiate into multinucleated syncytiotrophoblast, their inability to proliferate in vitro hampers experiments and prevents their use King et al. 2000;Kohli et al. 2016;Mannelli et al. 2015). ...
Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine that signals through a receptor complex containing a specific subunit, CNTF receptor α (CNTFRα). The two molecules are constitutively expressed in key structures for human placental growth and differentiation. The possible role of CNTF in enhancing cell proliferation and/or invasion during placental development and remodelling was investigated using HTR-8/SVneo and BeWo cells, taken respectively as cytotrophoblast and syncytiotrophoblast models. In both cell lines, treatment with human recombinant (hr) CNTF activated JAK2/STAT3 signalling and inhibited the ERK pathway. Interestingly, in HTR-8/SVneo cells, 50 ng hrCNTF induced significant downregulation of matrix metalloprotease (MMP)-1 and significant upregulation of MMP-9. Moreover, pharmacological inhibition of JAK2/STAT3 signalling by AG490 and curcumin resulted in MMP-9 downregulation; it activated the ERK signalling pathway and upregulated MMP-1 expression. Collectively, these data suggest a role for CNTF signalling in extravillous cytotrophoblast invasion through the modulation of specific MMPs.
... However, the precise definition and subtype of EnSCs are highly contradictory with other studies [21,[56][57][58][59]. Recently, EpCAM, E-cadherin, Mucin1, and CD44 have been identified as specific markers of endometrial epithelial stem cells in human and mouse [12,22,23]. However, culture and expansion of primary endometrial cells have been very difficult over decades [11,60]. In addition to the rapid senescence of cultured endometrial cells, these cells quickly lose their phenotype and hormone responsiveness [40,60]. ...
... However, culture and expansion of primary endometrial cells have been very difficult over decades [11,60]. In addition to the rapid senescence of cultured endometrial cells, these cells quickly lose their phenotype and hormone responsiveness [40,60]. In vivo, the endometrium expresses ER/ PR and maintains the exquisite balance in response to E2 and P4, which is essential for endometrial functions. ...
Background
There is unmet need for effective therapies of intrauterine adhesions (IUAs) that are common cause of menstrual disturbance and infertility, since current clinical procedures do not improve prognosis for patients with moderate to severe IUA, with a recurrence rate of 23–50%. Stem cell-based therapy has emerged as a therapeutic option with unsolved issues for IUA patients in the past few years. Primary endometrial epithelial cells for cell therapy are largely hampered with the extremely limited proliferation capacity of uterine epithelial cells. This study was to evaluate whether IUA is curable with conditionally reprogrammed (CR) endometrial epithelial cells.
Methods
Mouse endometrial epithelial cells (MEECs) were isolated from C57BL female mice, and long-term cultures of MEECs were established and maintained with conditional reprogramming (CR) method. DNA damage response analysis, soft agar assay, and matrigel 3D culture were carried out to determine the normal biological characteristics of CR-MEECs. The tissue-specific differentiation potential of MEECs was analyzed with air–liquid interface (ALI) 3D culture, hematoxylin and eosin (H&E) staining, Masson’s trichrome and DAB staining, immunofluorescence assay. IUA mice were constructed and transplanted with CR-MEECs. Repair and mechanisms of MEECs transplantation in IUA mice were measured with qRT-PCR, Masson’s trichrome, and DAB staining.
Results
We first successfully established long-term cultures of MEECs using CR approach. CR-MEECs maintained a rapid and stable proliferation in this co-culture system. Our data confirmed that CR-MEECs retained normal biological characteristics and endometrium tissue-specific differentiation potential. CR-MEECs also expressed estrogen and progesterone receptors and maintained the exquisite sensitivity to sex hormones in vitro. Most importantly, allogeneic transplantation of CR-MEECs successfully repaired the injured endometrium and significantly improved the pregnancy rate of IUA mice.
Conclusions
Conditionally reprogrammed physiological endometrial epithelial cells provide a novel strategy in IUA clinics in a personalized or generalized manner and also serve as a physiological model to explore biology of endometrial epithelial cells and mechanisms of IUA.
... Many models for the study of the placenta exist, from primary cells to trophoblastic cell lines such as HTR8/SVneo, BeWo, JEG and JAR [9]. HTR8/SVneo and BeWo cells are both trophoblastic cell lines originated from a first trimester placenta; BeWo cells are a representative of the villous pathway that remains within the placenta and HTR8/SVneo cells are a representative of the extravillious pathway, which migrate and infiltrate into the maternal decidua where they play important functions by interacting with cells in the stroma and modify blood vessels [10]. ZIKV is the first Flavivirus known to cause damages at this scale in the developing fetus, and the fact that only 20% of the infected population show any symptoms, makes this virus and the mechanisms that allow it to bypass the normally strict placental protection very important to understand. ...
... HTR8/SVneo cells and BeWo are representatives of two very distinct differentiation pathways of trophoblastic cells: BeWo cells are representatives of the villious pathway that remains in the outermost level of the placenta and can fuse to form the syncytiotrophoblast, secreting chorionic gonadotropin, which is responsible for the maternal recognition of pregnancy; HTR8/SVneo cells on the other hand, are representatives of the extravillious pathway, that migrate and infiltrate in the decidua, where they participate in arterial remodeling and invasion of veins, lymphatic and glands [19]. Based on these characteristics, any detrimental effect on BeWo cells may predict impairment of placental growth, while any effect towards HTR8/SVneo cells may explain unsatisfactory blastocyst implantation and placentation in vivo [10]. ...
Background
The uncontrollable spread of Zika virus (ZIKV) in the Americas during 2015–2017, and its causal link to microcephaly in newborns and Guillain-Barré syndrome in adults, led the World Health Organisation to declare it a global public health emergency. One of the most notable features of ZIKV pathogenesis was the ability of the virus to pass the placental barrier to infect the growing foetus. This pathogenic trait had not been observed previously for medically important flaviviruses, including dengue and yellow fever viruses.
Methods
In this study we evaluated the replication kinetics of ZIKV and the related encephalitic flavivirus West Nile strain Kunjin virus (WNV KUN ) in early-term placental cell lines.
Results
We have observed that WNV KUN in fact replicates with a greater rate and to higher titres that ZIKV in these cell lines.
Conclusions
These results would indicate the potential for all flaviviruses to replicate in placental tissue but it is the ability to cross the placenta itself that is the restrictive factor in the clinical progression and presentation of congenital Zika syndrome.
... Endometrium biopsy-derived epithelial and stromal cultures can be maintained in vitro and respond to hormonal stimuli 59,62,63 . Purified endometrial cell cultures have been used to understand physiological and pathological processes, including trophoblast invasion 64 . However, endometrium-derived primary culture offers limited potential for use in in vitro models since they cannot be propagated in vitro for many passages and tend to differentiate 62,63 . ...
The uterus is the organ for embryo implantation and fetal development. Most current models of the uterus are centred around capturing its function during later stages of pregnancy to increase the survival in pre-term births. However, in vitro models focusing on the uterine tissue itself would allow modelling of pathologies including endometriosis and uterine cancers, and open new avenues to investigate embryo implantation and human development. Motivated by these key questions, we discuss how stem cell-based uteri may be engineered from constituent cell parts, either as advanced self-organising cultures, or by controlled assembly through microfluidic and print-based technologies.
... Human trophoblast cell lines have been extensively used for investigating the in vitro effects of xenobiotics in the placenta (Guiñazú et al., 2012;Dominguez.-López et al., 2014;Mannelli et al., 2015). ...
... For example, explants retain elements of functional paracrine regulation between cells types through release of secreted signaling molecules. Therefore, explants more closely mimic in vivo conditions, than cell lines (Mannelli et al. 2015). ...
... Nevertheless, HTR-8/SVneo cells express a signature combination of molecular markers particular to extravillous trophoblasts, including cytokeratin 7 and HLA-G when grown on Matrigel (Irving et al. 1995;Khan et al. 2011;Kilburn et al. 2000;Takao et al. 2011). Despite the drawbacks of using these in vitro models, both have been extensively used to investigate placental biology and the complex processes involved in placental development (Mannelli et al. 2015). ...
Trichloroethylene (TCE) is an industrial solvent and widespread environmental contaminant. Although TCE exposure is prevalent, epidemiological studies of TCE exposure associations with adverse birth outcomes are inconclusive. Prior studies show that the TCE metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC) exhibits toxicity in a placental cell line. In the current study, genome-wide gene expression and gene set enrichment analyses were used to identify novel genes and pathway alterations in the HTR-8/SVneo human trophoblast cell line and human placental villous explants treated with DCVC at concentrations relevant to human exposures. In the cells, concentration- and time-dependent effects were observed, as evidenced by the magnitude of altered gene expression after treatment with 20 µM DCVC versus 10 µM, and 12-h versus 6-h of treatment. Comparing the two models for the transcriptional response to 12-h 20 µM DCVC treatment, no differentially expressed genes reached significance in villous explants, whereas 301 differentially expressed genes were detected in HTR-8/SVneo cells compared with non-treated controls (FDR < 0.05 + LogFC > 0.35 [FC > 1.3]). GSEA revealed five upregulated enriched pathways in common between explants and cells (FDR < 0.05). Moreover, all 12-h DCVC treatment groups from both models contained upregulated pathways enriched for genes regulated by the ATF4 transcription factor. The overrepresentation of ATF4 regulation of differentially expressed genes indicated activation of the integrated stress response (ISR), a condition triggered by multiple stress stimuli, including the unfolded protein response. DCVC-induced ISR activation was confirmed by elevated eIF2α phosphorylation, ATF4 protein concentrations, and decreased global protein synthesis in HTR-8/SVneo cells. This study identifies a mechanism of DCVC-induced cytotoxicity by revealing the involvement of a specific stress signaling pathway.
... Therefore, although these cell lines are easy to culture for a long time, there are general limitations of using such cell lines, including their genetic background, potential changes occurring during transformation and culture. [80] In addition, ethical and economic reasons limit the use of NHPs. Although rodent models are valuable in research, the research outcomes are critical in view of the differences between animals and humans. ...
Endometriosis is a prevalent chronic disease that affects approximately 6% to 10% of reproductive-aged women. Although numerous researchers have endeavored to explore the etiology of endometriosis over a century, its etiology still remains an enigma. The exploration of pathophysiologic mechanism and novel therapy for endometriosis depends on ideal endometriotic models. In the previous decade, various endometriotic models have been established; therefore, we made a conclusion for available information on these models. This review summarized the common experimental models used in endometriotic studies, including their origins, characteristics, applications, and limitations. Endometriotic models played an important role in studying etiologies and novel treatments of endometriosis during the last decades. Among them, animal models and endometriotic cell lines were viewed as most common studying tools to explore the intrinsic entities of endometriosis. In addition, endometrial organoid also emerged and was regarded as an ideal studying tool for endometriosis research. Different research models collectively complement each other to advance the endometriosis research. The successful establishment of endometrial organoids means that organoids are expected to become an ideal model for studying endometriosis in the future.
... Immortalized or carcinoma-derived cell lines, such as Ishikawa cells (derived from epithelial endometrial cells) or St-T1b cell (derived from stromal endometrial cells) are also available for in vitro investigations. While these cells are easily cultured for long periods (Table 1), there are general limitations of using such cell lines including their genetic background, the potential changes acquired following transformation and during their establishment in culture (Mannelli et al., 2015). Alternatively, primary endometrial cells can be isolated, but these are difficult to maintain and expand in long term culture. ...
... Alternatively, primary endometrial cells can be isolated, but these are difficult to maintain and expand in long term culture. Most of these cells lose their phenotype and hormone responsiveness in culture (Mannelli et al., 2015). Moreover, two dimensional (2D) cell cultures do not faithfully mimic in vivo three dimensional (3D) structure and function. ...
... Human trophoblast cell lines have been extensively used for investigating the in vitro effects of xenobiotics in the placenta (Guiñazú et al., 2012;Dominguez.-López et al., 2014;Mannelli et al., 2015). ...
The neonicotinoid (Neo) insecticide family is a relatively new class of pesticides of growing use. There is an increasing concern that human exposure to environmental pollutants in utero may be associated with diseases in adulthood. A functional placenta and trophoblasts are a requisite for a healthy pregnancy. The aim of this study was to investigate whether the Neo Acetamiprid (Ace) and one of its commercial formulations (Ace CF) display toxic features to a human first trimester trophoblast cell line. HTR-8/SVneo cells were cultured in the presence of Ace or Ace CF (0.1-100 µM) for 4 and 24 h, and changes in cell viability, reactive oxygen species, antioxidant system and macromolecule damage levels were evaluated. Ace and Ace CF are cytotoxic for HTR-8/SVneo trophoblasts. Cell viability loss and oxidative imbalance were triggered by Ace and Ace CF treatments. Impact in the antioxidant enzymes catalase, superoxide dismutase and gluthatione S-transferase activities were observed after 24 h exposure to Ace CF. Moreover, Ace CF caused oxidative damage in proteins, lipids and DNA, whereas Ace only damaged proteins. To test oxidative stress as a toxicity mechanism, cells were pre-incubated with the antioxidant N-acetyl-l-cysteine (NAC), prior Neo treatment. NAC protected trophoblasts from cell death and prevented oxidative damage. Results demonstrate that Ace (as active principle or CF) is cytotoxic for human trophoblasts, and oxidative stress is a toxicity mechanism. Ace CF exhibited a more toxic effect than the active principle, in an identical exposure scenario.
