Pauliina Damdimopoulou

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

Adult human and mouse ovaries lack DDX4-expressing functional oogonial stem cells.

The generally accepted viewpoint for more than 50 years has been that the number of oocytes is fixed in fetal or neonatal ovaries, and therefore, oocytes cannot renew themselves in postnatal or adult life. Over the past decade, however, the traditional viewpoint has been challenged by a number of investigators who have presented evidence that postnatal follicular renewal occurs in mammals, and that mitotically active oogonial stem cells (OSCs) exist in postnatal mouse ovaries. Health, Inc. All rights reserved. 30 Obstetrical and Gynecological Survey This letter to the editor presents experimental evidence that disputes the existence of mitotically active OSC in postnatal mouse ovaries. The results presented here are the summary of research conducted independently in 4 laboratories. A previous study (White et al.Nat Med. 2012;18:413–421) reported that OSCs could be purified from adult human and mouse ovaries by use of DEAD box polypeptide 4 (DDX4) antibody–based fluorescence-activated cell sorting (FACS), and that after in vitro manipulation, these isolated OSCs could form oocytes. Based on the well-established cytoplasmic location of DDX4, the use of this protein as a cell surface marker is controversial. Using the same DDX4 antibody–based FACS approach as in the White et al study, the investigators isolated a population of cells from human ovarian cortical tissue biopsied from 16 fertile reproductive-age women who had had at least 1 previous live birth. No DDX4 messenger RNA (mRNA) expression was detected using quantitative polymerase chain reaction in these cells or by a more sensitive single-cell mRNA sequencing analysis that could detect low expression of DDX4. In additional experiments, the sorted human ovarian cells were cultured as described in the previous study. Although no DDX4 expression was detected in the cultured DDX4-positive cells (cultured-POS) or cultured DDX4-negative cells (cultured-NEG) by immunofluorescence staining, the cultured-POS cells and cultured-NEG cells both bound tightly to the DDX4-specific antibody in FACS and became DDX4-positive after culture. The previous study had reported that oocytes enclosed in follicles regenerated 1 week after the DDX4-positive human OSCs were injected into human ovarian cortical tissues that were subsequently xenografted into female severe combined immunodeficient mice. The investigators repeated this experiment and labeled the cultured-POS cells with stable enhanced green fluorescent protein (EGFP) expression. After culturing and expanding this cell population, EGFP-expressing cultured-POS cells were injected into human ovarian cortical tissue biopsies, and these cortical tissues were then xenografted into female severe combined immunodeficient mice for further growth. Grafts were analyzed 1 week, 2 weeks, and 4 weeks after transplantation of the EGFP–cultured-POS cells into the human cortical tissues. The results of this experiment showed that EGFP-positive cells could be observed in the vicinity of the injection sites, but the absence of any EGFP-positive oocytes demonstrated that the DDX4-positive human cells obtained with the DDX4 antibody are not functional stem cells and cannot regenerate oocytes. To confirm these findings that the DDX4-specific antibody–based FACS does not select for a specific cell population expressing DDX4, the same FACS was performed with mouse cells from several organs (including adult liver, spleen, and kidney) that do not express DDX4. DDX4-positive cell populations were obtained from cells of these organs, which provide additional evidence that use of the DDX4-specific antibody using the FACS protocol (critical in purifying the reported OSCs) does not select for DDX4-expressing cells. These findings provide evidence that supports the traditional view that no postnatal follicular renewal occurs in mammals, and no mitotically active DDX4-expressing female germline progenitors exist in postnatal mouse ovaries.

Novel hydroxysteroid (17β) dehydrogenase 1 inhibitors reverse estrogen-induced endometrial hyperplasia in transgenic mice

Local estrogen production plays a key role in proliferative endometrial disorders, such as endometrial hyperplasia and cancer. Hydroxysteroid (17beta) dehydrogenase 1 (HSD17B1) is an enzyme that catalyzes with high efficiency the conversion of weakly active estrone into highly potent estradiol. Here we report that female transgenic mice expressing human HSD17B1 invariably develop endometrial hyperplasia in adulthood. These mice also fail to ovulate and have enhanced peripheral conversion of estrone into estradiol in a variety of target tissues, including the uterus. As in humans, endometrial hyperplasia in HSD17B1 transgenic female mice was reversible on ovulation induction, which triggers a rise in circulating progesterone levels, and in response to exogenous progestins. Strikingly, a treatment with an HSD17B1 inhibitor failed to restore ovulation yet completely reversed the hyperplastic morphology of epithelial cells in the glandular compartment, although less so in the luminal epithelium. The data indicate that human HSD17B1 expression enhances endometrial estrogen production, and consequently, estrogen-dependent proliferation. Therefore, HSD17B1 is a promising new therapeutic target in the management of estrogen-dependent endometrial diseases.

Biosensors paving the way to understanding the interaction between cadmium and the estrogen receptor alpha.

Cadmium is a toxic heavy metal ubiquitously present in the environment and subsequently in the human diet. Cadmium has been proposed to disrupt the endocrine system, targeting in particular the estrogen signaling pathway already at environmentally relevant concentrations. Thus far, the reports on the binding affinity of cadmium towards human estrogen receptor alpha (hERα) have been contradicting, as have been the reports on the in vivo estrogenicity of cadmium. Hence, the mode of interaction between cadmium and the receptor remains unclear. Here, we investigated the interaction between cadmium and hERα on a molecular level by applying a novel, label-free biosensor technique based on reflectometric interference spectroscopy (RIfS). We studied the binding of cadmium to hERα, and the conformation of the receptor following cadmium treatment. Our data reveals that cadmium interacts with the ligand binding domain (LBD) of the ERα and affects the conformation of the receptor. However, the binding event, as well as the induced conformation change, greatly depends on the accessibility of the cysteine tails in the LBD. As the LBD cysteine residues have been reported as targets of post-translational modifications in vivo, we present a hypothesis according to which different cellular pools of ERα respond to cadmium differently. Our proposed theory could help to explain some of the previously contradicting results regarding estrogen-like activity of cadmium.

Spider silk for xeno-free long-term self-renewal and differentiation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) can undergo unlimited self-renewal and have the capacity to differentiate into all somatic cell types, and are therefore an ideal source for the generation of cells and tissues for research and therapy. To realize this potential, defined cell culture systems that allow expansion of hPSCs and subsequent controlled differentiation, ideally in an implantable three-dimensional (3D) matrix, are required. Here we mimic spider silk - Natures high performance material - for the design of chemically defined 2D and 3D matrices for cell culture. The silk matrices do not only allow xeno-free long-term expansion of hPSCs but also differentiation in both 2D and 3D. These results show that biomimetic spider silk matrices enable hPSC culture in a manner that can be applied for experimental and clinical purposes.

Cadmium-induced effects on cellular signaling pathways in the liver of transgenic estrogen reporter mice.

Estrogen-like effects of cadmium (Cd) have been reported in several animal studies, and recent epidemiological findings suggest increased risk of hormone-dependent cancers after Cd exposure. The mechanisms underlying these effects are still under investigation. Our aim was to study the effects of Cd on cellular signaling pathways in vivo with special focus on estrogen signaling and to perform benchmark dose analysis on the effects. Transgenic adult ERE-luciferase male mice were exposed subcutaneously to 0.5-500 μg CdCl(2) per kg body weight (bw) or 17α-ethinylestradiol (EE2) for 3 days. These doses had no effects on organ and bw or testicular histology, indicating subtoxic exposure levels. The transgene luciferase, reporting genomic estrogen response, was significantly increased by EE2 but not by Cd. However, Cd significantly affected kinase phosphorylation and endogenous gene expression. Interestingly, gene expression changes displayed a traditional dose-response relationship, with benchmark dose levels for the expression of Mt1, Mt2, p53, c-fos, and Mdm2 being 92.9, 19.9, 7.6, 259, and 25.9 μg/kg bw, respectively, but changes in kinase phosphorylation were only detected at low exposure levels. Phosphorylation of Erk1/2 was significantly increased even in the lowest dose group, 0.5 μg/kg bw, rendering pErk1/2 a more sensitive sensor of exposure than changes in gene expression. Collectively, our data suggest that the effects triggered by Cd in vivo are markedly concentration dependent. Furthermore, we conclude that the estrogen-like effects of Cd are likely to result from a mechanism different from steroidal estrogens.

The human thioredoxin reductase-1 splice variant TXNRD1_v3 is an atypical inducer of cytoplasmic filaments and cell membrane filopodia

Thioredoxin reductases are important selenoproteins maintaining cellular redox balance and regulating several redox dependent processes in apoptosis, cell proliferation and differentiation. Specific functions of dedicated splice variants may add further complexity to the functions of these proteins. We show here that a splice variant of human thioredoxin reductase 1, TXNRD1_v3, forms both dynamic cytoplasmic filaments and provokes instantaneous formation of dynamic cell membrane protrusions identified as filopodia. Using truncated versions of the protein we found that both the cytoplasmic filaments and the filopodia formation were exclusively dependent on the glutaredoxin domain of the protein. Interestingly, actin polymerization was required for filopodia formation triggered by TXNRD1_v3, but not for generation of cytoplasmic filaments. We conclude that the glutaredoxin domain of TXNRD1_v3 is an atypical regulator of the cell cytoskeleton that potently induces formation of highly ordered cytoplasmic filaments and cell membrane filopodia.

A Single Dose of Enterolactone Activates Estrogen Signaling and Regulates Expression of Circadian Clock Genes in Mice

Enterolactone (EL) is an enterolignan produced by gut microbiota from dietary plant lignans. Epidemiological and experimental studies suggest that EL and plant lignans may reduce the risk of breast and prostate cancer as well as cardiovascular disease. These effects are thought to at least in part involve modulation of estrogen receptor activity. Surprisingly little is known about the in vivo estrogenicity of EL. In the present study, we investigated the target tissues of EL, the genes affected by EL treatment, and the response kinetics. Following a single dose of EL, luciferase was significantly induced in reproductive and nonreproductive tissues of male and female 3xERE-luciferase mice, indicating estrogen-like activity. Microarray analysis revealed that EL regulated the expression of only 1% of 17β-estradiol target genes in the uterus. The majority of these genes were traditional estrogen target genes, but also members of the circadian signaling pathway were affected. Kinetic analyses showed that EL undergoes rapid phase II metabolism and is efficiently excreted. In vivo imaging demonstrated that the estrogen response followed similar, fast kinetics. We conclude that EL activates estrogen signaling in both male and female mice and that the transient responses may be due to the fast metabolism of the compound. Lastly, EL may represent a link among diet, gut microbiota, and circadian signaling.

Emerging role of PLAG1 as a regulator of growth and reproduction

Pleomorphic adenoma gene 1 (PLAG1) belongs to the PLAG family of zinc finger transcription factors along with PLAG-like 1 and PLAG-like 2. The PLAG1 gene is best known as an oncogene associated with certain types of cancer, most notably pleomorphic adenomas of the salivary gland. While the mechanisms of PLAG1-induced tumorigenesis are reasonably well understood, the role of PLAG1 in normal physiology is less clear. It is known that PLAG1 is involved in cell proliferation by directly regulating a wide array of target genes, including a number of growth factors such as insulin-like growth factor 2. This is likely to be a central mode of action for PLAG1 both in embryonic development and in cancer. The phenotype of Plag1 knockout mice suggests an important role for PLAG1 also in postnatal growth and reproduction, as PLAG1 deficiency causes growth retardation and reduced fertility. A role for PLAG1 in growth and reproduction is further corroborated by genome-wide association studies in humans and domestic animals in which polymorphisms in the PLAG1 genomic region are associated with body growth and reproductive traits. Here we review the current evidence for PLAG1 as a regulator of growth and fertility and discuss possible endocrine mechanisms involved.

Estrogen-like effects of diet-derived cadmium differ from those of orally administered CdCl2 in the ERE-luc estrogen reporter mouse model

Cadmium (Cd), an environmental and dietary contaminant, has been described to mimic the effects of 17β-estradiol (E(2)) in selected model systems when studied as an inorganic salt. However, inorganic Cd salts do not represent the main form of Cd exposure in general human populations. The aims of this study were to compare the estrogen-like effects and the bioavailability of dietary Cd to inorganic CdCl(2). Adult ovariectomized ERE-luc reporter mice were administered two bread based diets containing different concentrations of Cd (17.57 and 49.22μg/kg, corresponding to oral intakes of 1.8 and 5.1μg/kg body weight (bw) per day, respectively), inorganic CdCl(2) (1μg/kg bw per day by gavage) or E(2) (5μg/kg bw per day pellet) for 21 days. The effects on estrogen signaling were investigated by studying the uterine weights, luciferase activation, and expression of endogenous estrogen target genes. The uterine weight was significantly increased by both CdCl(2) and E(2) but not by the Cd containing diets. All treatments modulated the expression of luciferase and the endogenous estrogen target genes; however, there was no consistent overlap between the responses triggered by the bread diets and the responses stimulated by CdCl(2) or E(2). Oral exposure to Cd was calculated and the concentrations in liver and kidneys quantified to estimate the amount of absorbed Cd retained in tissues. The results suggest significantly lower absorption and/or tissue retention of dietary Cd compared to CdCl(2) following oral exposure. Altogether, our results support previous reports on in vivo estrogenicity of CdCl(2) but do not suggest the same activity for diet bound Cd. This study calls for caution when extrapolating results from pure compound studies (e.g. estrogenicity of CdCl(2)) to dietary exposure scenarios (e.g. estrogenicity of diet bound Cd). Further basic research is needed on the mechanisms of interaction between Cd and the estrogen signaling, biologically active species of Cd, and biomarkers of estrogen-like effects of Cd in vivo before human health risk assessment on the hormone disruptive effects of Cd can be carried out.