Generation of Human Female Reproductive Tract Epithelium from Human Embryonic Stem Cells

BACKGROUND: Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT). METHODOLOGY/PRINCIPAL FINDINGS: We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1(+) mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA). CONCLUSIONS/SIGNIFICANCE: These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT

Ovine multiparity is associated with diminished vaginal muscularis, increased elastic fibres and vaginal wall weakness: implication for pelvic organ prolapse

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Pelvic Organ Prolapse (POP) is a major clinical burden affecting 25% of women, with vaginal delivery a major contributing factor. We hypothesised that increasing parity weakens the vagina by altering the extracellular matrix proteins and smooth muscle thereby leading to POP vulnerability. We used a modified POP-quantification (POP-Q) system and a novel pressure sensor to measure vaginal wall weakness in nulliparous, primiparous and multiparous ewes. These measurements were correlated with histological, biochemical and biomechanical properties of the ovine vagina. Primiparous and multiparous ewes had greater displacement of vaginal tissue compared to nulliparous at points Aa, Ap and Ba and lower pressure sensor measurements at points equivalent to Ap and Ba. Vaginal wall muscularis of multiparous ewes was thinner than nulliparous and had greater elastic fibre content. Collagen content was lower in primiparous than nulliparous ewes, but collagen organisation did not differ. Biomechanically, multiparous vaginal tissue was weaker and less stiff than nulliparous. Parity had a significant impact on the structure and function of the ovine vaginal wall, as the multiparous vaginal wall was weaker and had a thinner muscularis than nulliparous ewes. This correlated with “POP-Q” and pressure sensor measurements showing greater tissue laxity in multiparous compared to nulliparous ewes

Nominalphrasen in literarischen Texten : Strukturtypen und Funktionen beim Figurenentwurf in Werken des 20. und 21. Jahrhunderts

Nominalphrasen und ihre Teile tragen wesentlich dazu bei, Wissen über literarische Figuren einzuführen und eingeführtes figurenbezogenes Wissen an relevanten Stellen zu aktualisieren. Das vorliegende Buch bewegt sich an der Schnittstelle von Grammatik und Textlinguistik: Anhand von ausgewählten Werken des 20. und 21. Jahrhunderts wird systematisch und detailliert dargestellt, welche Strukturtypen von Nominalphrasen eingesetzt werden, um bei der Figureneinführung bzw. beim Weiterreden über literarische Figuren bestimmte Dimensionen der Figurencharakterisierung anzusprechen. In einer Fallstudie wird darüber hinaus nach der Dynamik des Wissensaufbaus im Textstrom gefragt

Mitotic Arrest in Teratoma Susceptible Fetal Male Germ Cells

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27KIP1, p15INK4B, activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility

Development of a pipeline for automated, high-throughput analysis of paraspeckle proteins reveals specific roles for importin α proteins

We developed a large-scale, unbiased analysis method to measure how functional variations in importin (IMP) α2, IMPα4 and IMPα6 each influence PSPC1 and SFPQ nuclear accumulation and their localization to paraspeckles. This addresses the hypothesis that individual IMP protein activities determine cargo nuclear access to influence cell fate outcomes. We previously demonstrated that modulating IMPα2 levels alters paraspeckle protein 1 (PSPC1) nuclear accumulation and affects its localization into a subnuclear domain that affects RNA metabolism and cell survival, the paraspeckle. An automated, high throughput, image analysis pipeline with customisable outputs was created using Imaris software coupled with Python and R scripts; this allowed non-subjective identification of nuclear foci, nuclei and cells. HeLa cells transfected to express exogenous full-length and transport-deficient IMPs were examined using SFPQ and PSPC1 as paraspeckle markers. Thousands of cells and >100,000 nuclear foci were analysed in samples with modulated IMPα functionality. This analysis scale enabled discrimination of significant differences between samples where paraspeckles inherently display broad biological variability. The relative abundance of paraspeckle cargo protein(s) and individual IMPs each influenced nuclear foci numbers and size. This method provides a generalizable high throughput analysis platform for investigating how regulated nuclear protein transport controls cellular activities

Evaluating the impact of human amnion epithelial cells on angiogenesis

The effects of human amnion epithelial cells (hAECs) on angiogenesis remain controversial. It is yet unknown if the presence of inflammation and/or gestational age of hAEC donors have an impact on angiogenesis. In this study, we examined the differences between term and preterm hAECs on angiogenesis in vitro and in vivo. Conditioned media from term hAECs induced the formation of longer huVEC tubules on Matrigel. Both term and preterm hAECs expressed VEGFA, PDGFB, ANGPT1, and FOXC1, which significantly increased after TNFα and IFNγ stimulation. In the presence of TNFα and IFNγ, coculture with term hAECs reduced gene transcription of Tie-2 and Foxc1 in huVECs, while coculture with preterm hAECs increased gene transcription of PDGFRα and PDGFRβ and reduced gene transcription of FOXC1 in huVECs. In vivo assessment of angiogenesis using vWF immunostaining revealed that hAEC treatment decreased angiogenesis in a bleomycin model of lung fibrosis but increased angiogenesis in a neonatal model of hyperoxia-induced lung injury. In summary, our findings suggested that the impact of hAECs on angiogenesis may be influenced by the presence of inflammation and underlying pathology

Tissue response to collagen containing polypropylene meshes in an ovine vaginal repair model

Pelvic Organ Prolapse (POP) is the herniation of pelvic organs into the vagina. Despite broad acceptance of mesh use in POP surgical repair, the complication rate is unacceptable. We hypothesized that collagen-containing polypropylene (PP) mesh types could modulate mesh-tissue integration and reduce long-term inflammation, thereby reducing mesh-associated complications. This study compared the long-term tissue response to an unmodified PP mesh and two collagen containing meshes in an ovine model which has similar pelvic anatomy and vaginal size to human. Three commercially available macroporous PP meshes, uncoated PP mesh (Avaulta Solo) (PP), the same textile PP mesh layered with a sheet of cross-linked porcine acellular matrix (Avaulta Plus) (PP-ACM) and a different yet also macroporous PP (Sofradim) mesh coated with solubilized atelocollagen (Ugytex) (PP-sCOL) were implanted in the ovine vagina and tissue explanted after 60 and 180 days. The macrophage phenotype and response to implanted meshes, and vascularity were quantified by immunostaining and morphometry. We quantified changes in extracellular matrix composition biochemically and collagen organization and percentage area around the interface of the mesh implants by Sirius Red birefringence and morphometry. PP-ACM induced a more sustained inflammatory response, indicated by similar CD45(+) leukocytes but reduced CD163(+) M2 macrophages at 60 days (P<0.05). PP-sCOL increased Von Willebrand Factor (vWF)-immunoreactive vessel profiles after 60 days. At the micro-molecular level, collagen birefringence quantification revealed significantly fewer mature collagen fibrils (red, thick fibrils) at the mesh-tissue interface than control tissue for all mesh types (P<0.001) but still significantly greater than the proportion of immature (green thin fibrils) at 60 days (P<0.05). The proportion of mature collagen fibrils increased with time around the mesh filaments, particularly those containing collagen. The total collagen precent area at the mesh interface was greatest around the PP-ACM mesh at 60 days (P<0.05). By 180 days the total mature and immature collagen fibres at the interface of the mesh filaments resembled that of native tissue. In particular, these results suggest that both meshes containing collagen evoke different types of tissue responses at different times during the healing response yet both ultimately lead to physiological tissue formation approaching that of normal tissue.publisher: Elsevier articletitle: Tissue response to collagen containing polypropylene meshes in an ovine vaginal repair model journaltitle: Acta Biomaterialia articlelink: http://dx.doi.org/10.1016/j.actbio.2016.05.010 content_type: article copyright: © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.status: publishe