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

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

Quantitative analysis of the microtubule interaction of rabies virus P3 protein:Roles in immune evasion and pathogenesis

Although microtubules (MTs) are known to have important roles in intracellular transport of many viruses, a number of reports suggest that specific viral MT-associated proteins (MAPs) target MTs to subvert distinct MT-dependent cellular processes. The precise functional importance of these interactions and their roles in pathogenesis, however, remain largely unresolved. To assess the association with disease of the rabies virus (RABV) MAP, P3, we quantitatively compared the phenotypes of P3 from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derivative strain, Ni-CE. Using confocal/live-cell imaging and dSTORM super-resolution microscopy to quantify protein interactions with the MT network and with individual MT filaments, we found that the interaction by Ni-CE-P3 is significantly impaired compared with Ni-P3. This correlated with an impaired capacity to effect association of the transcription factor STAT1 with MTs and to antagonize interferon (IFN)/STAT1-dependent antiviral signaling. Importantly, we identified a single mutation in Ni-CE-P3 that is sufficient to inhibit MT-association and IFN-antagonist function of Ni-P3, and showed that this mutation alone attenuates the pathogenicity of RABV. These data provide evidence that the viral protein-MT interface has important roles in pathogenesis, suggesting that this interface could provide targets for vaccine/antiviral drug development

A single-cell transcriptome atlas of the adult human retina

The retina is a specialized neural tissue that senses light and initiates image processing. Although the functional organization of specific retina cells has been well studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile the human retina, we performed single-cell RNA sequencing on 20,009 cells from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct cell populations representing all known neural retinal cells: rod photoreceptors, cone photoreceptors, Müller glia, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, astrocytes, and microglia. Our data captured molecular profiles for healthy and putative early degenerating rod photoreceptors, and revealed the loss of MALAT1 expression with longer post-mortem time, which potentially suggested a novel role of MALAT1 in rod photoreceptor degeneration. We have demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to understanding retinal biology and disease.This work was supported by funding from the Ophthalmic Research Institute of Australia (RW, SL), the University of Melbourne De Brettville Trust (RW), and the Kel and Rosie Day Foundation (RW) and GOSHCC (JCS). The Centre for Eye Research Australia receives operational infrastructure support from the Victorian Government

The generation of live offspring from vitrified oocytes

Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M) and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P < 0.05). As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P < 0.05). Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10). When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and thus the propagation of specific genetic traits

Following in vitro fertilization and culture, both fresh (A1) and vitrified oocytes in 0.1 M trehalose (B1) were capable of developing to the two-cell (A2; B2), four-cell (A3; B3) and blastocyst (A4; B4) stages, respectively.

<p>Furthermore, blastocysts were able to hatch (A5; B5) without chemical or mechanical assistance and some were used to derive embryonic stem cells.</p

Survival, and live offspring rates for vitrified oocytes.

<p>Fisher's test indicated no significant differences between the treatments within same column (P&gt;0.05). Oocyte and live offspring data collected from 5 and 2 replicates respectively.</p

Survival, vitrified two-cell embryo and live offspring rates from either fresh or vitrified oocytes.

<p>Fisher's test indicated no significant differences between the treatments within same column (P&gt;0.05).</p