A defect in the NOG gene increases susceptibility to spontaneous superficial chronic corneal epithelial defects (SCCED) in boxer dogs

Background Superficial chronic corneal epithelial defects (SCCEDs) are spontaneous corneal defects in dogs that share many clinical and pathologic characteristics to recurrent corneal erosions (RCE) in humans. Boxer dogs are predisposed to SCCEDs, therefore a search for a genetic defect was performed to explain this susceptibility. DNA was extracted from blood collected from Boxer dogs with and without SCCEDs followed by whole genome sequencing (WGS). RNA sequencing of corneal tissue and immunostaining of corneal sections from affected SCCED Boxer dogs with a deletion in the NOG gene and affected non-Boxer dogs without the deletion were performed. Results A 30 base pair deletion at a splice site in Noggin (NOG) (Chr 9:31453999) was identified by WGS and was significantly associated (P < 0.0001) with Boxer SCCEDs compared to unaffected non-Boxer dogs. NOG, BMP4, MMP13, and NCAM1 all had significant fold reductions in expression and SHH was significantly increased in Boxers with the NOG deletion as identified by RNA-Seq. Corneal IHC from NOG deletion dogs with SCCEDs had lower NOG and significantly higher scores of BMP2. Conclusions Many Boxer dogs with SCCED have a genetic defect in NOG. NOG is a constitutive protein in the cornea which is a potent inhibitor of BMP, which likely regulate limbal epithelial progenitor cells (LEPC). Dysregulation of LEPC may play a role in the pathogenesis of RCE.

Delayed maturation of thymic epithelium in mice with specific deletion of β-catenin gene in FoxN1 positive cells

Wnt signalling pathways have been reported to be involved in thymus development but their precise role in the development of both thymic epithelium (TE) and thymocytes is controversial. Herein, we examined embryonic, postnatal and adult thymi of mice with a specific deletion of β-catenin gene in FoxN1+ thymic epithelial cells (TECs). Together with a high postnatal mouse mortality, the analysis showed severe thymic hypocellularity, largely due an important reduction in numbers of developing thymocytes, and delayed, partially blocked maturation of mutant TECs. Affected TECs included largely cortical (c) TEC subsets, such as immature MTS20+ TECs, Ly51+ cTECs and a remarkable, rare Ly51+MTS20+MHCIIhi cell subpopulation previously reported to contain thymic epithelial progenitor cells (TEPCs) (Ulyanchenko et al., Cell Rep 14:2819–2832, 2016). In addition, altered postnatal organization of mutant thymic medulla failed to organize a unique, central epithelial area. This delayed maturation of TE cell components correlated with low transcript production of some molecules reported to be masters for TEC maturation, such as EphB2, EphB3 and RANK. Changes in the thymic lymphoid component became particularly evident after birth, when molecules expressed by TECs and involved in early T-cell maturation, such as CCL25, CXCL12 and Dll4, exhibited minimal values. This represented a partial blockade of the progression of DN to DP cells and reduced proportions of this last thymocyte subset. At 1 month, in correlation with a significant increase in transcript production, the DP cell percentage increased in correlation with a significant fall in the number of mature TCRαβhi thymocytes and peripheral T lymphocytes.

O-065 The naughty cells of the endometriumxx

Stem/progenitor cells are the naughty cells of the endometrium! The term “naughty” has a number of connotations, one being immaturity which I will apply to the rare stem/progenitor cell populations hiding in the endometrium, where they have eluded scientists for so long. Despite their rarity, these immature cells have the capability of growing up and differentiating into the functional cells of the endometrium, producing their progenies in the process. The self-willed human endometrial epithelial progenitor cells (eEPC) and mesenchymal stem cells (eMSC) first revealed themselves through their clonogenic activity, shunning their mates and setting up clones of cells on their own. Their risqué production of identical copies of themselves ensures their continuity, much to the chagrin of their mature counterparts. They are sneaky and can produce large numbers of mature progeny, but rarely proliferate themselves preferring to take life easy and do little. They also spit out viability dyes (Hoechst) at a greater rate than mature endometrial cells to become Side Population (SP) cells. A number of approaches have been used to tame these naughty endometrial stem/progenitors. In order to determine the identity and location of these elusive cells, specific markers had to be found. The immature endometrial epithelial progenitor cells play tricks with the specific markers they express. For example, clonogenic eEPC are N-cadherin+, an epithelial mesenchymal transition marker, found by unbiassed gene profiling, revealed their hiding place in the bases of glands deep in the endometrial basalis. Similarly, SSEA-1+ basalis epithelial progenitors pirated their marker from mature neutrophils and differentiating human pluripotent stem cells. In mice the stem/progenitor cells like to play chase, with lineage tracing of individual genetically marked cells revealing their location in the intersection zone of the glands and luminal epithelium, and also in the gland bases (Axin2+ and Lgr5+). The identity of eMSCs has also been determined by discovery of specific markers, but even here the eMSC play games in human endometrium where sometimes they are pericytes (CD140b and CD146 double positive cells), sometimes perivascular cells (SUSD2+) and sometimes CD34+ cells in the adventitia of blood vessels. They are also adventitial perivascular cells in ovine endometrium, but this time they are CD271+. Mature endometrial stromal cell progeny are also naughty, often pretending to be eMSC, particularly when shed into menstrual fluid, confusing many of their status. Adding further to their misbehaviour, they express the same official MSC surface markers. To get even immature endometrial MSC strike back, claiming immunomodulatory properties in attempt to upstage their mature stromal progeny, also endowed with these properties. Finally, other endometrial cells such as macrophages may also be naughty as their mischievousness in evading detection can trick us to consider them as stem cells from the bone marrow, masquerading as endometrial epithelial or stromal cells. Naughty implies behaving badly and I will show data suggesting that stem/progenitor cells may escape the endometrium to cause a nasty disease, endometriosis. They may also become wayward and unruly, invading the myometrium to form adenomyosis. Some naughty epithelial progenitors defiantly pick up mutations to become cancer stem cells and initiate endometrial cancer. They may also malfunction because they do not obey estrogen signalling instructions, failing to proliferate and causing thin unresponsive endometrium. In their naughtiness, they may run away or get totally lost, thereby resulting in Asherman’s syndrome. Therefore, for numerous reasons, stem/progenitor cells are the naughty cells of the endometrium. © The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: [email protected].

Acquisition of alveolar fate and differentiation competence by human fetal lung epithelial progenitor cells

Variation in lung alveolar development is strongly linked to disease susceptibility. However, the cellular and molecular mechanisms underlying alveolar development are difficult to study in humans. Using primary human fetal lungs we have characterized a tip progenitor cell population with alveolar fate potential. These data allowed us to benchmark a self-organising organoid system which captures key aspects of lung lineage commitment and can be efficiently differentiated to alveolar type 2 cell fate. Our data show that Wnt and FGF signalling, and the downstream transcription factors NKX2.1 and TFAP2C, promote human alveolar or airway fate respectively. Moreover, we have functionally validated cell-cell interactions in human lung alveolar patterning. We show that Wnt signalling from differentiating fibroblasts promotes alveolar type 2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. Our organoid system recapitulates key aspects of human lung development allowing mechanistic experiments to determine the underpinning molecular regulation.

Fibrin-Plasma Rich in Growth Factors Membrane for the Treatment of a Rabbit Alkali-Burn Lesion

The purpose of this work is to describe the use of Fibrin-Plasma Rich in Growth Factors (PRGF) membranes for the treatment of a rabbit alkali-burn lesion. For this purpose, an alkali-burn lesion was induced in 15 rabbits. A week later, clinical events were evaluated and rabbits were divided into five treatment groups: rabbits treated with medical treatment, with a fibrin-PRGF membrane cultured with autologous or heterologous rabbit Limbal Epithelial Progenitor Cells (LEPCs), with a fibrin-PRGF membrane in a Simple Limbal Epithelial Transplantation and with a fibrin-PRGF membrane without cultured LEPCs. After 40 days of follow-up, corneas were subjected to histochemical examination and immunostaining against corneal or conjunctival markers. Seven days after alkali-burn lesion, it was observed that rabbits showed opaque cornea, new blood vessels across the limbus penetrating the cornea and epithelial defects. At the end of the follow-up period, an improvement of the clinical parameters analyzed was observed in transplanted rabbits. However, only rabbits transplanted with cultured LEPCs were positive for corneal markers. Otherwise, rabbits in the other three groups showed positive staining against conjunctival markers. In conclusion, fibrin-PRGF membrane improved the chemically induced lesions. Nonetheless, only fibrin-PRGF membranes cultured with rabbit LEPCs were able to restore the corneal surface.

Mechanogenetic role of actomyosin complex in branching morphogenesis of epithelial organs

The actomyosin complex plays crucial roles in various life processes by balancing the forces generated by cellular components. In addition to its physical function, the actomyosin complex participates in mechanotransduction. However, the exact role of actomyosin contractility in force transmission and the related transcriptional changes during morphogenesis are not fully understood. Here, we report a mechanogenetic role of the actomyosin complex in branching morphogenesis using an organotypic culture system of mouse embryonic submandibular glands. We dissected the physical factors arranged by characteristic actin structures in developing epithelial buds and identified the spatial distribution of forces that is essential for buckling mechanism to promote the branching process. Moreover, the critical genes required for the distribution of epithelial progenitor cells were regulated by YAP/TAZ through a mechanotransduction process in epithelial organs. These findings are important for our understanding of the physical processes involved in the development of epithelial organs and provide a theoretical background for developing new approaches for organ regeneration.