Purpose: Retinal degenerative diseases lead to the death of retinal neurons causing visual impairment and blindness. In lower order vertebrates, the retina and its surrounding tissue contain stem cell niches capable of regenerating damaged tissue. Here we examine these niches and review their capacity to be used as retinal stem/progenitor cells (RSC/RPCs) for retinal repair. Recent Findings: Exogenous factors can control the in vitro activation of RSCs/PCs found in several niches within the adult eye including cells in the ciliary margin, the retinal pigment epithelium, iris pigment epithelium as well as the inducement of Müller and amacrine cells within the neural retina itself. Recently, factors have been identified for the activation of adult mammalian Müller cells to a RPC state in vivo. Summary: Whereas cell transplantation still holds potential for retinal repair, activation of the dormant native regeneration process may lead to a more successful process including greater integration efficiency and proper synaptic targeting.

Interactions of anaerobic gut bacteria, such as Clostridium difficile , with the intestinal mucosa have been poorly studied due to challenges in culturing anaerobes with the oxygen-requiring gut epithelium. Although gut colonization by C. difficile is a key determinant of disease outcome, precise mechanisms of mucosal attachment and spread remain unclear. Here, using human gut epithelial monolayers co-cultured within dual environment chambers, we demonstrate that C. difficile adhesion to gut epithelial cells is accompanied by a gradual increase in bacterial numbers. Prolonged infection causes redistribution of actin and loss of epithelial integrity, accompanied by production of C. difficile spores, toxins, and bacterial filaments. This system was used to examine C. difficile interactions with the commensal Bacteroides dorei , and interestingly, C. difficile growth is significantly reduced in the presence of B. dorei . Subsequently, we have developed novel models containing a myofibroblast layer, in addition to the epithelium, grown on polycarbonate or three-dimensional (3D) electrospun scaffolds. In these more complex models, C. difficile adheres more efficiently to epithelial cells, as compared to the single epithelial monolayers, leading to a quicker destruction of the epithelium. Our study describes new controlled environment human gut models that enable host–anaerobe and pathogen–commensal interaction studies in vitro .

A three-dimensional thermoresponsive fibrous scaffold system for the subsequent extended culture and enzyme-free passaging of a range of mammalian cell types is presented. Poly(PEGMA188) was incorporated with poly(ethylene terephthalate) (PET) via blend-electrospinning to render the fibre thermoresponsive. Using primary human corneal stromal stem cells as an therapeutically relevant exemplar, cell adhesion, viability, proliferation and phenotype on this fibrous culture system over numerous thermal enzyme-free passages is described. We also illustrate the versatility of this system with respect to fabricating thermoresponsive fibres from biodegradable polymers and for the culture of diverse mammalian cell types including mesenchymal stem cells, colon adenocarcinoma cells, and NIH-3T3 fibroblasts. This thermoresponsive scaffold system combines the advantages of providing a physiologically relevant environment to maintain a desirable cell phenotype, allowing routine enzyme-free passaging and expansion of cultured cells, whilst offering mechanical support for cell growth. The system described in this study presents a versatile platform for biomedical applications and more specfically for the expansion of mammalian cells destined for the clinic.

33Interactions of anaerobic gut bacteria with the intestinal mucosa have been poorly 34 studied due to challenges in culturing anaerobes with the oxygen-requiring gut 35 epithelium. Gut colonization by the anaerobic pathogen Clostridium difficile, a leading 36 cause of hospital-acquired diarrhea, is a key determinant of disease outcome. However, 37 precise mechanisms of mucosal attachment and spread remain unclear. Here, using 38 human gut cells co-cultured within controlled dual environment chambers, we describe 39 the dynamics of C. difficile infection. We demonstrate that C. difficile adheres and 40 multiplies as micro-communities when grown on a mucin-producing Caco-2/HT29-MTX 41 gut epithelial cell layer. Prolonged infection causes redistribution of actin and loss of 42 epithelial integrity, accompanied by production of C. difficile spores, and interestingly C. 43 difficile filamentation, a potentially new mechanism of C. difficile persistence. 44Intriguingly, we find that even in the absence of major C. difficile toxins, the bacterium is 45 able to disrupt epithelial barrier function. To examine C. difficile-commensal interactions, 46 we co-cultured C. difficile with the anaerobic gut commensal Bacteroides dorei. B. dorei 47 adheres to gut cells and inhibits C. difficile multiplication. Furthermore, we have 48 developed novel multilayer and three-dimensional gut models containing gut 49 myofibroblasts. C. difficile adheres more efficiently to epithelial cells in these models 50 compared to the monolayer systems, inducing a stronger chemokine response, 51 indicating a role for myofibroblasts in early host responses to C. difficile. Our study 52 describes new human gut models which effectively recreate the C. difficile infection 53 process, and enable host-anaerobe and pathogen-commensal interaction studies in 54 vitro. 55 . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/269035 doi: bioRxiv preprint first posted online Feb. 21, 2018; 3 56 57 IMPORTANCE 58 59Understanding the host responses to anaerobic gut pathogens or commensals, and 60 gaining mechanistic insights into the three-way interaction between pathogens, 61 commensals and the gut epithelium is fundamental to advancing our knowledge of gut 62 microbiome function. Host-pathogen interaction studies of the human anaerobic 63 pathogen, C. difficile and other gut-associated anaerobes is challenging due to the lack 64 of appropriate in vitro systems. In this study, employing novel two and three-65 dimensional models, we have tracked C. difficile infection over longer time scales, 66 examining the attachment and invasion of the gut epithelium and the effects of key 67 virulence factors. Furthermore, we demonstrate interactions of other important gut 68 commensals like Bacteroides with the epithelium and with C. difficile. This study 69 provides new insight into the molecular and cellular events that occur at the ...

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