Purification of Retinal Ganglion Cells from Differentiation Through Adult via Immunopanning and Low-Pressure Flow Cytometry.

The isolation and culturing of rodent retinal ganglion cells (RGC) is a key step in studying the function and cellular response of this crucial cell type. Typical methods used for isolation of RGCs include immunopanning or magnetic bead separation with antibodies targeting RGC specific protein markers. However, in developmental research, many of the most common markers, such as Thy-1, are not expressed in early stages of development. To help study these crucial early stage RGCs, we have developed a novel method that utilizes a transgenic mouse with a GFP tag on the protein BRN3 and a low-pressure fluorescence-activated cell sorter (FACS) system.

Purification of retinal ganglion cells using low-pressure flow cytometry.

Purified Retinal Ganglion Cells (RGCs) for in vitro study have been a valuable tool in the study of neural regeneration and in the development of therapies to treat glaucoma. Traditionally, RGCs have been isolated from early postnatal rats and mice, and more recently from human in vitro derived retinal organoids using a two-step immunopanning technique based upon the expression of Thy-1. This technique, however, limits the time periods from which RGCs can be isolated, missing the earliest born RGCs at which time the greatest stage of axon growth occurs, as well as being limited in its use with models of retinal degeneration as Thy-1 is downregulated following injury. While fluorescence associated cell sorting (FACS) in combination with new optogenetically labeled RGCs would be able to overcome this limitation, the use of traditional FACS sorters has been limited to genomic and proteomic studies, as RGCs have little to no survival post-sorting. Here we describe a new method for RGC isolation utilizing a combined immunopanning-fluorescence associated cell sorting (IP-FACS) protocol that initially depletes macrophages and photoreceptors, using immunopanning to enrich for RGCs before using low-pressure FACS to isolate these cells. We demonstrate that RGCs isolated via IP-FACS when compared to RGCs isolated via immunopanning at the same age have similar purity as measured by antibody staining and qRT-PCR; survival as measured by live dead staining; neurite outgrowth; and electrophysiological properties as measured by calcium release response to glutamate. Finally, we demonstrate the ability to isolate RGCs from early embryonic mice prior to the expression of Thy-1 using Brn3b-eGFP optogenetically labeled cells. This method provides a new approach for the isolation of RGCs for the study of early developed RGCs, the study of RGC subtypes and the isolation of RGCs for cell transplantation studies.

Adult Stem Cells, Tools for Repairing the Retina.

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.

A thermoresponsive three-dimensional fibrous cell culture platform for enzyme-free expansion of mammalian cells.

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 specifically for the expansion of mammalian cells destined for the clinic. STATEMENT OF SIGNIFICANCE: The lack of three-dimensional (3D) cell culture environments significantly impacts mammalian cell morphology, proliferation and phenotype in vitro. A versatile, 3D fibrous scaffold system for the extended culture and passaging of a range of clinically-relevant cell types is presented herein. This methodology can be used to fabricate thermoresponsive fibres from polymer blends of any polymer amenable to electrospinning and with a thermoresponsive component. A variety of mammalian cells cultured on the thermoresponsive system were detached from the surface solely by lowering the temperature whilst retaining high viability, a desirable cell phenotype, and supported long-term cell proliferation over numerous thermal enzyme-free passages. This is a significant advance for in vitro expansion of diverse cell types destined for the clinic.

Probing Clostridium difficile Infection in Complex Human Gut Cellular Models.

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.