PEDF Promotes Retinal Neurosphere Formation and Expansion In Vitro

The retina is subject to degenerative conditions leading to blindness. Although retinal regeneration is possible in lower vertebrates, it does not occur in the adult mammalian retina. Retinal stem cell (RSC) research offers unique opportunities for developing clinical application for therapy. The ciliary body of adult mammals represents a source of quiescent RSC. These neural progenitors have a limited self-renewal potential in vitro but this can be improved by mitogens. Pigment Epithelium Derived Factor (PEDF), a member of the serpin gene family, is synthesized and secreted by retinal pigment epithelium (RPE) cells. We tested combinations of PEDF with fibroblast growth factor (FGF) during RSC growth to evaluate self-renewal and subsequent differentiation into retinal-like neuronal cell types. Medium supplemented with FGF + PEDF enhanced the RSC yield and more interestingly allowed expansion of the culture by increasing secondary retinal neurospheres after the 1st passage. This effect was accompanied by cell proliferation as revealed by BrdU incorporation. PEDF usage did not affect rod-like differentiation potential. This was demonstrated by immunofluorescence analysis of Rhodopsin and Pde6b that were found similarly expressed in cells derived from FGF or FGF + PEDF cultured RSC. Our studies suggest a possible application of PEDF in Retinal Stem Cell culture and transplantation

BIOCOMPATIBILITY OF COLLAGEN MEMBRANES ASSESSED BY CULTURING HUMAN J111 MACROPHAGES CELLS.

We have carried out an in vitro study on the interactions of human macrophages (J111) with two different membranes made of collagen type I and II, isolated from horse tendon and from horse articular and trachea cartilagene in order to obtain data on their biocompatibility. We have described the morphology of cell seeded on collagen films, and we have evaluated their proliferation as well as cytokine production as indicator of macrophage activation. The inflammatory response may in fact induce the destruction of collagen membranes and may interfere with cell and tissue behaviour. Results might be relevant for in vivo application such ad “tissue engeneering” and /or specialized cells implantation

Biocompatibility of Collagen Membranes Assessed by Culturing Human J111 Macrophage Cells

We have carried out an in vitro study on the interactions of human macrophages (J111 cell line) with different scaffolds made of type I and II collagen, isolated from horse tendon and from horse articular and trachea cartilage, in order to assess growth properties and biocompatibility of these membranes. We have therefore evaluated cell adhesion and proliferation as well as cytokine production considered an indicator of macrophage activation. The inflammatory response is in fact one of the major causes of collagen destruction thus interfering with cell and tissue behaviour. Moreover, the morphology of cells, seeded on membranes selected for the best characteristics, was described. Results might be relevant for in vivo application such ad “tissue engineering” and/or specialized cells implants

Differentiation of retinal pigment epithelium from retinal neurospheres of the adult ciliary body

Transplantation of retina pigment epithelium (RPE) has great potentials for the cure of several retinal degenerative diseases such as age-related macular degeneration (AMD). The source of cells for this type of therapy is still under evaluation even if clinical trials with RPE derived from human embryonic stem cells have recently started. We evaluated the ciliary body as a source of cells to differentiate RPE in vitro. The ciliary body is an accessible tissue that can be easily obtained from post-mortem donors. Retinal neurospheres can be generated from ciliary body cells after dissociation and growth in clonal conditions. We deeply characterized differentiation of RPE cells from retinal neurospheres derived from murine eyes. We will also discuss the differentiation protocol developed for the human tissue

In vitro differentiation of retinal pigment epithelium from adult retinal stem cells.

One of the limitations in molecular and functional studies of the retinal pigment epithelium (RPE) has been the lack of an in vitro system retaining all the features of in vivo RPE cells. Retinal pigment epithelium cell lines do not show characteristics typical of a functional RPE, such as pigmentation and expression of specific markers. The present study was aimed at the development of culture conditions to differentiate, in vitro, retinal stem cells (RSC), derived from the adult ciliary body, into a functional RPE. Retinal stem cells were purified from murine eyes, grown as pigmented neurospheres and induced to differentiate into RPE on an extracellular matrix substrate using specific culture conditions. After 7-15 days of culture, pigmented cells with an epithelial morphology showed a polarized organization and a capacity for phagocytosis. We detected different stages of melanogenesis in cells at 7 days of differentiation, whereas RPE at 15 days contained only mature melanosomes. These data suggest that our protocol to differentiate RPE in vitro can provide a useful model for molecular and functional studies