P2. As shown in Fig. 70.1c, immunohistochemistry revealed that nanoparticle transferred protein (3B6) is detected at PI-2 after P2 injection, while endogenous protein is not apparent this early. Interestingly, when we used this same IRBP construct to generate stable transgenic mice, the transgenic RDS protein was not detected before the endogenous protein (Fig. 70.1d). Our data shown here and previously (Farjo et al. 2006) demonstrate that CK30PEG nanoparticles can efficiently transfect both mitotic and post-mitotic cells and induce rapid-onset and sustain gene expression. When the Rds gene is delivered, nanoparticles are capable of driving gene expression earlier than the native RDS without ectopic expression outside the photoreceptor OS layer. Our current studies are focused on determining whether these nanoparticles can rescue the rds+/– adRP disease phenotype, thus making them potential candidates for clinical use in the eye.

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Chapter 71

PEDF Promotes Retinal Neurosphere Formation

and Expansion In Vitro

Anna De Marzo, Claudia Aruta, and Valeria Marigo

Abstract 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.

71.1 Introduction

Many forms of blindness arise from photoreceptor degeneration and to date have no satisfactory solutions to rescue retinal tissues.

Mammalian eyes do not have a regenerative capability characteristic of lower vertebrates but recent evidences have demonstrated that ciliary body (CB), a

A. De Marzo (B)

Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy e-mail: anna.demarzo@unimore.it

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_71,C Springer Science+Business Media, LLC 2010

structure analogous to the CMZ of lower vertebrates, contains retinal stem cells (RSC) (Moshiri et al. 2004; Nishiguchi et al. 2008; Tropepe et al. 2000; Ahmad et al. 2000; Coles et al. 2004; Inoue et al. 2005).

Multipotent retinal stem or progenitor cells can be isolated from the ciliary body of an adult mammalian retina using a neurosphere culture. Tropepe et al. have demonstrated that one out of 500 CB cells gives rise to a clonal aggregate called neurosphere consisting of 12,000 pigmented cells and that each neurosphere can generate six to eight daughter neurosphere colonies.

Although the presence and the function of retinal stem cells (RSCs) in vivo remains unknown RSCs proliferate in vitro; they differentiate into cell subtypes expressing markers of certain mature retinal neurons such as bipolar cells, photoreceptor cells or Mueller glia suggesting the possibility that these cells may represent a potential cell source in transplantation therapy for retinal diseases (Tropepe et al. 2000; Giordano et al. 2007). Differentiation is induced by plating the cells on an extracellular matrix substrate and exposing the cells to 1% Fetal Bovine Serum (FBS). In these conditions the percentage of cells that undertake a rod-like fate is about 30–40% as assessed by rhodopsin and the Pde6b co-expression. Growth of neurospheres with FGF without EGF before differentiation favors rod-like differentiation (Giordano et al. 2007).

Primary cultures of cells collected from the CB are in FBS free medium and the formation of neurospheres takes about 5–7 days. The number of neurospheres is dependent upon the growth factors to which cells are exposed, such as FGF or EGF. A limitation in the culture of RSC comes from low ability of these cells to be expanded in vitro. In fact, a single retinal neurosphere composed of about 12,000 cells upon passaging does not give rise to 12,000 new spheres but only an average of 3. Furthermore, no expansion of the culture has been obtained from the third passage (Giordano et al. 2007). Amelioration of the culture condition is therefore a fundamental aspect that needs to be addressed if we want to bring RSC to therapeutic applications.

Previous works have shown that pigment epithelium-derived factor (PEDF) secreted by the murine subventricular zone (SVZ) promotes self-renewal and activation of slowly dividing adult neural stem cells (NSC) in vitro (Ramirez-Castillejo et al. 2006). PEDF is a neurotrophic antiangiogenic factor initially purified from conditioned media of retinal pigment epithelial cells (Tombran-Tink et al. 1991; Becerra et al. 1995). PEDF is secreted from many different cells and can modulate cell cycle progression (Pignolo et al. 2003). Thus PEDF may have positive effects on RSC culture.

Here we show that the treatment of RSC with PEDF together with FGF increases the neurosphere yield and, ameliorates self-renewal in neurosphere passaging. We then tested whether PEDF could affect differentiation to rod-like fate. This improvement does not decrease the number of cells expressing rhodopsin and Pde6b. Moreover, cells differentiated from a FGF+PEDF culture did not express bipolar cell markers.