Stably transduced cells were selected in media containing puromycin

ipotent cells differentiate into a specific cell type after reaching the target site after transplantation. For instance, previous studies have found that rod precursors can successfully integrate into adult or degenerating retina and form classic triad synaptic connections with second-order bipolar and horizontal cells. In the second paradigm, cells are able to secret NTFs in culture media or in the target location leading to the intended effects in a paracrine manner with mild direct cellular integration. Studies regarding this paradigm confirm that RGC and axon survival can be increased both in vitro and in vivo by transplanting human dental pulp stem cells or bone marrow-derived mesenchymal stem cells by intravitreal injection. In general, grafted cells remain viable for a relatively short period within the target PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783706 area. A similar concept has been applied to retinal neuronal stem/progenitor cells, which can be used for direct replacement of lost cells such as photoreceptors, or to enhance retinal survival after injury through delivery of NTFs. Progenitor-like cells of the retina generally include cells from the ciliary marginal zone and Mller glia. We have previous described PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783858 a retinal neuronal cell line whose lineage is strictly restricted to a neuronal and not glial phenotype. Upon differentiation, these cells develop RGC-like characteristics in vitro and in vivo after induction by retinoic acid. After intravitreal injection, hNPs penetrate and integrate into the host’s inner retina, mostly within the RGC and nerve fiber layers, and extend up to the inner nuclear layer. We investigated whether hNPs could fulfill one or both SNDX-275 paradigms in a glaucomatous model of RGC injury. To enhance their trophic effects, we stably transfected hNPs with a vector to secrete IGF-1, a known NTF, in the form of a fusion protein with TD. It has been shown that intravitreal injection of IGF-1 inhibits secondary cell death in axotomized RGCs. In addition, in vitro and in vivo studies have showed that IGF-1 is developmentally-regulated and its expression in the retina dramatically decreases after birth. Based on these observations, we postulated that IGF-1 would enhance the survival of RGCs and maintain regional density of axons despite the glaucomatous environment. For this purpose, we utilized a model in which elevation of intraocular pressure induced by injection of microbeads in the anterior chamber of eyes yields a reproducible loss of RGCs. Given that IGF-1 has a very short half-life of about half day, without a delivery system, it would require multiple intravitreal injections to maintain a therapeutically relevant level that would elicit its trophic effects. To overcome this, we opted for a cell-based system that provided sustained delivery of IGF-1. hNPs were used to locally deliver biologically active IGF-1 in the form of a fusion protein with TD to facilitate its detection in situ. The purpose of these experiments was to test the hypothesis that hNPs could be used as a means of local delivery for IGF-1 to the host retina. We evaluated whether hNPs could be stably transfected to express sustained levels of biologically active IGF-1 and explored visualization of the secreted protein and assess whether secretion of IGF-1 could confer global neuroprotection of RGCs both in vitro and in experimentally induced stress such as that observed in a model of rodent glaucoma. In this study, we show that hNPs that secrete biologically active IGF-1 in the form