Human neural progenitor cells promote photoreceptor survival in retinal explants.

Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-beta1 and TGF-beta2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of growth factors secreted from the HNPCs.

Progenitor cell-derived factors enhance photoreceptor survival in rat retinal explants.

Explantation of postnatal rat retinas is associated with degenerative events that show morphological similarities to human retinal degenerative disorders. The most evident morphological features are photoreceptor apoptosis involving caspase-3 and Müller cell activation. The purpose of the present study was to determine the content of protective factors in rat retinal progenitor cells and analyze the influence of the identified factors on the survival of photoreceptor cells and retinal gliosis. Tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF) were identified as putative beneficial factors, and their combined effect was examined in rat retinal explant cultures. Photoreceptor apoptosis was estimated by cell counts of cleaved caspase-3 and caspase-12 immunolabeled as well as TUNEL labeled cells. TIMP-1 and VEGF in combination significantly suppressed photoreceptor apoptosis involving caspase-3 activation. Cell counts of caspase-12 and TUNEL labeled photoreceptors showed no significant difference between the experiment and control retinas. TIMP-1 and VEGF appeared to have no effect on Müller cell activation as measured by GFAP and Ki-67 immunohistochemistry. Our data suggest that TIMP-1 and VEGF in combination promote the survival of photoreceptor cells in rat retinal explants, possibly by affecting a caspase-3 signaling pathway.

Further assessment of neuropathology in retinal explants and neuroprotection by human neural progenitor cells.

Explanted rat retinas show progressive photoreceptor degeneration that appears to be caspase-12-dependent. Decrease in photoreceptor density eventually affects the inner retina, particularly in the bipolar cell population. Explantation and the induced photoreceptor degeneration are accompanied by activation of Müller and microglia cells. The goal of this study was to determine whether the presence of a feeder layer of human neural progenitor cells (hNPCs) could suppress the degenerative and reactive changes in the explants. Immunohistochemical analyses showed considerable sprouting of rod photoreceptor axon terminals into the inner retina and reduced densities of cone and rod bipolar cells. Both sprouting and bipolar cell degenerations were significantly lower in retinas cultured with feeder layer cells compared to cultured controls. A tendency toward reduced microglia activation in the retinal layers was also noted in the presence of feeder layer cells. These results indicate that hNPCs or factors produced by them can limit the loss of photoreceptors and secondary injuries in the inner retina. The latter may be a consequence of disrupted synaptic arrangement.