RPE and neuronal differentiation of allotransplantated porcine ciliary epithelium-derived cells.

PURPOSE: Cell replacement has the potential to be applied as a therapeutic strategy in retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration (AMD) for which no adequate pharmacological and surgical treatments are currently available. Although controversial, the use of ciliary epithelium (CE)-derived cells is supported by evidence showing their differentiation into retinal phenotypes. This study examines the differentiation potential of porcine CE-derived cells in vitro and their survival, migration, morphological characteristics, and immunohistochemical phenotype in vivo, upon transplantation into the subretinal space of normal pigs. METHODS: Cells were isolated from the CE of postnatal pigs and were grown in a suspension sphere culture. Differentiation was assessed in vitro after exposure to laminin and the addition of serum. For transplantation, CE-derived spheres were dissociated, labeled with CM-DiI vital dye, and the cells were injected subretinally into one eye of eight week-old allorecipients. The eyes were examined at eight days and at two and four weeks after transplantation. RESULTS: Cells positive for neuronal and retinal pigment epithelium (RPE) markers were detected by immunohistochemistry in differentiation cultures. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) revealed upregulation of neuronal markers after in vitro differentiation. CM-DiI dye-labeled CE-derived cells dissociated from primary spheres survived for up to four weeks after transplantation in vivo. Some of the surviving cells migrated distantly from the injection site. Large clusters of transplanted cells integrated into the RPE layer and multilayered RPE-like structures positive for RPE65 were often observed. Grafted cells were also identified in the neuroretina where 5%-10% were positive for recoverin, protein kinase C alpha (PKCα), and calbindin. CONCLUSIONS: The efficient conversion to an RPE-like phenotype suggests that CE-derived cells could be a potential source of RPE for cell replacement. Our data also suggest that the ability of these cells to acquire neuronal phenotypes is influenced by the environment. Thus, pre-differentiated or (re)programmed CE-derived cells may be more amenable for retinal repair.

Occurrence of alpha- and beta-nortestosterone residues in the urine of injured male cattle.

The administration of anabolic steroids, for the purposes of growth promotion, to food-producing animals is banned in the EU. Among the compounds covered by this prohibition is ss-nortestosterone (beta-NT). This hormone is known to occur naturally in stallions and boars, and its main bovine metabolite, alpha-nortestosterone (alpha-NT), occurs naturally in pregnant cows and neonatal calves. However, neither compound is believed to occur naturally in male cattle. During 2006, the presence of alpha-NT and, on occasion, beta-NT was confirmed in male cattle (bulls and steers) slaughtered in Northern Ireland on welfare grounds, as a result of acute injury. Subsequent investigations revealed no evidence of abuse at any of the farms involved and revealed that the phenomenon also occurred in three other regions of the EU, in similarly injured animals. A hypothetical link to release of the adrenal steroid, dehydroepiandrosterone (DHEA), in response to the stress of the injury was tested. Following the intravenous administration of DHEA to two normal steers, beta-NT (but not alpha-NT) was confirmed in the urine of one steer. Thus, it may be concluded that both beta-NT and, by implication, alpha-NT can occur naturally in male cattle (or a specific cohort thereof) in contrast to previously accepted scientific knowledge.