of a fusion protein with TD selectively enhance survival and neurite outgrowth when co-cultured with P0 mouse RGCs, and that this effect can be abrogated with selective inhibitors. Furthermore, using an established and reproducible model of glaucoma, we show that sustained delivery of IGF-TD by hNPIGF-TD cells effectively protect against loss of RGCs. This 2 / 24 Progenitor Cells Expressing IGF-1 on Retinal Ganglion Cell Survival neurotrophic effect was not observed in untransfected hNPs and hNPs that secrete only TD. Analysis of signal pathways by RT-PCR suggests that at least some of the neurotrophic mechanisms of IGF-1 may be related to its anti-inflammatory activity. These order Seliciclib findings provide experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Materials and Methods Ethics Statement and Animals This study was approved by the IACUC of the Schepens Eye Research Institute/Mass. Eye and Ear Infirmary for use of animals and by the committee on microbial safety, COMS, at Harvard University. This study adheres to the Helsinki Agreement for clinical studies and use of clinical materials for research. This study was also reviewed and approved by the IRB of Schepens Eye Research Institute /Massachusetts Eye and Ear Infirmary, Harvard Medical PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786681 School. The study proposal, consent form and method of obtaining consent were approved by the IRB. Each participant was given ample time to read and understand the IRB-approved consenting form prior to his/her surgical procedure. Each subject’s questions and concerns were addressed. A written consent was obtained from each participating subject and each subject received a copy of the signed consent form. We carefully followed the protocol to perform our animal experiments. After the microbead injection and cell transplantation, all animals were closely monitored to ensure no observable signs of inflammatory responses or overt damage in the anterior segment or cataract formation. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. Transfection of hNPs hNPs were previously isolated from human persistent fetal vasculature retrolental membranes dissected during vitreoretinal surgery from a few young donors. These membranes were cultured according to an established protocol. The coding sequences of IGF-TD or TD were inserted into a pJ603-neo plasmid backbone, generating a fusion protein with TD PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19785045 tagged to the C-terminus of IGF-1, or generating TD protein alone, respectively. Gaussia luciferase signal peptide connected at the N-terminus was used to improve IGF-TD or TD expression and secretion. Plasmids were transfected into DH5 Competent E. Coli cells, expanded and purified using the EndoFree Plasmid Maxi Kit. Cells were seeded onto 6-well plates at 1 105 cells/well. The next day, the culture 
medium in each well was replaced with 1 ml the transfection complex, 240 l plasmid, and serum-free X-vivo medium. The transfection medium was replaced with regular growth medium comprised of X-vivo medium supplemented with 10% of fetal bovine serum, 1:50 B27, 1:100 N2, 10 ng/ml basic fibroblast growth factor, 20 ng/ml epidermal growth factor, and 50 g/ml nystatin after a 5 hr incubation at 37C. Immunocytochemical Analysis hNPIGF-TD, hNPTD, and untransfected hNPs were grown and maintained in X-vivo media supplemented with FGF-2 and EGF. Cells were washed in plain X-vivo med
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