Erythropoietin produced by genetic‐modified NIH /3T3 fibroblasts enhances the survival of degenerating neurons

Background Erythropoietin ( EPO ) has potent neuroprotective effects. The short‐term delivery of high‐dose EPO seemed to improve patients’ neuromuscular functions; however, excessive EPO resulted in systematically high hematocrit and thrombotic risk. In our study, we established a cellular material for future in vivo studies of neurodegenerative diseases based on EPO provided regionally at a nontoxic level. Methods A mouse EPO cDNA was subcloned into the pCMS ‐ EGFP vector and transfected into NIH /3T3 fibroblasts to design a biological provider that can regionally release EPO for the treatment of neurological diseases. After G418 selection, a stable EPO ‐overexpressing cell line, EPO ‐3T3‐ EGFP , was established. To further confirm the neuroprotective abilities of secreted EPO from EPO ‐3T3‐ EGFP cells, a cell model of neurodegeneration, PC 12‐ INT ‐ EGFP , was applied. Results The expression level of EPO was highly elevated in EPO ‐3T3‐ EGFP cells, and an abundant amount of EPO secreted from EPO ‐3T3‐ EGFP cells was detected in the extracellular milieu. After supplementation with conditioned medium prepared from EPO ‐3T3‐ EGFP cells, the survival rate of PC 12‐ INT ‐ EGFP cells was significantly enhanced. Surprisingly, a fraction of aggregated cytoskeletal EGFP ‐tagged α ‐internexin in PC 12‐ INT ‐ EGFP cells was disaggregated and transported into neurites dynamically. The immunocytochemical distribution of IF proteins, including NF ‐M, phosphorylated‐ NF ‐M, and the α ‐ INT ‐ EGFP fusion protein, were less aggregated in the perikaryal region and transported into neurites after the EPO treatment. Conclusion The established EPO ‐overexpressing NIH /3T3 cell line, EPO ‐3T3‐ EGFP , may provide a material for future studies of cell‐based therapies for neurodegenerative diseases via the secretion of EPO on a short‐term, high‐dose, regional basis.