title: Erythropoietin produced by genetic-modified NIH/3T3 fibroblasts enhances the survival of degenerating neurons.
abstract: 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, PC12-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 PC12-INT-EGFP cells was significantly enhanced. Surprisingly, a fraction of aggregated cytoskeletal EGFP-tagged α-internexin in PC12-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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University Taipei, Taiwan.
authors.forename: Yi-Chin
authors.initials: YC
authors.lastname: Li
authors.affiliation: Department of Medicine, Mackay Medical College New Taipei, Taiwan.
authors.forename: Shiu-Jau
authors.initials: SJ
authors.lastname: Chen
authors.affiliation: Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University Taipei, Taiwan.
authors.forename: Chung-Liang
authors.initials: CL
authors.lastname: Chien
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009410:Nerve Degeneration / Q000188:drug therapy* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D018696:Neuroprotective Agents; D011994:Recombinant Proteins; D004921:Erythropoietin
keywords: EPO; NIH/3T3 fibroblasts; neurodegeneration; neuronal survival