ABSTRACT
OBJECTIVE: To demonstrate and compare polycationic-mediated cochlear gene transfer with linear polyethylenimine (PEI) via cochleostomy and osmotic pump infusion method. DESIGN: A dissociated cochlear culture was used to select the optimum nitrogen to phosphate ratio of PEI/DNA complexes to be used in vivo. The PEI-enhanced green fluorescent protein reporter gene DNA complex was introduced with single inoculation (cochleostomy) or with sustained delivery (osmotic pump method) into guinea pig cochleas and examined for transgene expression. SUBJECTS: Male Albino Hartley guinea pigs (250-350 g). RESULTS: The relatively low transfection efficiency of PEI limits its potential when compared with viral counterparts; however, sustained release of the vector solution was able to improve PEI's transfection efficiency. The PEI-infused cochleas maintained intact cellular and tissue architecture with absence of inflammation. Transfection confined to the perilymphatic space highlights the need to target the gene vector into the scala media if transfection is targeted at cells within the organ of Corti. CONCLUSION: These findings indicate that PEI is able to transfect the cochlea in vivo with sustained delivery and present an alternative for nonviral cochlear gene therapy.
Subject(s)
Cochlea/cytology , Cochlea/metabolism , Gene Transfer Techniques , Genetic Therapy/methods , Polyethyleneimine , Transfection/methods , Animals , Basilar Membrane/metabolism , Basilar Membrane/pathology , Cochlea/pathology , Genes, Reporter , Green Fluorescent Proteins/genetics , Guinea Pigs , Male , Microscopy, ConfocalABSTRACT
The high degree of bone marrow cell (BMC) plasticity has prompted us to test its restoration possibility in inner ear repair. Our aim was to determine the potential of these cells to transdifferentiate into specialized cochlea cell types after acoustic injury and BMC mobilization. Lethally irradiated mice were transplanted with BMCs from green fluorescent protein (GFP) transgenic mice and subjected to acoustic deafening 3 months later. In a separate experiment, stem cell factor and granulocyte colony-stimulating factor were administered to test the effect of BMC mobilization on bone marrow-derived cell (BMDC) transdifferentiation. All mice showed almost complete chimerism 3 months after bone marrow transplantation. Upon acoustic trauma, robust BMDC migration was observed in the deafened cochlea. GFP+ cell migration was most prominent during the first week after acoustic deafening, and these cells accumulated significantly at the spiral ligament, perilymphatic compartment walls, and limbus regions. Most of the BMDCs expressed CD45 and CD68 and were identified as macrophages. Upregulation of stromal-derived factor 1 (SDF-1) was also observed in the spiral ligament during the first week after acoustic deafening. Cytokine treatment resulted in increased BMC mobilization in the systemic circulation. However, the presence of any stem cell progenitors or the differentiation of BMDCs into any cell types expressing cochlea sensory, supporting, fibrocytic, or neuronal markers were not detected in the deafened cochlea. In conclusion, we have demonstrated the homing capability of BMDCs to the deafened cochlea, and these cells displayed mature hematopoietic properties without spontaneous transdifferentiation to any cochlea cell types after acoustic trauma or bone marrow mobilization.
