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Jie Ma, Chenying Guo, Guochun Chen, Desirée Cyr, Kameran Lashkari; Transfection of IGF-1 and IGFBPL-1 in Neuronal Progenitor Cells from Human Persistent Fetal Vascular for Neuroprotection. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5915.
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© ARVO (1962-2015); The Authors (2016-present)
Our laboratory has previously shown that cells isolated from human persistent fetal vasculature (hPFV) membranes are retinal progenitor cells, which differentiate into retinal neurons after intravitreal transplantation. We have also observed that in the DBA/2J pigmentary glaucoma mice model which exhibits profound loss of retinal ganglion cells (RGC), transplanted hPFVs integrated into the inner retinal layer. Based on these observations we hypothesized that hPFVs produced neuroprotective factors that would allow survive the ocular hypertensive environment of the DBA/2J mouse. We determined whether transfection of a known neuroprotective factor, insulin-like growth factor-1 (IGF-1) and its binding protein (IGFBPL-1) could confer more robust and global neuroprotection to host RGCs.
Plasmid for IGF-1 and IGFBPL-1 were constructed and spliced into pJ603-neo vectors expressing red (RFP) and green (GFP) fluorescent proteins, respectively. Cells were transfected by electroporation using one or both vectors and examined under fluorescence microscopy. Transfected cells were enriched by antibiotic selection and subjected to RT-qPCR and ELISA for quantitation of IGF-1 and IGFBPL-1. Transfected cells, hPFVIGF-1, hPFVIGFBLP-1 and hPFVIGF-1/IGFBLP-1 were studied under co-culture condition with cultured RGCs and evaluated for neuronal morphology, axonal growth and apoptosis.
Transfection of the vector expressing GFP-IGFBPL-1 was more efficient than the vector expressing RFP-IGF-1 (60% vs. 20%). Double transfection of these genes decreased the yield of expression to < 5% RT-qPCR showed that naive hPFVs expressed low levels of IGF-1 and IGFBPL-1; while transfected cells expressed significantly levels of these genes (the fold increased 3.7 and 15.8, respectively). ELISA examination also indicated that the protein expression of these genes in the transfected hPFVs was significantly increased. hPFVIGF-1 and hPFVIGFBLP-1 were differentiated into neurons by retinoic acid and exhibited better neuronal morphology and longer axonal lengths than control cells. In co-culture condition, these cells improved morphology of resident RGCs and decreased apoptosis signals.
Our data indicate that neuroprotective genes (such as IGF-1 and IGFBPL-1) could be successfully transfected and expressed into neuronal protenitor cells such as hPFVs. Expression of these proteins improves neuronal morphology, axonal growth and improves survival of RGCs. Transfected hPFVs may be good candidate cells for transplantation of the inner retinal to confer increased neuroprotection to host RGCs in disease condition.
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