Abstract
Purpose:
We have shown that neural progenitor cells isolated from human persistent fetal vasculature (herein, called hNPPFV cells) can differentiate into retinal neurons and specifically, retinal ganglion-like cells (RGC) after transplantation into the mouse vitreous. Based on our observations, we assessed whether hNPPFV cells could be good candidate cells to locally delivery neuroprotective factors to the inner retina and confer significantly global neuroprotection against stress-induced RGC loss.
Methods:
IGF-1 cDNA were cloned into pJ603-neo vector expressing a red fluorescence reporter gene (tdTomato) to generate fluorescent fusion proteins and tranfected into hNPPFV cells. hNPPFVs expressing IGF-1-tdTomato and tdTomato empty vector (hNPPFVIGF-1/tdTomato and hNPPFVtdTomato) were subjected to qRT-PCR, Western blot analysis, ELISA, co-culture and immunostaining for quantitation of IGF-1 expression. The effects of IGF-1 on apoptosis and axonal growth of RGCs were also studied in presence of two IGF-1 analogs (H-1356 and NBI-31772), as well as in presence of a blocking antibody against the mouse IGF-1 receptor in the co-culture system.
Results:
Plasmids were successfully transfected to over 90% of hNPPFV cells. Expression of IGF-1tdTomato mRNA and protein significantly increased after the transfection compared to controls. The concentration of secreted IGF-1tdTomato in the conditioned medium reached the peak of 220 ng/ml at day 3 post-transfection. In co-culture condition, hNPPFVIGF-1/tdTomato cells improved morphology of RGCs and decreased apoptotic signals. Specifically, IGF-1tdTomato significantly increased the survival rate of RGCs (22 ± 13% vs. 11 ± 5% for control), and enhanced the outgrowth (93 ± 45 µm vs. 17 ± 9 µm for control) and branching of axons (1 - 5/cell vs. 1 - 3/cell for control). Our data indicate that IGF-1tdTomato protein significantly improves neuronal morphology, axonal growth and survival of RGCs compared to tdTomato (control) alone (P < 0.05, Independent Samples t-test).
Conclusions:
Transfected hNPPFVIGF-1/tdTomato cells can secret clinically-significant concentrations of IGF-1tdTomato protein to improve survival and axonal outgrowth of RGCs in vitro. These findings also indicate that IGF-1 plays a key role in neuroprotection of RGCs and should be considered as a key neuroprotective factor for future studies.
Keywords: 615 neuroprotection •
531 ganglion cells •
533 gene/expression