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J. Yang, P. Gu, S. Tao, T. Desai, H. Klassen; Sequential Quantitative Profiling of Porcine CNS Progenitor Cells on Biodegradable Nanowire Scaffolds. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4777. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously documented the potential benefits of transplanting CNS progenitor cells to the retina on biodegradable PLGA scaffolds and found that PLGA tended to differentiate the co-cultured progenitor cells. We now investigate whether this finding generalizes to polymers made with different material and structure.
GFP-transgenic pig brain-derived progenitor cells (gpBPCs) were isolated at gestational day 45 and cultured in DMEM:F12 containing N2 supplement and 20ng/ml bFGF and EGF. Cells were seeded onto poly(e-caprolactone) (PCL) biodegradable scaffolds with 4 different surface topographies: unmodified (plain), smoothened (smooth), short nanowire (SNW), and long nanowire (LNW). Expression of 7 genes was compared for seeded vs. unseeded controls. Morphology was documented via Nikon fluorescence microscope. Total RNA was extracted (Qiagen isolate kit) and treated with RNase-free DNase I before reverse transcription. Quantitative real-time PCR was performed with 7500 fast system (ABI).
Seeded gpBPCs showed varied morphologies, generally similar to proliferating controls. Changes in gene expression were less than an order of magnitude for all 7 porcine transcripts across all PCL scaffolds. The largest increase was for PKC-alpha on plain PCL (2x), followed by nestin on LNW (1.8x). The greatest decrease was for GFAP on plain (0.14x) followed by GFAP on smooth (0.43x). Looked at by gene, nestin was consistently up-regulated and Sox2 down-regulated on all surface topographies. GFAP was generally down-regulated. PKC-alpha was up-regulated or unchanged while beta3-tubulin, MAP2, and vimentin were either unchanged or down-regulated. Considered by polymer, LNW showed a net increase in expression across genes examined, while SNW showed a net decrease. Plain and smooth PCL tended towards controls, with smooth showing less variability.
PCL-based polymers generally favor the progenitor state based on morphology, consistent up-regulation of the BPC marker nestin, general down-regulation of the glial marker GFAP, and general maintenance of the pro-neuronal markers beta3-tubulin and MAP2. Plain PCL and LNW might promote neural lineage. Together with other studies, these data illustrate that both the chemistry and topography of polymer scaffolds can effect cellular gene expression and thus specific constructs differ in potential application to retinal diseases.
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