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M. A. Beaumont, E. Pashuck, B. Kovacs, S. Xu, S. J. Stupp, V. P. Sarthy; Growth of Retinal Stem Cells in Three-Dimensional Peptide-Amphiphile Nanofiber Matrices. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4799.
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Self-assembling peptide-amphiphile (PA) materials are designed to contain a long, hydrophobic alkane (C16) tail to which peptide head groups are attached and form elongated micelles, or nanotubes, with the addition of media or biological fluids. Our group has assessed the ability of PA nanofiber matrices containing epitopes from laminin-1 (IKVAV) to sustain growth and viability of adult retinal stem cells (RSCs) isolated from the mouse ciliary epithelia.
RSCs were isolated from the ciliary body of adult mice (1-3 mo.) and cultured in serum free media (SFM) containing FGF-2 and EGF for 10-12 days. Neurospheres were then embedded within the gels and cultured in differentiation media containing FGF-2 and 1% FBS for an additional period (2-28 days). Laminin-coated coverslips were employed for control differentiation cultures. Brightfield and confocal fluorescence microscopy was used to assess the cell morphology and viability (calcein/ethidium homodimer cytotoxicity assay). Immunofluorescence was used to monitor presence of RSC and neuronal markers.
It was found that the RSC spheres survive the self-assembly process and begin to migrate out from the spheres in all directions. Immunocytochemical assays show expression of retinal stem cell and early neuronal markers (Pax6 and β3-tubulin, respectively) within the RSC neurospheres after 5-7 days in differentiation culture. At further time points, cells can be observed to migrate up to 2-3 sphere diameters into the matrices. While the time-course for migration and expression of β3-tubulin was similar to that of laminin controls, the three-dimensional nature of the PA matrices conferred a morphology upon the RSCs that was distinct from the control 2-D surfaces. We have also begun to characterize the response of the RSCs to the unique epitope-density presentation found in these matrices. This is accomplished by titration of the active ligand content with a biologically inert, control sequence (EQS).
Laminin-1 epitope-containing PAs support RSC survival and growth and show promise for influencing differentiation.
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