Purchase this article with an account.
J. Phillips, D. C. Otteson; Neuronal Gene Expression Profiling of Muller-Derived Stem Cells in 2- and 3-Dimensional Cultures. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3216.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To determine whether growth in 3-dimensional (3D) cultures promotes expression of neuronal genes by Muller-glia derived retinal stem cells.
Conditionally immortalized mouse Muller cells were grown in uncoated dishes (2D) or encapsulated in Puramatrix (BD) hydrogels (3D) in transwell inserts. Neurosphere formation was induced with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Differentiation was induced by transient exposure to FGF2 prior to growth factor withdrawal. Gene expression was assayed by immunocytochemistry, quantitative RT-PCR (QPCR) with retinal specific primers and a neurogenesis PCR array (SABiosciences). Statistical analysis used Relative Expression Software Tool (REST) or RT2 Profiler PCR Array Data Analysis software (SABiosciences). Phalloidin conjugated with rhodamine was used to image cytoskeletal changes.
Following serum withdrawal and EGF+FGF2 stimulation, many Muller cells in 3D cultures formed neurospheres that were smaller than those formed in 2D cultures. Following growth factor withdrawal, cells in 3D culture migrated out of the spheres, extending processes into the matrix as revealed by phalloidin labeling and DIC images. Expression of genes associated with neuronal differentiation (NeuroD, Mef2c), neurite growth, axon guidance and synaptogenesis (BMP2, Neuronal Pentraxin, Robo1, Nrcam) were significantly higher in 3D compared to 2D cultures. Expression of M-cone opsin was greater in 3D culture, although rhodopsin was equally expressed in both 2D and 3D cultures. Rhodopsin kinase, CRX and the bipolar gene mGluR6 were only expressed in 3D cultures, whereas the bipolar gene Cabp5 and the ganglion cell gene melanopsin were only expressed in 2D cultures.
The neuronal characteristics of Muller-derived stem cells in vitro are generally enhanced in 3D cultures, although some genes are preferentially upregulated in 2D conditions. Experiments are underway to replicate these studies using primary Muller cells. Future studies will focus on effects of matrix density on differentiation and survival of Muller-derived stem cells.
This PDF is available to Subscribers Only