Abstract
Abstract: :
Purpose: It has been observed in several tissues that direct isolation of cells in serum–free media and on nonadhesive substrates results in the formation of spherical clusters of cells known as free–floating spheres. Such free–floating spheres have been hypothesized to contain undifferentiated progenitor cells capable of differentiating into different cell types. Our goal was to isolate and characterize such free–floating spheres from HTM cell primary cultures. Methods: HTM cells were incubated in serum–free media and on a nonadhesive substrate. Individual free–floating spheres generated in these conditions were cloned in 96–well plates and their proliferative capacity was evaluated by monitoring their size increase over time. Cell differentiation was induced by culturing the spheres onto adhesive substrates in the presence of serum. The expression of the TM markers, MGP and CHI3L1, was examined using recombinant adenoviruses containing the respective promoters. Ultrastructure and gene expression profile of the free–floating spheres were compared to that of the differentiated cells generated from the spheres as well as to HTM cells grown in standard cell culture conditions. Results: HTM cells incubated in serum–free media and on nonadhesive substrate generated free–floating spheres that could be grown for more than three months. Addition of serum to the culture media promoted the attachment of the spheres to the substrate, migration of cells from the spheres, and differentiation into cells phenotypically similar to normal TM cells. Both infection with the recombinant adenoviruses and gene array analysis demonstrated the expression of CHI3L1 and MGP. Ultrastructural analysis indicated that these TM free–floating spheres were highly dynamic structures demonstrating a distinct radial gradient of cell proliferation, survival, apoptosis, and phagocytosis. Gene profiling analysis of the spheres demonstrated upregulation of nestin, LIF, BDNF, and several interleukins and chemokine ligands compared to HTM cells. Conclusions: The expression of CHI3L1 and MGP indicates that free–floating spheres likely originate from HTM cells. The expression of a marker for precursor neural cells (nestin), as well as genes involved in the regulation of progenitor cell differentiation (LIF and BDNF), together with their high replicative potential, suggests that these free–floating spheres might contain relatively undifferentiated or progenitor HTM cells. The availability of TM progenitor cell cultures could constitute a useful tool to investigate cell therapy approaches targeting the TM in glaucoma.
Keywords: trabecular meshwork • gene/expression • microscopy: electron microscopy