Characterization of free-floating spheres from human trabecular meshwork (HTM) cell culture in vitro

Abstract

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 multipotent progenitor cells. Our goal was to isolate and characterize such free-floating spheres from HTM cell primary cultures.For this purpose, HTM cells were incubated in serum-free media and on a nonadhesive substrate. Individual free-floating spheres generated in these conditions were isolated in 96-well plates, and their proliferative capacity was evaluated by monitoring their size increase over time. The expression of the TM markers, MGP and CHI3L1, was examined using recombinant adenoviruses containing the respective promoters. Morphology of the free-floating spheres was analysed in semithin sections, and the gene expression profile was obtained using Human Genome U133 Plus 2.0 Affymetrix microarrays.HTM cells incubated in serum-free media and on nonadhesive substrate generated free-floating spheres that could be grown for more than 3 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. Gene profiling analysis demonstrated strong similarities between the gene expression profiles of the spheres and HTM cell monolayers. Both infection with the recombinant adenoviruses and gene array analysis demonstrated the expression of CHI3L1 and MGP, indicating that free-floating spheres likely originate from HTM cells. Gene array analysis also showed expression of the marker for neural precursor cells nestin, as well as leukemia inhibitory factor, a gene involved in the maintenance of the undifferentiated state of progenitor cells. Analysis of semithin sections indicated that these TM free-floating spheres were highly dynamic structures demonstrating a distinct radial gradient of cell proliferation, survival, and apoptosis. Extensive up-and down-regulation of gene expression was associated with the processes of sphere attachment and cell migration after the addition of serum.These results suggest that HTM primary cultures might contain relatively undifferentiated or progenitor cells. The availability of TM progenitor cell cultures could constitute a useful tool to investigate cell therapy approaches targeting the TM in glaucoma.

Bovine Posterior Limbus: An Evaluation of an Alternative Source for Corneal Endothelial and Trabecular Meshwork Stem/Progenitor Cells

“…The evidence suggesting the presence of stem cells in the human TM has been accumulating for some time [6,7,9,12,15]. To date, there have been five studies that successfully cultivated the TM stem cells in vitro [16][17][18]72,73]. These data proved that stem/progenitor cells from the human TM could be cultivated and home-in to their tissue of origin.…”

“…Published works on the cultivation of CE or TM stem cells have been largely confined to the human species [16][17][18][19][20][21][22]. Given the scarcity of human donor eyes, a supplementary animal model for studying the PET cells would be hugely advantageous.…”

Stem Cells in Ophthalmology

“…trabecular meshwork progenitor cells can be expanded in three-dimensional sphere-like cultures (Gonzalez et al, 2006). Cells from these cultures express markers and gene expression profiles associated with monolayer cultures of TM stem cells (Gonzalez et al, 2006). This raises the question, would transplanting trabecular meshwork cells or trabecular meshwork progenitor cells back to the trabecular meshwork enhance outflow, thereby lowering IOP and protecting RGCs from damage in glaucoma?…”

“…Whether this proliferative population is derived from insert cells or a subpopulation of trabecular meshwork cells remains to be determined. trabecular meshwork progenitor cells can be expanded in three-dimensional sphere-like cultures (Gonzalez et al, 2006). Cells from these cultures express markers and gene expression profiles associated with monolayer cultures of TM stem cells (Gonzalez et al, 2006).…”

Biomaterials for Regenerative Medicine Approaches for the Anterior Segment of the Eye

“…[12] Following argon laser trabeculoplasty of ex vivo organ cultures there was an increase in cell division within cells in the nonfiltering insert region of the trabecular meshwork [13] and the cells migrated into the posterior trabecular meshwork. Isolation and amplification of progenitor cells from the trabecular meshwork using a floating sphere culture method [14] or cell sorting of a side population [15] showed the expression of stem cell characteristics and differentiation of these cells subsequently resulted in the expression of trabecular meshwork markers. The progenitor cells of the insert region offer a potential source of cells to regenerate both the corneal endothelium and the trabecular meshwork.…”