Purchase this article with an account.
Marie A. Shatos, Robin R. Hodges, Rachel A. Scott, David McNay, Sharmila Masli, Darlene A. Dartt; Comparative Analysis of Progenitor Cells from Lacrimal Glands of Wild Type and Thrombospondin-1 Null Mice. Invest. Ophthalmol. Vis. Sci. 2012;53(14):634.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To identify and compare progenitor cells from lacrimal glands (LG) of WT and thrombospondin (TSP)-1 null mice.
LG frozen sections from 11 week old female C57B6 (WT) and TSP-1 null mice and cultured progenitor cells from TSP-1 null mice were prepared. LG sections and progenitor cells grown on coverslips were double labeled using immunocytochemistry (ICC) with antibodies (AB) to alpha-smooth muscle actin (alpha-SMA), a myoepithelial cell (MEC) marker and with progenitor cell markers ABCG2, CHX 10, Nestin, SOX 2, delta-N P63, Musashi Factor-1 and PAX 6 and incubated with secondary AB CY2 and CY3. Slides were analyzed using confocal and fluorescence microscopy. Western blotting using AB to nestin, ABCG2 and delta-N P63 were performed on homogenized WT and TSP-1 null LG. In vivo LG cell proliferation was determined by injecting WT and TSP-1 null mice with BrdU 5 times. After 72 hrs, LG were removed, sections cut, processed and incubated with anti-BrdU AB. BrdU positive cells were counted in 8 sections (5 fields per section.
ICC showed that WT LG expressed ABCG2, CHX 10, nestin, SOX 2, delta-N P63, Musashi Factor-1 and PAX 6 located in acinar, ductal and in a few MEC, the putative LG progenitor cells. In TSP-1 null LG, these same markers were exclusively expressed in MEC, except for Musashi Factor-1, which was observed in acinar, ductal and MEC. Progenitor cells could only be isolated from TSP-1 null LG and when differentiated expressed nestin, ABCG2, Mushashi Factor-1, CHX 10 and PAX 6 that co-localized with alpha-SMA. Western blotting of WT and TSP-1 null LG showed no significant difference between them in amounts of nestin, ABCG2 and delta-N P63. BrdU analysis indicated that WT LG had more proliferating cells than TSP-1 null LG (1.86±0.27 and 0.75±0.19 cells per section, respectively.
We conclude that even though progenitor cells are present and more easily isolated from TSP-1 null compared to WT LG, cell proliferation is decreased in the mice lacking TSP-1. TSP-1 appears to regulate LG cell proliferation and its loss increases progenitor cells.
This PDF is available to Subscribers Only