Purchase this article with an account.
Paul Knollman, Matt Bailey, Rhonda Newman, Chris Langsdorf, Bradley Dubbels, Jolene Bradford, David Kuninger; Identification And Initial Characterization Of Limbal Stem Cells Isolated From Human Cornea. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5125.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Limbal stem cells play a central role in repopulating the cornea during wound healing. To identify putative progenitor populations in adherent cultures derived from human limbal tissue, several approaches were taken including qPCR and flow cytometry. Transcript levels of limbal stem and corneal cell markers including ABCG2 and ΔNp63alpha were assessed as a function of culture level in various media systems. In addition, side population analysis was performed using flow cytometry.
Gibco® HCEC are derived from trimmed normal human corneal-scleral buttons. Limbal epithelial cells were subsequently released via enzymatic digestion, expanded in growth media and are cryopreserved at the end of the secondary culture. For subsequent expansions, HCEC were cultured in the indicated growth media at a seeding density of 5,000 cells/cm2. Total RNA was isolated, reverse transcribed, and used in qPCR with the indicated TaqMan probesets and normalized to GAPDH levels. For flow cytometry, cells were cultured as described above, harvested, and incubated with LIVE/DEAD® Fixable Far Red dead cell stain to gate on living cells and with Vybrant® DyeCycleTM Violet stain to identify limbal stem cell side populations using an Attune ® Acoustic Focusing Cytometer.
qPCR analysis of the limbal progenitor markers, ΔNp63α and ABCG2, in Gibco® HCEC demonstrate a significant decrease in mRNA transcript levels when comparing third (3’) and seventh (7’) cultures: ΔNp63α 3’ CT of 28.68 ± 0.03 vs. 7’ CT of 32.06 ± 0.07, ABCG2 3’ CT of 33.64 ± 0.13 vs. 7’ CT of 38.66 ± 1.34. Attune® Acoustic Focusing Cytometer data identified a distinct Vybrant® DyeCycleTM Violet dye-excluding side population (SP) in early passage cultures (3’) which was reduced (5% to 1%) as the cultures were expanded.
Expression of ABCG2 and ΔNp63alpha declined during continuous culture (3’ to 7’), which we attribute to differentiation and senescence. The SP cells, identified by flow cytometry, appeared smaller in size (based on forward and side scatter) when compared to the population as a whole, and did not overlap with the population expressing CK3/12 in flow cytometry analysis. Taken together, this data suggests that qPCR and SP analysis using flow cytometry are effective methods to identify and quantify characteristics of stem cell populations in ex vivo expansion of limbal epithelial cells.
This PDF is available to Subscribers Only