Rare Stem Cells Detected Using the Attune® Acoustic Focusing Cytometer
by Jennifer Hornstein - 05/10/12
It is widely accepted that the stem cells which maintain and renew the corneal epithelium reside in the limbus, located on the periphery of the cornea. They are commonly referred to as limbal stem cells (LSCs) and are useful for basic research into various aspects of corneal biology such as ocular injuries that impair vision.
Progress in this research has been hampered, however, by the lack of markers and assays that can be used to identify LSCs. Here we present a method for identification and quantification of LSCs derived from primary human tissue, using the Attune® Acoustic Focusing Cytometer.
Side Population Analysis Facilitated with the Attune® Cytometer
Many types of stem cells can be identified using a technique known as side population (SP) analysis. This approach takes advantage of stem cells’ relatively high expression of membrane transporter proteins, such as ABCG2, that can specifically efflux various compounds, including fluorescent dyes, from live cells. Once stem cells begin to differentiate, expression of these transporters is reduced and cells retain the dye molecules.
Adapting the SP Analysis Technique
The SP analysis technique for identification of limbal stem cells in populations of human corneal epithelial cells had to be adapted by taking advantage of the ability of Vybrant® DyeCycle™ Violet stain to be effluxed by the ABCG2 transporter. Cells were labeled with this cell-permeant, fluorescent DNA-binding dye, then the population was analyzed using the rare-event detection capability of the Attune® Acoustic Focusing Cytometer.
Human corneal epithelial cells (HCECs) were isolated via enzymatic digestion of limbal sections from which the central cornea and most of the sclera were trimmed.
Epithelial cells released from these sections were subsequently expanded through two passages in Gibco® Keratinocyte-SFM, then cryopreserved.
HCECs were then thawed and passaged twice in Keratinocyte-SFM. Subconfluent cells were detached from the culture dish, resuspended at 1 x 106 cells/mL in culture medium and incubated with 5 µM Vybrant® DyeCycle™ Violet stain for 60 min at 37°C with or without the ABCG2 inhibitor fumitremorgin C (1 µM).
Cells were subsequently washed with cold HBSS with 2% FBS and resususpended in culture media with 5 µM Vybrant® DyeCycle™ Violet stain with or without 1 µM fumitremorgin C for an additional 30 min at 37°C.
Cells were washed once and resuspended in culture medium prior to analysis.
The Attune® Acoustic Focusing Cytometer was used to analyze ~120,000 cells per experimental replicate. Vybrant® DyeCycle™ Violet stain was excited using the 405 nm laser and emission was collected in 450/40 and 603/48 bandpass filters.