Abstract
Purpose::
Flow cytometry of cells stained with the DNA-binding dye Hoechst 33342 often reveals the presence of a cohort of poorly stained cells, presently known as a side population (SP). The SP cells have been proposed to represent a subset of stem cells. Consistent with this proposal we have previously identified SP cells within the limbal (Li) and conjunctival (CNJ) epithelia and have demonstrated that these cells exhibit a number stem cell (SC) features. Now we sought to establish the molecular signatures of the Li and CNJ SP cells by comparing their mRNA expression (molecular signature) with that of a non-SP population.
Methods::
Single epithelial cell suspensions, generated by sequential Dispase-trypsin digestions of fresh or briefly stored human (h) CNJ and porcine (p) or rabbit CNJ and Li tissues, were cultured for 16 hr in SHEM. Adherent (i.e., pure basal) cells were incubated for 2 hr with 5 µg/ ml Hoechst 33342, collected and analyzed for SP content. In all cases SP cells amounted to ~ 1 % of the total viable cell count. Triplicate collections of equal amounts of SP and non-SP cells were completed for hCNJ, pCNJ and pLi epithelia (using 50 corneas/run). Cells were sorted into Tri-Reagent for RNA purification. Isolated RNA (36 ng) was subjected to two RNA amplification rounds, labeled and hybridized to h (54, 614 transcripts derived from ~ 30,000 different genes) or p (23,936 transcripts from ~ 17,000 genes) Affymetrix microarrays. The triplicate results were used to build sets of SP expressed genes comprising only transcripts a) that were present in all three experiments and b) whose average signal intensity (SI) exceeded 1 % of that for ß-actin. These sets were used to identify transcripts over-expressed in the SP sets (SI SP > SI non-SP; SP transcripts).
Results::
Our protocol identified 2519 hCNJ, 519 pCNJ and 592 pLi SP transcripts at the 2x and 918, 138 and 95 and 3x thresholds, respectively. The over-represented sets include genes that are a) recognized as stem cells genes in other cellular systems and/or suggested to mark ocular surface stem cells; b) involved in detoxification and cytoprotection (e.g., cytochrome P450 enzymes); c) anti-apoptotic; and d) can be predicted to elicit main properties of stem cells such as slow cycling and stem cell ‘niche’ homing. About half of the pCNJ SP transcripts were also included in the pLi SP set.
Conclusions::
The results support the notion that the ocular surface SP cells are stem cells. They yield a trove of valuable information for the characterization of stem cells. Importantly, the results also indicate that many hCNJ SC genes may also be Li SC genes.
Keywords: cornea: epithelium • conjunctiva • differentiation