Abstract
Purpose: :
Limbal and conjunctival epithelia (LiE, CNJE) Hoechst 33342- excluding (side population; SP) cells display features consistent with a stem cell phenotype. We sought to define the molecular signatures of these SP cells and identify shared genes that maybe central genes for stem cell function.
Methods: :
Freshly isolated pig LiE (pools of 50) and CNJE (pools of 6) and human CNJE (from single donors) cells were cultured for 16 hr, incubated with 5 µg Hoechst 33342 and SP and a non-SP cell cohorts were sorted by flow cytometry into a RNA isolation solution. Isolated RNA was twice amplified and hybridized to large oligonucleotide spoted arrays. Paired, normalized SP and non-SP expression levels and present/absent ‘calls’ (MAS5 software, Affymetrix) of triplicate pCNJE and pLiE experiments were set on a single spreadsheet and filtered to preserve only those transcripts that are present in all six (3 LiE, 3 CNJE) SP samples. This common SP-present list was filtered using numerical (SP/nonSP expression ratio is > 1.33 in every replicate) or statistical criteria to establish a) individual SP over-expressed transcript lists for each tissue type and b) shared transcript lists where the criteria applies simultaneously to both phenotypes. This shared list was also probed against the human CNJE list.
Results: :
The SP-present and the pLiE and pCNJE SP over-expressed lists contained 9987, 971 (0.109 of total) and 487 (0.055 of total) transcripts, respectively. Of the latter, 231 were shared by both tissues. This number is 5 times larger than that expected from transcript overlap by random chance (0.109 x 0.055 x 8,897 = 53 transcripts). Modifications of the filtering criteria changed list compositions but the shared LiE and CNJE over-expressed list was in all cases 5 times larger than that expected from random chance. When sorted according to average n-fold expression ratios between both tissues, the ten most over-expressed genes were, CXCR4, HISTH2AD, CEBPD, VAV3, PITX2, GPX2, HS3ST6, RHBDD2, VAV1, ZZEF1 and FXB032. Finally, 85 % of the genes identified of the shared porcine list were also found in the human CNJE SP over-expressed list. Over-expression of several shared list genes was confirmed by PCR or, at the protein level, by immunohistology.
Conclusions: :
Side by side molecular signature analysis for the SP cells of pLiE, pCNJE and human CNJE allows to identification of those genes that are specific to each tissue type and those common to the SP stem cells in ocular epithelia.
Keywords: cornea: basic science • conjunctiva • gene/expression