Transcription factor gene expression profiling and analysis of SOX gene family transcription factors in human limbal epithelial progenitor cells

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Doctoral Thesis
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Menzel-Severing, Johannes

To function as our “window to the world”, the cornea requires an intact epithelial surface. Epithelial stem/progenitor cells at the corneoscleral limbus are a reservoir for corneal epithelial homeostasis and repair. When these cells are lost, delayed wound healing, vascularisation and scarring may lead to painful loss of vision. Current treatment options include expansion of limbal epithelial progenitor cells (LEPCs) from a healthy donor eye through ex vivo culture and transplantation of these cells to the diseased ocular surface. However, the availability of autologous LEPCs for transplantation is limited in cases of systemic and/or bilateral disease. This has raised interest in the use of induced pluripotent cells or direct transdifferentiation of non-ocular cells towards a corneal epithelial phenotype. Transcription factors (TFs) are key players both in establishing pluripotency and in direct reprogramming. Understanding TF regulation of LEPCs and corneal epithelial homeostasis may aid in successfully using non-ocular cell sources to regenerate the corneal surface. Hence, this study aimed to identify differentially expressed TF genes in human limbal and corneal epithelial cells and to characterise their role for proliferation and differentiation of limbal epithelial cells. LEPC clusters and central corneal epithelial cells were excised from cryosections of snap-frozen human post-mortem donor eyes using laser capture microdissection (LCM). RNA extracted from these specimens underwent linear amplification. Limbal and central corneal samples were screened for levels of expression of a panel of stem cell TF genes using real-time polymerase chain reaction (PCR) arrays. Four genes showed preferential limbal expression (at least two-fold elevated in limbal specimens compared to central cornea): DACH1, HOXA11, SOX9, and PPARG. Eleven genes were preferentially expressed in central corneal epithelial cells: FOXP2, RB1, MSX2, JUN, PCNA, SP1, SIX2, PAX6, FOXP3, SMAD2, and FOXP1. Validation experiments using real-time PCR hydrolysis probe assays confirmed statistically significant preferential limbal expression of SOX9 (with the highest fold change value of 428), DACH1 (272.5), HOXA11 (104.7) and PPARG (29.3).
Because SOX genes (Sry-related high mobility group box) encode TFs known to regulate cell fate and differentiation, a complete screen of SOX transcription factor gene expression was performed on LCM samples using real-time PCR. Preferential limbal expression was found for a number of SOX family genes, including all members of the SoxE, SoxF and SoxH groups. Intracellular localization of their respective gene products was assessed using in-situ immunofluorescence. Here, SoxE proteins showed distinctive staining patterns, with predominantly cytoplasmic staining in basal limbal epithelial cells suggesting inhibition of its transcriptional program, and predominantly nuclear localisation in suprabasal and central corneal epithelial cells suggesting DNA binding and transcriptional activity. SOX9 was selected for further analyses given its strong expression and taking into consideration previous reports from other progenitor cell types. Using double-labeling, partial co-localisation was observed between SOX9 and putative limbal progenitor cell markers (Bmi1, OCT4, p63α, N-cadherin and Keratin 15). SOX9 protein expression was also assessed in human corneas that had been subjected to a central epithelial wound in vitro. Increased expression and nuclear translocation of SOX9 was found in activated LEPCs and re-grown corneal epithelial cells compared to unwounded control eyes. Next, mRNA expression of SOX9 was analyzed in primary cultures of limbal epithelial cells at different passages. Expression levels increased from P0 to P1 and P2. Immunofluorescent labeling of SOX9 in LEPC clones showed a nuclear staining pattern, with immunopositive cells being located predominantly towards the proliferating periphery of the clones. Knockdown of SOX9-expression in cultured LEPCs was achieved using RNAi, and at 24 hours after transfection, effects on target gene regulation were monitored by real-time PCR. Expression of the progenitor cell marker gene KRT15 (Cytokeratin 15) was significantly reduced in cells after knockdown of SOX9. No significant changes were seen in expression of other progenitor cell marker genes such as CEBPD, ABCG2, p63α or CDH2. We did observe a trend towards upregulation of differentiation markers KRT3 and IVL but found no effect on expression of KRT12, PAX6 or MUC1. Also, we observed a trend towards upregulation of cyclin-dependent kinase inhibitors p21 and p57 and a trend towards downregulation of proliferation marker PCNA (Proliferating cell nuclear antigen). Using Western blot, reduced levels of Cytokeratin 15 and PCNA were detected in cultured cells following siRNA-mediated knockdown of SOX9. In line with downregulation of PCNA, proliferation rates (analyzed by BrdU incorporation) significantly decreased following knockdown of SOX9, in comparison to cells transfected with scramble siRNA. In a nutshell, this study identified a number of TFs not previously known to be preferentially expressed in LEPCs. It also provided some evidence that SOX9, and potentially other SOX-family TFs, are expressed in LEPCs and may regulate corneal epithelial homeostasis. Our results suggest that SOX9 promotes proliferation and differentiation in transient amplifying cells following nuclear translocation, while supporting a progenitor cell phenotype and the continued expression of marker genes of putative LEPC by means of its cytoplasmic retention. Activation of SOX9 may assist clonal expansion, proliferation and differentiation of limbal epithelial cells in vitro for clinical applications. Therefore, SOX9 is a strong candidate gene, which, in combination with other factors, may form part of a strategy to achieve transdifferentiation and expansion of cells from non-ocular sources towards a corneal epithelial phenotype. The functional mechanisms underlying cytoplasmic retention and nuclear shuttling of SOX9 require further investigations.

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