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PhD Seminar – Role of Transcription Factor SOX2 and Mechanosensing in Maintaining Corneal Stem Cell Identity

August 22 @ 10:00 - 12:00 IDT

Corneal blindness affects 50 million individuals worldwide; a significant proportion of them require a cell or tissue-based repair or replacement strategy to mend their damaged or diseased cornea. Furthermore, the disease mechanisms involved are largely unknown. Mutations in key transcription factors SOX2 and P63 were linked with developmental defects and post-natal abnormalities such as corneal opacification, neovascularization and blindness. The latter phenotypes suggested that SOX2 and P63 may be involved in corneal epithelial regeneration. While P63 has been shown to be a key regulator of limbal stem cells (LSCs), the expression pattern and function of SOX2 in the adult cornea remained unclear. Moreover, recent studies from the field provided evidence that epithelial stem cell’s decision to self-renew or differentiate is tightly controlled by the neighboring mesenchymal stroma. Interestingly, the biomechanical properties of the extracellular matrix (ECM) of the corneal stroma, was shown to enhance epithelial stem cell differentiation signals while stromal remodeling has been linked with severe corneal pathologies and blindness. However, the mechanisms by which biomechanical information is sensed and translated into cellular signals is unclear.

The aim of my thesis was to identify the mechanisms by which LSC maintain their identity and to unravel the signaling cues that lead to their differentiation. In the first part of my study, we discovered that SOX2 regulates P63 to control corneal epithelial stem/progenitor cell function. SOX2 and P63 were co-expressed in the stem/progenitor cell compartments of the murine cornea in vivo and in undifferentiated human limbal epithelial stem/progenitor cells in vitro. In line, a new consensus site that allows SOX2 mediated regulation of P63 enhancer was identified while repression of SOX2 reduced P63 expression, suggesting that SOX2 is upstream to P63. Importantly, knockdown of SOX2 significantly attenuated cell proliferation, long-term colony-forming potential of stem/progenitor cells and induced robust cell differentiation. However, this effect was reverted by forced expression of P63, suggesting that SOX2 acts, at least in part, through P63. Moreover, miR-450b was identified as a direct repressor of SOX2 that was required for SOX2/P63 down-regulation and cell differentiation.

In second part of my study, I focused on how stromal mechanobiology influences LSC differentiation. We observed that confined changes in limbal and central corneal stromal stiffness during post-natal corneal maturation coincided with the formation of spatially segregated compartments for limbal epithelial stem cells and corneal differentiated cells in the murine cornea. Interestingly, the transcriptional co-activator YAP was mainly localized to the nucleus of LSCs while corneal epithelial progenitor cells expressed cytosolic YAP in vivo. Inline, induction of human LSC differentiation by increased matrix rigidity or by elevating calcium levels in vitro, diminished nuclear localization of YAP. Knockdown of YAP induced robust cell differentiation and reduced LSC colony-forming efficiency, indicating that Acto-Myosin/YAP mechanotransduction pathways play a key role in LSC differentiation. In agreement, pharmacological inhibition or forced activation of mechanotransduction significantly influenced nuclear YAP localization and cell differentiation on gels as well as on the petri dish.

Taken together, these data suggest that SOX2/P63 pathway is an essential regulator of corneal stem/progenitor cells while mutations in SOX2 or P63 may disrupt epithelial regeneration, leading to loss of corneal transparency and blindness. In parallel, mechano-sensing of the low rigidity of the limbus positively regulate YAP localization to the nucleus, to prevent LSC differentiation. Traditional LSC growth on plastic dishes that are extremely stiff in comparison to the limbus, allows very limited expansion and self-renewal. However, a better understanding of molecular circuitry controlled by transcription factors and modulation of mechanotransduction pathways may improve LSC cultivation for research and cell therapy.

Details

Date:
August 22
Time:
10:00 - 12:00
Event Category: