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Gary S L Peh, Matthew Lovatt, Gary Yam, Ray Dunn, Jodhbir S Mehta; Directed differentiation of periocular mesenchyme from human embryonic stem cells.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1372. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To better understand the directed differentiation of periocular mesenchyme (POM) from pluripotent human embryonic stem (ES) cells for the derivation of corneal endothelial cells (CECs) and stromal keratocytes.
Human ES cells (HES-3) were maintained in its undifferentiated state on growth factor reduced matrigel in mTesR1. Cells were passaged at a 1:10-1:20 ratio every 5-7 days using gentle cell dissociation, with colony size adapted by gentle passing through a p1000 pipette.Differentiation were induced by dissoication of HES-3 colonies into single cells, plated on matrigel-coated plates in mTesR1 with 10μm ROCK inhibitor. At 50-70% confluence, media was changed to KO-DMEM containing 15% serum replacer (SR) wtih 10μm SB431542 (SB) (KOSR media). From day 4, differentiation media was adjusted by gradual inclusion of DMEM/F12 containing 1x N2 MAX supplement (N2 media) to a composition of 75% KOSR:25% N2. Subsequently, on day 6, 50% KOSR:50% N2; day 8, 25% KOSR:75% N2; and on day 10, 100% N2 medium. In some studies, cultures were supplemented with 3μm all-trans retinoic acid (RA).Genes of interests of differentiated cells, such as FOXC1, LMX1B, PITX2, SOX9, SOX10, SNAI2 and TFAP2, were assessed using real-time PCR, and protein expressions of p75, HNK1, FOXC1, PITX2, SOX10 and TFAP2 were assayed via either immunofluosrescence or western blots.
Results indicated that the inhibition of TGF-β signalling with SB alone promoted the expression of FOXC1, as well as the induction of neural crest (NC) transcripts such as SOX10 and TFAP2, SOX9 and SNAI2, together with POM marker LMX1B. With the addition of RA on day 6 of differentiation, transcripts of PITX2 were found to be at its highest. However, PITX2 protein was not detected. Interestingly, RA treatment resulted in selective down-regulation of SOX10 at both the mRNA and protein level, but did not affect TFAP2. Taken together, it appeared that SB in combination with RA alters the fate of NC cells and favours the induction of POM markers with the selective down-regulation of SOX10.
In embryonic development, POM gives rise to a variety of cells within the developing anterior segment of the eye. In this study, the differentiation of POM from human ES cells is an important first step in the derivation of cells that may be directed into clinically relevant cells such as CECs and stromal keratocytes, which form the basis of future studies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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