Abstract
Purpose :
Whilst TM progenitor cells (TMPCs) have the potential to repopulate the trabecular meshwork (TM), there is loss and dysfunction of TM cells which occurs with increasing age and in primary open-angle glaucoma (POAG). The profibrotic cytokine transforming growth factor-β (TGFβ) plays key role in the regulation of extracellular matrix and pathobiology of the trabecular meshwork (TM) in POAG. The role of TGF-β-1 and -2 on TMPSC differentiation are currently unknown and we sought to determine the effect of TGF-β-1 and -2 on an in vitro model of human TMPC differentiation.
Methods :
Human primary TM cells (PTM) were isolated from the TM tissue of donated cadaver globes. The TMPCs isolated from the PTM utilising a sphere culture assay and were further differentiated to TM cells (DTM) on ECM derived from TM cells in the presence or absence of TGF-β-1 and -2 (5ng/ml) for up to 5 days. Phase contrast microscopy, mRNA and protein expression were evaluated at days, 1, 3 and 5 post differentiation with qPCR, immunofluorescence and western blotting for stem cell and known differentiated TM cell markers.
Results :
TMPCs proliferated, formed spheres, and could differentiate into TM cells in the presence or absence of TGF-β-1 and -2. TM sphere differentiation and outgrowth area was increased in all donors (n=3) by day 5 in the presence of TGFβ1 or TGFβ2 spheres compared to ECM alone. TM progenitor cell markers (SOX2, FOXC1, SPARCL1, PDGFRA, ANKG and KDR) mRNA expression was reduced in TMPCs differentiated in the presence of either TGF-β-1 and -2 compared to ECM only controls. TGFβ1/2 treated groups had a higher expression of TAGLN and SPARC or compared to non-treated groups. Expression of the cell contractility related markers α-SMA and CTGF were upregulated in the TGFβ1 (α-SMA: p=0.06; CTGF: p= p=0.032) and TGFβ2 (α-SMA: p=0.07; CTGF: p=0.016) treated groups compared with the ECM control group.
Conclusions :
This study found that the TGFβ1/2 could promote the differentiation of the TM progenitor cells by upregulating the TM cell markers and downregulating the TM progenitor cell markers. Elevated levels of TGFβ1/2 in the aqueous humour of the glaucomatous eyes may promote the differentiation of the TMPCs, which may lead to the depletion of the TM progenitor cells observed in POAG eyes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.