Investigating the Role of TGF-β Signaling Pathways in Human Corneal Endothelial Cell Primary Culture

Abstract

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial–mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-βs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-β signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-β1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-β-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-β1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-β receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-β1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD.

Femtosecond Laser Cutting of Human Crystalline Lens Capsule and Decellularization for Corneal Endothelial Bioengineering

“…CD166 is a basolateral transmembrane protein involved in cell-cell adhesion. NCAM is a specific endothelial marker localized on the lateral membrane of CECs [30,31].…”

TGF-β-Based Therapies for Treating Ocular Surface Disorders

“…In a study of corneal transplantation, TGF-β has been shown to play a crucial role in triggering the immune response that can lead to graft rejection by reducing the density of endothelial cells [192]. To address this, the authors utilized an in vitro treatment scheme with a TGF-β neutralizing antibody, fresolimumab.…”

“…This treatment aimed to prevent the loss of endothelial cells by inhibiting the transition from endothelial to mesenchymal states, a process stimulated primarily by TGF-β1 and, to a lesser extent, TGF-β3. The findings highlighted that while fresolimumab effectively suppresses this transition, its efficacy varies across different TGF-β isoforms, which could influence the overall success of preventing graft rejection [192].…”

Advancements in Bioengineering for Descemet Membrane Endothelial Keratoplasty (DMEK)

“…Advanced cell culture media formulations containing growth factors, cytokines, and extracellular matrix components have been designed to support CEC proliferation while preserving cellular morphology and function. Optimized culture media contains the following components [66][67][68][69]:…”