Toward Corneal Limbus In Vitro Model: Regulation of hPSC‐LSC Phenotype by Matrix Stiffness and Topography During Cell Differentiation Process

Abstract

A functional limbal epithelial stem cells (LSC) niche is a vital element in the regular renewal of the corneal epithelium by LSCs and maintenance of good vision. However, little is known about its unique structure and mechanical properties on LSC regulation, creating a significant gap in development of LSC‐based therapies. Herein, the effect of mechanical and architectural elements of the niche on human pluripotent derived LSCs (hPSC‐LSC) phenotype and growth is investigated in vitro. Specifically, three formulations of polyacrylamide gels with different controlled stiffnesses are used for culture and characterization of hPSC‐LSCs from different stages of differentiation. In addition, limbal mimicking topography in polydimethylsiloxane is utilized for culturing hPSC‐LSCs at early time point of differentiation. For comparison, the expression of selected key proteins of the corneal cells is analyzed in their native environment through whole mount staining of human donor corneas. The results suggest that mechanical response and substrate preference of the cells is highly dependent on their developmental stage. In addition, data indicate that cells may carry possible mechanical memory from previous culture matrix, both highlighting the importance of mechanical design of a functional in vitro limbus model.

Squishy matters – Corneal mechanobiology in health and disease

“…It has been well-established that cell behavior is directly influenced by both the stiffness of the substrates upon which they reside; however, relatively few studies have focused on the corneal epithelium ( Alauzun et al, 2010 ; Kauppila et al, 2023 ; Klenkler et al, 2010 ). A recent study demonstrated that corneal epithelial cells cultured on stiff substrates (~1.0 MPa) composed of polydimethylsiloxane (PDMS) demonstrated higher proliferative indices (phosphorylated ERK, Ki67) and higher expression of cytokeratin 3, a marker of epithelial cell maturity ( Masterton and Ahearne, 2019 ).…”

Deciphering the heterogeneity of differentiating hPSC-derived corneal limbal stem cells through single-cell RNA-sequencing

“…In this study, we used one human embryonic stem cell (hESC) line Regea08/017 (hereafter, "hESC") and two human induced pluripotent stem cell (hiPSC) lines WT001.TAU.bB2 and WT003.TAU.bC (hereafter, "hiPSC1" and "hiPSC2", respectively) for differentiation toward LSCs with previously established protocol (Hongisto et al, 2017) (Figure 1A). The hESC line has been used for the initial establishment of the method (Hongisto et al, 2017;Mikhailova et al, 2014) and since then, has served as our golden standard line with consistently good performance in various experimental settings (Kauppila et al, 2023;Koivusalo et al, 2019;Sorkio et al, 2018;Vattulainen et al, 2019Vattulainen et al, , 2021. The hiPSC1 line has been used for hPSC-LSC differentiation in one previous study (Vattulainen et al, 2021), while hiPSC2 is a newly established line (characterization described in Supplementary File 1).…”