Abstract
Purpose :
The objective of this study was to determine whether canine adipose derived mesenchymal stromal cells (adMSC) express keratocyte-specific phenotypic markers when cultured under conditions inducing differentiation of corneal stromal stem cells to keratocytes in 2D.
Methods :
AdMSC were differentiated in keratocyte differentiation media (KDM) containing basic fibroblastic growth factor (bFGF) and ascorbic acid in the absence of serum for 3 weeks and compared to cells pre-cultured in corneal stromal stem cell media (CSSCM) containing ascorbic acid, platelet derived growth factor (PDGF), epidermal growth factor (EGF) and low serum for 7 days followed by 3 weeks KDM. This was performed in a biological triplicate and cells were characterized using quantitative RT–PCR and immunostaining. Statistical analysis included Shapiro-Wilk normality test, ANOVA, followed by the Tukey's post hoc test.
Results :
AdMSC differentiated into KDC showed keratocyte-like morphology, expressed keratocyte associated proteins Keratocan, Lumican and ALDH1A3 and but not the stem cell marker Pax6 when cultured in KDM.
Pre-culturing adMSC in CSSCM before differentiating in KDM increased the level of Keratocan and Lumican gene expression significantly but not ALDH1A3. AdMSC cultured in CSSC media showed a trend of upregulated Pax6 and N-cadherin, which was downregulated after keratocyte differentiation. Expression of differentiation markers was quantitatively lower than following the keratocyte differentiation of primary canine CSSC.
BFGF increased the cell expansion of adMSC-KDCs but retained a fibroblastic cell morphology. AdMSCs express α-smooth muscle actin (α-SMA) at the protein and mRNA level in the undifferentiated state and at all stages of differentiation.
Conclusions :
Keratocyte-like cells derived from canine adMSCs may serve as a source of keratocytes but expressed myofibroblastic features when cultured in 2D.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.