Purchase this article with an account.
Dimitrios Karamichos, Audrey E. Hutcheon, James D. Zieske, Martha L. Funderburgh, James L. Funderburgh; Comparison of ECM Secretion by Corneal Stem Cells and Corneal Fibroblasts in a 3D Model. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5147.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
This study compared the ability of two different cell types, human corneal stem cells (hCSSC) and human corneal fibroblasts (HCF), to generate a cornea-like extracellular matrix (ECM) in response to transforming growth factor beta 3 (TGF-β3) in the presence of ascorbate.
hCSSC and HCF were cultured in minimal essential medium (MEM) containing fetal bovine serum (FBS) and stabilized ascorbate (sAsc) for 4 weeks: a) Controls: MEM+10%FBS+sAsc and b) Experimental: MEM+10%FBS+sAsc+0.1ng/ml TGF-β3. Indirect-immunofluorescence microscopy (IF) and transmission electron microscopy (TEM) were used to characterize the constructs.
After 4 weeks in culture, hCSSC constructs stratified to a mean thickness of ~8µm, only 30% as thick as the HCF cultured under the same conditions (30µm). In the presence of TGF-β3, hCSSC construct thickness was 25µm and HCF reached 60µm. Long aligned fibrillar collagen was observed by TEM in both HCF and hCSSC constructs. IF showed type I collagen to be present in all conditions in both cell types. Type III collagen was found in controls of both hCSSCs and HCF, but was barely detectable in cultures containing TGF-β3. Smooth muscle actin (SMA) expression and cells with phalloidin-stained stress fibers were reduced in TGF-β3-containing cultures compared to controls; however, some cells were still positive. TGF-β3 treatment had a more pronounced effect on HCFs than hCSSC. Thrombospondin-1, similar to type III collagen, was present throughout the control cultures, but was not detected in cultures with TGF-β3 of both cell types. Fibronectin (cFN) expression was also seen throughout the controls, but in the presence of TGF-β3, cFN expression was not induced.
Overall, our data show a very similar ability of the hCSSCs and HCFs to express and organize an ECM that resembles that of the corneal stroma. Furthermore, stimulation with TGF-β3 in both hCSSC and HCF increased the secretion of an ECM with reduced fibrotic markers. HCF appeared to accumulate more ECM than hCSSC under similar conditions. Our results showed that fibroblasts and stem cells both hold a potential for use in corneal tissue engineering.
This PDF is available to Subscribers Only