Adult Stem Cells Secrete Corneal Specific Matrix Components

Abstract

Abstract: : Purpose: Cornea has a unique structure and unique extracellular components which are essential for tissue integrity and transparency. Cells used for corneal tissue engineering must have the ability to secret such a cornea–specific matrix. Keratocytes are the major cellular constituents of corneal stroma, but in vitro human keratocytes change into fibroblasts losing their unique phenotype. Multipotent adult progenitor cells (MAPC) have recently been identified in a number of tissues. These cells are considered candidates for cellular therapy and tissue engineering. In this study, we investigated whether corneal stromal derived stem cells (CSSC) and adipose derived stem cells (ADSC) can adopt the keratocyte phenotype and secret unique components of the corneal stromal extracellular matrix. Methods: CSSCs were isolated by cell sorting using Hoechst 33342 dye exclusion and ADSCs were isolated by collagenase digestion and differential centrifugation. Each cell type was cultured and passaged in medium containing 2% fetal bovine serum (FBS) with growth factors. For keratocyte differentiation, the stem cells were cultured in defined medium using Advance MEM (In Vitrogen) with addition of horse serum or of bovine corneal extract (Exp Eye Res 2003 77:273). Cells were cultured either as pellet or in fibrin gels. Keratocan and keratan sulfate were detected by immunostaining, immunoblotting, and RT–PCR. Pellets and fibrin gel were fixed, sectioned, and stained for keratocan and keratan sulfate. Results: Stem cells from human corneal stroma and from human subcutaneous adipose tissue can express keratocan and keratan sulfate after culture in defined medium. After 14 days in pellet culture, both cells types secret an amorphous connective tissue containing keratocan and keratan sulfate. In fibrin gels, the cells also secret keratocan and keratan sulfate, and exhibit extensive intracellular connection via extended cellular processes similar to those observed in the corneal stroma in vivo. Conclusions: Stem cells from human corneal stroma and from adipose tissue can differentiate into cells showing the characteristic morphology and connective tissue secretion of human keratocytes. Ability to expand cultures of human stem cells and transform them into functional keratocytes may represent a key step development of a bioengineered corneal bioprostheses.

Keywords: proteoglycans/glycosaminoglycans • cornea: stroma and keratocytes • extracellular matrix