Purchase this article with an account.
J. C. Pardo, J. C. Grandin, M. C. Yoder, M. Price, F. W. Price; Method for Detecting Corneal Endothelial Cell Identity From Contaminating Keratocytes and Epithelial Cells in Co-Culture. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1820.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Transplantation of cultured human corneal endothelial cells (HCEC) to treat endothelial dysfunction could help alleviate a worldwide shortage of donor corneas, but a means of definitively proving culture purity must be developed. The purpose of this study was to assess the specificity of protein markers that have previously been identified in corneal endothelium, keratocytes or epithelium.
HCEC were isolated by stripping Descemet’s membrane with endothelium from the posterior surface of corneoscleral tissue. Subsequently the epithelium was removed gently with a scalpel and the tissue was cut with a trephine to isolate stromal keratocytes. Messenger RNA was isolated from cultured HCEC, keratocytes, and whole stromal pieces. mRNA was transcribed into cDNA and then used in PCR analysis with primers specific to S100b protein, neural cadherin (ENO2), neural specific enolase (NSE), collagen 8 alpha one (CVIII1), keratin 12(K-12), alpha smooth muscle actin (SMA). Angiopoietin like protein 7 (ANGPTL7) has been determined to be specific to the corneal stroma so we tested its specificity on cultured cells along with the expression of other members of the angiopoietin like family (ANPTL1-6). Also, Immunocytochemistry was performed for S100b, NSE, ENO2, and K-12.
PCR results showed that both HCEC and keratocytes were positive for S100b, ENO2, NSE, CVIII1, ANGPTL1, 2, 4, and 5, and negative for K-12 and ANGPTL3. SMA was expressed in cultured keratocytes but not in whole stromal samples or HCEC. ANGPTL6 and 7 were both expressed in keratocytes but not in HCEC. Immunocytochemistry results corroborated with the PCR analysis and none of the investigated protein markers were uniquely positive for HCEC.
This PDF is available to Subscribers Only