A Putative Role for RHAMM/HMMR as a Negative Marker of Stem Cell-Containing Population of Human Limbal Epithelial Cells

¹ North East Stem Cell Institute and Institute of Human Genetics, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, United Kingdom.; Department of Ophthalmology, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne, NE1 4LP, United Kingdom
² Ocular Surface Center, Department of Ophthalmology, Baylor College of Medicine, Houston, USA
³ North East Stem Cell Institute, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, United Kingdom.; School of Biological Sciences, University of Durham, UK
⁴ North East Stem Cell Institute and Institute of Human Genetics, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, United Kingdom
⁵ North East Stem Cell Institute, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, United Kingdom.; Department of Ophthalmology, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne, NE1 4LP, United Kingdom

^* To whom correspondence should be addressed. E-mail: Majlinda.Lako{at}ncl.ac.uk.

	Abstract

The corneal epithelium is maintained by stem cells located at the periphery of the cornea in a region known as the limbus. Depletion of limbal stem cells (LSCs) results in limbal stem cell deficiency. Treatments for this disease are based on limbal replacement or transplantation of ex vivo expanded LSCs. It is therefore crucial to identify cell surface markers for LSCs that can be used for their enrichment and characterisation. Aldehyde dehydrogenases (ALDHs) are enzymes which protect cells from the toxic effects of peroxidic aldehydes. In this manuscript we show for the first time that ALDH1 is absent from the basal cells of the limbal and corneal epithelium. We separated limbal epithelial cells on the basis of ALDH activity and showed that ALDH^dim cells expressed significantly higher levels of Np63 and ABCG2 as well as having a greater colony forming efficiency (CFE) when compared to ALDH^bright cells. Large scale transcriptional analysis of these two populations led to identification of a new cell surface marker, RHAMM/HMMR, which is located in all layers of corneal epithelium and in the suprabasal layers of the limbal epithelium but is completely absent from the basal layer of the limbus. Our studies indicate that absence of RHAMM/HMMR expression is correlated with properties associated with LSCs. RHAMM/HMMR- limbal epithelial cells are smaller in size, express negligible CK3, have higher levels of Np63 and have a higher CFE compared to RHAMM/HMMR+ cells. Taken together these results suggest a putative role for RHAMM/HMMR as a negative marker of stem cell containing limbal epithelial cells. Cell selection based on Hoechst exclusion and lack of cell surface RHAMM/HMMR expression resulted in increased colony forming efficiency compared to negative selection using RHAMM/HMMR alone or positive selection using Hoechst on its own. Combination of these two cell selection methods presents a novel method for LSC enrichment and characterisation.