A putative role for RHAMM/HMMR as a negative marker of stem cell-containing population of human limbal epithelial cells.
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 characterization. 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 DeltaNp63 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 DeltaNp63 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 characterization. Disclosure of potential conflicts of interest is found at the end of this article.
Ahmad
Sajjad
S
North East Stem Cell Institute and, Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Newcastle upon Tyne, United Kingdom.
Kolli
Sai
S
Li
De-Quan
DQ
de Paiva
Cintia S
CS
Pryzborski
Stefan
S
Dimmick
Ian
I
Armstrong
Lyle
L
Figueiredo
Francisco C
FC
Lako
Majlinda
M
D016475:3T3 Cells; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000080924:Aldehyde Dehydrogenase 1 Family; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods; D003114:Colony-Forming Units Assay; D003315:Cornea / Q000166:cytology / Q000201:enzymology / Q000502:physiology; D019573:Epithelium, Corneal / Q000166:cytology / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000502:physiology*; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics; D006801:Humans; D018960:Hyaluronan Receptors / Q000502:physiology*; D007150:Immunohistochemistry; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics; D050697:Retinal Dehydrogenase; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015415:Biomarkers; D016326:Extracellular Matrix Proteins; D018960:Hyaluronan Receptors; D009363:Neoplasm Proteins; C098946:hyaluronan-mediated motility receptor; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)