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M.T. Budak, M.A. Akinci, J.M. Wolosin; Ocular Surface Epithelial Cells Express the MDR Transporter ABCG2 and Thus Can Be Isolated as a Side Population Following Staining With Bisbenzimide . Invest. Ophthalmol. Vis. Sci. 2003;44(13):859.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Multidrug resistance (MDR) pump-mediated efflux of the dye bisbenzimide (Ho33342) from cells results in an spectral shift which causes such cells to appear as a side population (SP) in FACS emission plots. This feature is routinely exploited for the isolation of hematopoietic stem cells by FACS. Recent studies demonstrate that, a) this efflux is accomplished by the MDR transporter BCRp1/ABCG2 (Nat Med.; 7:1028); and b) ABCG2 is expressed in other somatic stem cells. The purpose of this study was to identify ABCG2 in ocular surface epithelial cells and thereby provide the means for their isolation. Methods: RNA was isolated from human (Hu), conjunctiva (Cnj), limbus(Li) and cornea (Co). cDNA was prepared by reverse transcription (RT). PCR was performed using established primers for ABCG2. Expression and spatial distribution of ABCG2 were determined by immunofluorescence using a monoclonal anti-human ABCG2 Ab. HuCnj and rabbit (Rb) Cnj and Li cells were isolated by sequential Dispase/Trypsin treatments and incubated with 0.004 mg/ml Ho made in 2% FCS/HBSS for 90 min at 37° C. SP cells presence and content and their light scattering properties were determined using a MoFlo FCAS instrument equipped with UV and argon laser excitations. FITC-conjugated Annexin V staining was used to exclude apoptotic cells. The contribution of ABCG2 to the SP population was established with the specific inhibitor fumitremorgin C (FTC; BBA 1512: 171). Results: The expected PCR amplicon for ABCG2 was generated from the HuCnj and HuLi material but not in HuCo or in Li and Cnj samples in which the RT step was omitted. ABCG2 immunofluorescence was present in the plasma membrane of isolated cells or cell patches of basal Li and Cnj cells (<10% of total basal cells). SP cells were present in the Cnj and Li but not in the Co. FTC-sensitive SPs represented 1.2 % (n =4) in the RbCnj, 0.34 % in the HuCnj and 0.2% in the RbLi, respectively. In the light scatter plot the SP cells displayed extremely low side scattering (indicates a scarcity of organelles) and low forward scattering (indicates small size). Following sorting, when grown over 3T3 feeder layers, the SP cells started to proliferate after a 72 hr delay and yielded only long term proliferating colonies (holoclones). In contrast, non-SP cells started to proliferate within 24 hr and yielded both holo- and paraclones Conclusions: A subset of ocular surface cells express the MDR transporter ABCG2. These cells can be isolated as SP cells by FACS following incubation with bisbenzimide and display physical features and growth properties consistent with a stem cell nature.
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