Fresh and Cryopreserved Umbilical Cord Blood Side Population Cells Have Comparable Immunophenotype and Function but Are Not Characterised by ABCG2 Expression.

The side population (SP) phenotype resulting from efflux of the fluorescent dye Hoechst 33342 has been associated with high quality stem cells from both hematopoietic and non-hematopoietic organs. While expression of the transporter proteins MDR1 and ABCG2 has been strongly implicated in the SP phenotype of murine bone marrow stem cells, relatively little is known about the SP in human umbilical cord blood. The aim of this study was to investigate the relationship between the expression of common stem cell markers, the SP phenotype and the expression of the MDR1 and ABCG2 transporters in both fresh and cryopreserved umbilical cord blood. Assays of long-term Cobblestone Area Forming Cell (CAFC) activity at week 13 confirmed a 22 fold enrichment (p=0,002) of CAFC potential in the sorted SP (0,11% +/−0,06%, n=7) compared to the control living lymphocyte population subjected to Hoechst-free preparation and sorting (0,005% +/−0,0023%, n=6). SP cells from fresh (n=3) and cryopreserved (n=4) blood samples performed equally well in CAFC assays (p=0,28). A multiparameter FACS analysis involving a combination of four colors plus SP controlled with verapimil revealed the CD133⁺ and CD34⁺ cells to be distributed towards the upper part of the SP plot and to extend into the verapimil-sensitive SP. However, only 20,5% +/−15,6% (n=9) of the SP cells expressed either CD34 or CD133. ABCG2⁺ cells were found to be distributed throughout the main population but to be specifically absent from the side population. Fresh and cryopreserved blood samples were similar in all of these respects. In contrast to the SP cells, the MACS-purified ABCG2⁺ population failed to grow in cytokine-supplemented media and yielded no colonies in the CAFC assay. Semi-quantitative RT-PCR assays comparing cDNA prepared from the SP to that from the entire mononuclear population revealed a moderate but consistent enrichment of MDR1 mRNA in the SP, but no enrichment of ABCG2 mRNA. A similar comparison of the MACS-separated CD133⁺ (n=4) and CD133⁻ (n=3) cells revealed the CD133⁺ population to be enriched (4 fold) for MDR1 but depleted (19-fold) for ABCG2 mRNA. We conclude that ABC transporters other than ABCG2 are responsible for the SP phenotype in human UCB; that MDR1 remains a potential candidate in this respect and that the frequency, immunophenotype and CAFC function of SP cells from fresh and cryopreserved blood are indistinguishable.