Title:Accuracy and Reproducibility of Stem Cell Side Population Measurements on Clinically Relevant Products
Volume: 9
Issue: 6
Author(s): Ariadna Avendano, Irene Sales-Pardo, Maria Dolores García-Godoy, Laura G. Rico, Pedro Marin and Jordi Petriz
Affiliation:
Keywords:
ABCG2, CD34, flow cytometry, side population, stem cell.
Abstract: Introduction: In 1996, Goodell et al. first described a rare subpopulation of bone marrow stem cells termed the
Side Population (SP). SP cells are known to be CD34 negative and to have a high repopulating capability. The SP was
identified by ultraviolet excitation based on the efflux of the DNA binding dye, Hoechst 33342 (Ho342). ABCG2, a halftransporter
that belongs to the ATP binding cassette transporter superfamily, is the major contributor to the SP phenotype
by actively pumping Ho432 selectively from stem cells. To date, very little is known about the identification of the SP in
peripheral blood samples, and about its peripheral circulation, enrichment or isolation to evaluate its therapeutic potential.
Due to the SP potential role in tissue regeneration, we studied the numbers of the SP in bone marrow and peripheral blood
samples in regard to count accuracy and reproducibility. Materials and methods: Bone marrow (BM) and apheresis (AP)
specimens were obtained from healthy donors and patients undergoing stem cell transplantation. Bone marrow samples
were obtained by aspiration. Peripheral blood cells after granulocyte colony stimulating factor (G-CSF) mobilization with
or without chemotherapy, were obtained by apheresis. All samples were prepared for identification of SP cells by flow cytometry.
Results: SP cells were detected in only 19 of 111 apheretic products, with relative frequency ranging from 0.01 to
4.75% of cells by the Ho342 exclusion method and flow cytometry analysis. Cell preparations used for these measurements
consisted of 5 x 106 cells. However, no SP cells were detected when aliquots from the same positive specimens,
consisting of previously stained 55 x 106 cells and fractionated into independent aliquots with 5 x 106 cells were used.
Conclusions: In this study, we show that there is great variability in SP cell numbers when aliquots obtained either from
leukapheresis or bone marrow products represent about 1% of the total product volume. In contrast, when aliquots represented
about 12% of the total product volume SP cells measurements were consistent. The high cell number of some
specimens can be a limitation for the accurate identification and isolation of the SP compartment. Aliquots containing a
minimum of 55 × 106 cells should be used for statistical significance.
