Side-population cells from different precursor compartments

The rapid efflux of the fluorescent DNA-binding dye Hoechst 333,42 identifies a rare, so-called side population (SP), which rapidly expels the dye, can reconstitute the bone marrow (BM) of lethally irradiated mice, and has proven negative for most lineage markers including CD34. Because SP cells from human cell sources, such as mobilized peripheral blood [apheresis products (AP)], cord blood (CB), or BM have not been extensively characterized to date, we sought to analyze SP cells from various cell sources. We detected murine SP cells with a median frequency of 0.04% (n=23) and a 52-fold colony-forming units (CFU) increase compared to unsorted cells (p=0.028). The median frequency of human SP cells was 0.02% (n=90), with highest numbers in donor AP, and lower in CB and BM. Human SP cells were mostly CD34(-) and lineage marker-negative. These showed no enrichment in CFU before expansion; however, they displayed a CFU increase after 5-7 days of cytokine-supported suspension culture (10.7-fold at day 5, 7.2-fold at day 7; n=17) that was significant compared to both input (day 0) SP and to non-SP cells before and after expansion (P<0.05). SP cells demonstrated a significant long-term culture-initiating cell (LTC-IC) increase of 167-fold (n=17) as compared to non-SP cells (p=0.002), with the highest numbers from AP specimens. We conclude that human primitive hematopoietic cells can be isolated via Hoechst staining and that SP cells of various human sources show substantial differences and represent a rare CD34(-) population with stem cell potential.