Most of the Short Term Repopulating Cells in Human Mobilized Peripheral Blood Do Not Have a Side Population (SP) Phenotype.
Authors: Fischer M. , Schmidt M. , Klingenberg S. , Eaves C. , von Kalle4 C. , Glimm4 H. 
Published: April 2020

Abstract
The multidrug resistance transporter, ABCG2, is expressed in primitive hematopoietic stem cells from a variety of sources. These cells are detected in dual wave-length fluorescent FACS profiles as a “side population” (SP cells) on the basis of their ability to efflux the fluorescent dye, Hoechst 33342. We have previously shown that 2 types of human short term repopulating cells (STRC) can be enumerated by limiting dilution analysis of their efficient ability to regenerate exclusively myeloid cells after 3 weeks (STRC-Ms), or both myeloid and lymphoid cells after 6–12 weeks (STRC-MLs) in NOD/SCID-b2microglobulin-/- (b2m-/-) mice. Previous findings also implicated these STRCs as determinants of the rapidity of early hematologic recovery in patients transplanted with cultured mobilized peripheral blood (mPB) cells. Here we asked whether any human STRCs have an SP phenotype and hence whether the isolation of SP cells would retain the rapid repopulating activity of a clinical transplant. CD3- SP and non-SP cells were isolated by FACS from low-density (LD) mPB cells after Hoechst staining and transplanted at limiting dilutions into 117 sublethally irradiated b2m-/- mice. The numbers and types of human hematopoietic cells present in the bone marrow of these mice were subsequently monitored by FACS analysis of bone marrow cells aspirated serially, 3, 8 and 12 wks post-transplant. A verapamil-sensitive SP population was reproducibly detected in all 5 patients’ samples studied (0.039 ± 0.012% of the CD3- LD cells). The in vivo assays failed to detect either STRC-Ms or STRC-MLs in the SP fraction and all these activities were obtained from the non-SP cells. If even a single recipient of the largest dose of SP cells transplanted had been positive, this would have detected 10% of the STRCs present. Thus, &gt;90% of all STRC-M and STRC-ML in mPB are non-SP cells. However, 4 of 40 mice transplanted with SP mPB cells produced some B-lymphoid cells only starting 12 wks post-transplant. However, this result is difficult to interpret since subjecting the STRC-Ms to the Hoechst 33342 staining and FACS isolation procedure alone eliminated their ability to generate megakaryocytic progeny in vivo, although this did not occur when these cells were just stained for CD34 and then isolated by FACS. In addition, the differentiation behaviour of STRC-MLs was not affected by the Hoechst staining and subsequent FACS isolation procedure. In summary, we demonstrate that purification of SP cells depletes human mPB transplants of STRCs, thereby raising serious concerns about the safety of any clinical use of SP cell-enriched transplants as stem cell support after myeloablation. Our results also suggest that the staining and enrichment procedure for isolating SP human cells may differentially affect the lineage potential of some types of STRCs, including those whose activity may be indispensable for rapid and multi-lineage hematologic recovery.