Characterisation of Side Population (SP) Cells Obtained from Different Hematopoietic Progenitor/Stem Cell Compartments in Human Bone Marrow and Peripheral Blood.
Authors: Josefsen Dag , Forfang Lise , Dyrhaug Marianne , Kvalheim Gunnar 
Published: April 2020

Abstract
Side population (SP) cells are characterised by their ability to exclude Hoechst 33342 dye from the cells. Using this method, it has been demonstrated that cells within the SP+ fraction of mononuclear cells from both murine and human hematopoietic systems are enriched for primitive hematopoietic stem- and progenitor cells. Moreover, most of the SP+ cells did not express CD34, indicating the presence of a CD34 negative hematopoietic stem cell population. To explore this further, we have examined SP+ cells obtained from different cell compartments in human bone marrow and peripheral blood.
Human bone marrow (BM) was obtained from healthy volunteer donors by iliac crest aspiration after informed consent. Mononuclear cells (MNC) were obtained by Ficoll grade centrifugation. CD34+ cells were then isolated from MNC. Highly enriched CD34+ cells were isolated from PBPC obtained from patients with Hodgkin lymphoma. To identify the SP+ cells, the cells were stained with Hoechst 33342 dye. Using flowcytometric techniques (FACStar+, FACSDiva, Becton Dickinson, San Jose, CA) we were able to visualize the dye efflux in SP+ cells. SP+ cells were functionally confirmed using Verapamil.
Phenotypical characterisation of the different cell populations using flow cytometric methods was performed. The level of SP+ cells in BM-MNC was 1,3% (mean, n=3) In line with previous findings, we observed that SP+ cells obtained from BM-MNC lack expression of several lineage committed markers, including CD15 and CD19. Most of the cells were CD34− (mean=2,2%), which was lower than in the main population (MP; mean=5%). The level of CD133 expression was low and similar in both populations. Furthermore we found a higher fraction of CD3+ T-cells in the SP fraction than in the MP fraction (mean: 69% vs 51%).
To further investigate the SP+CD34+ cell fraction, we examined CD34+ cells isolated from both human bone marrow and peripheral blood. The percentage of SP+CD34+ cells varied from 0,4 up to 18% of the total CD34+ cell population obtained from PBPC (n= 16), whereas the level of SP+CD34+ cells obtained from bone marrow was 5% of the total CD34+ cell population (n=3). Expression of lineage committed markers, including CD10, CD15 and CD19 was less then 10% of the whole CD34+ cell population obtained from PBPC, whereas we found a higher level of expression of these markers in CD34+ cells isolated from bone marrow. However, when we examined the SP+CD34+ cells from either PBPC or bone marrow, we observed that the phenotypic profile of these cells were similar with almost no expression of lineage markers.
The frequency of LTC-IC was markedly increased in SP+MNC, in line with previous findings. In addition we also observed a marked increase in LTC-IC in SP+CD34+ cells compared to SP-CD34+ cells in both BM and PB (BM: 7-fold increase; PB: 3–4 fold).
In conclusion, SP cells are present in different hematopoietic progenitor cell populations, including BM-MNC, BM-CD34+ cells and PB-CD34+ cells. In SP+CD34+ cell fractions from both BM and PB we observed an increased expression of stem cell markers like CD90 and CD133, whereas in SP+MNC we found low levels of CD34, CD90 and CD133 expression. However, the LTC-IC frequency was markedly higher in all SP+fractions compared to MP fractions, suggesting that sorting of SP+ cells from different hematopoietic stem- and progenitor cell compartments identify immature hematopoietic cells.