Further phenotypic characterization of the primitive lineage- CD34+CD38-CD90+CD45RA- hematopoietic stem cell/progenitor cell sub-population isolated from cord blood, mobilized peripheral blood and patients with chronic myelogenous leukemia

Abstract

The most primitive hematopoietic stem cell (HSC)/progenitor cell (PC) population reported to date is characterized as being Lin-CD34+CD38-CD90+CD45R. We have a long-standing interest in comparing the characteristics of hematopoietic progenitor cell populations enriched from normal subjects and patients with chronic myelogenous leukemia (CML). In order to investigate further purification of HSCs and for potential targetable differences between the very primitive normal and CML stem/PCs, we have phenotypically compared the normal and CML Lin-CD34+CD38-CD90+CD45RA- HSC/PC populations. The additional antigens analyzed were HLA-DR, the receptor tyrosine kinases c-kit and Tie2, the interleukin-3 cytokine receptor, CD33 and the activation antigen CD69, the latter of which was recently reported to be selectively elevated in cell lines expressing the Bcr-Abl tyrosine kinase. Notably, we found a strikingly low percentage of cells from the HSC/PC sub-population isolated from CML patients that were found to express the c-kit receptor (<1%) compared with the percentages of HSC/PCs expressing the c-kitR isolated from umbilical cord blood (50%) and mobilized peripheral blood (10%). Surprisingly, Tie2 receptor expression within the HSC/PC subset was extremely low from both normal and CML samples. Using in vivo transplantation studies, we provide evidence that HLA-DR, c-kitR, Tie2 and IL-3R may not be suitable markers for further partitioning of HSCs from the Lin-CD34+CD38-CD90+CD45RA- sub-population.

Figure 1

CB- enriched CD34-positive cells were analyzed for the expression of lineage markers CD34, CD38, CD90, CD45RA and HLA-DR by flow cytometry. The left panel (a) is gated on lineage-negative (Lin−) viable cells whereas the center panel (b) is gated on the Lin−CD34+CD38− cells. The right panel (c) is gated on the Lin−CD34+CD38−CD90+CD45RA− HSC/PC sub-population and shows the distribution of the HLA-DR antigen (blue histogram) within this primitive subset. The histogram in red is the fluorescence histogram obtained for the gated cells (CD90+CD45RA−) in the absence of staining with an Alexa 700-conjugated MoAb and is referred to as the FMO (Fluorescence Minus One) control. The number under each descriptive sub-population represents the percentage of positive cells within the total population of Lin−CD34+ enriched (a) or the CD34+CD38− (b) cells. Data shown represent one of three separate MPB experiments.

Figure 2

CB-, MPB- and CML-enriched CD34+ cells were gated on the Lin−CD34+CD38−CD90+CD45RA− sub-population (as demonstrated in Figure 1) and analyzed for their expression of HLA-DR, c-kitR, Tie2, CD69, CD33 and the IL-3R by flow cytometry using Alexa Fluor 700-conjugated MoAbs to each of these antigens. Each data point in the figure represents the percentage of cells in an individual experiment that was positive for the antigen shown. Control samples represent percentage of positive cells obtained in the absence of any Alexa 700 MoAb (FMO). Crossbars represent the arithmetic means of the data points in each column.

Figure 3

Representative expression profiles of six surface markers in the Lin− CD34+CD38−CD90+CD45RA− sub-population isolated from CB, MPB and CML patient samples. The six markers were (a) HLA-DR, (b) c-kitR, (c) Tie2, (d) CD69, (e) CD33 and (f) IL-3R. Red histograms are the control FMOs (see Figure 1) whereas those in blue are the expression profiles obtained by staining the cells with the Alexa 700-conjugated MoAbs.