How to cite this article: Sharma S, Shariatmadar S, Krishan A. Electronic volume, aldehyde dehydrogenase, and stem cell marker expression in cells from human peripheral blood apheresis samples. Cytometry Part B 2010; 78B: 123–129.
Over-expression of aldehyde dehydrogenase and other stem cell markers is characteristic of cells with tumorigenic potential in NOD/SCID mice. Most of these studies have focused on metastatic cells in bone marrow and on solid tumors. There are no studies on correlation of marker expression with ALDH1 expression in cells from human peripheral blood apheresis (HPC-A) samples.
HPC-A samples from 44 patients were incubated with Aldefluor® with or without the presence of aldehyde dehydrogenase inhibitor DEAB. Cells with high aldehyde dehydrogenase expression (ALDH1bright) were analyzed for stem/progenitor markers CD34, CD90, CD117, and CD133. Electronic volume measured by Coulter principal in a Quanta flow analyzer was correlated with ALDH1 and marker expression.
In ALDH1bright/SSClow cells, 0.13% of the cells had CD34+ expression and three distinct populations were seen. Expression of CD90 was dim and the frequency of ALDH1bright/SSClow/CD90dim cells amongst the nonlineage depleted samples was 0.04%. CD117dim-bright expression was seen in 0.17% of the samples. Three distinct populations of cells with CD133 expression were seen in ALDH1bright/SSClow nonlineage depleted cells with a frequency of 0.28%. The ALDH1bright/CD90dim cells had the smallest mean electronic volume of 264.9 μm3 when compared with cells with CD34bright expression (270.2 μm3) and ALDH1dim/CD90dim cells (223 μm3).
Aldehyde dehydrogenase isoform 1 (ALDH1) belongs to a family of enzyme involved in metabolism of aldehydes to their corresponding carboxylic acids (1). ALDH1 plays an important role in vitamin A metabolism and in resistance to alkylating agents. ALDH1 as a detoxifying enzyme protects stem cells and may have a role in early stem cell differentiation, by oxidizing retinol to retinoic acid (2). Several studies have shown that murine and human hematopoietic and cord blood and neural stem cells have high ALDH1 expression (3–5).
Storms et al. (6) designed a fluorescent substrate BODIPY aminoacetaldehyde (BAAA), which after intracellular diffusion is converted by ALDH1 into BODIPY aminoacetate (BAA), a green fluorescent marker. Flow cytometric detection of ALDH1 expression by staining with Aldefluor® with or without the presence of ALDH1 inhibitor, diethylaminobenzyaldehyde (DEAB) is used for stem cell analysis.
Several workers have characterized the ALDH1 positive cells from human and murine hematopoietic and cord blood samples. Hess et al. (7) purified cells with high ALDH1 activity from human cord blood and showed that they coexpressed stem cell markers such as, CD34 and CD133. Pearce and Bonnet (8) purified ALDH1 positive, lineage negative cells from murine bone marrow and showed that they overlapped with the side population (SP) cells. Gentry and coworkers (9, 10) reported that sorted ALDH1bright cells from cord blood and bone marrow were highly enriched in hematopoietic colony forming cells (CFC-H) and those in human bone marrow formed endothelial and fibroblast colonies in culture.
This study was undertaken to monitor the coexpression of stem cell markers such as CD34, CD90, CD117, and CD133 in ALDH1bright/SSClow cells from human apheresis samples. We have also used a Quanta flow cytometer (Beckman Coulter, Miami, FL) to determine the electronic volume of cells expressing these markers.
MATERIALS AND METHODS
This study was based on the analysis of human peripheral blood apheresis (HPC-A) samples from 44 patients with hematological malignancies referred for autologous hematopoietic progenitor cell transplantation. Our apheresis population consists of multiple myeloma 47.4%, Non-Hodgkin's Lymphoma 31.6%, and Hodgkin's Lymphoma 21%. The HPC-A samples were from patients in complete remission, who had received chemotherapy or immunomodulatory treatment. The mononuclear cell population examined consisted of normal cells from the peripheral blood. Large volume leukapheresis was performed after 5 days of mobilization with subcutaneous administration of granulocyte colony stimulating factor (G-CSF), 5–10 μg/kg/day. At the completion of leukapheresis, 1 ml of the final HPC-A product was used for flow cytometric analysis. HPC-A samples were collected by the University of Miami Miller School of Medicine Institutional Review Board approved IRB.
Processing of Leukapheresis Samples
One milliliter of the leukapheresis sample diluted with phosphate buffered saline (PBS) was layered on 15 ml of Histopaque-1077 (Sigma Diagnostics, St Louis, MO). After centrifugation at 400× relative centrifugal force (RCF) for 30 min at room temperature, cells were aspirated and washed twice with ice-cold PBS. The cell pellet was resuspended and number of dye excluding cells was determined by the trypan blue dye exclusion method.
Samples were enriched for lineage negative cells by magnetic depletion of the lineage positive cells, labeled with a cocktail of biotin-conjugated monoclonal antibodies (CD2, CD3, CD11b, CD14, CD16, CD19, CD56, CD123, and CD235a), followed by the addition of anti biotin monoclonal antibody conjugated to metal colloid microbeads. Briefly, a total of 1 × 107 cells/ml was washed with PBS, centrifuged, and the cell pellet resuspended in 40 μl of PBS buffer (containing 0.5% BSA and 2 mM EDTA). After centrifugation, 10 μl of Biotin-Antibody Cocktail was added and the cells were incubated for 10 min at 4–8°C. Cell pellet retrieved by washing with PBS buffer, and centrifugation was resuspended in 80 μl of PBS buffer and 20 μl of Anti Biotin Microbeads (Miltenyi Biotech GmBH, Auburn, CA). After 15 min of incubation at 4–8°C, cells were washed, centrifuged, and the cell pellet was resuspended in 500 μl of PBS buffer. The cell suspension was transferred to a DYNAL®MPC®L (Dynal Biotech ASA, Oslo, Norway) magnetic rack for 20 min at 4–8°C. The supernatant was decanted, centrifuged, and the cell pellets washed twice with PBS buffer.
One milliliter aliquot containing 2 × 106cells was diluted with 2 ml of ice-cold PBS, centrifuged, and the cell pellet was resuspended in 1 ml of ice-cold Aldefluor® buffer. Five microliter of activated Aldefluor® substrate (Stem Cell Technologies, Durham, NC) was added to the suspension, and after mixing, 500 μl were transferred to a control tube containing 5 μl of DEAB. After incubation for 60 min at 37°C, antibodies were added and the tubes stored in a refrigerator for 20 min. The following antibodies were used for the experiments: CD34-PE-CY5 (Clone 581, catalog number IM2648U, Beckman Coulter, Hialeah), CD90-PE (Clone 5E10, catalog number R7275, Dako, Carpinteria, CA), CD117-PE (Clone 104d2, catalog number 340867, BD Pharmingen, San Jose), and CD133/2-PE (Clone 293C3, catalog number 130-090-853, Miltenyi Biotech GmBH, Auburn, CA) according to the manufactures instructions. Following incubation, centrifugation, and washing, cells were resuspended in 0.5 ml of ice-cold Aldefluor® assay buffer for flow analysis.
Flow Cytometric Analysis
A Cell Lab Quanta™ SC flow analyzer (Beckman Coulter, Miami, FL) was used for the determination of electronic volume and marker expression. Cells were analyzed for side scatter, electronic cell volume, and two-color fluorescence of Aldefluor® and CD34-PE-CY5, CD90-PE, CD117-PE, or CD133-PE. Cells incubated with the Aldefluor® with or without the DEAB blocker were analyzed to identify populations with ALDH1bright fluorescence and low side scatter (SSClow). Electronic gates were used to determine the expression of CD34, CD90, CD117, and CD133 in the ALDH1bright/SSClow cells. A total of 50,000 events were collected for each list mode file.
Electronic volume was calibrated with the National Institute of Standards and Technology (NIST) certified beads obtained from Beckman Coulter (Standard L5, 5.15 μm, L10, 10.24 μm). The coefficient of variation of electronic volume measurement averaged between 4 and 7% depending on the lot number of the beads. The correlation coefficient (r2) of the mean channel value (MCV) with the mean volume/diameter of the certified beads was 0.998, indicating that linearity of the measurement was excellent. Calibration data was either expressed as volume (μm3) or diameter (μm). Winlist 3D (Version 5.0) and WinMDI (Version 2.9) were used for analysis of the list mode data and for auto compensation of the two color dot plots.
MedCalc (Version 188.8.131.52) software was used for data analysis. Results were expressed as mean ± standard deviation (SD). Student's paired t-test was used for statistical evaluation of data.
Single Marker Expression in ALDH1bright/SSClow Cells
Table 1 lists the mean frequency of the ALDH1bright and ALDH1dim cells with SSClow with CD34, CD90, CD117, and CD133 expression. In mononuclear cells from a HPC-A sample incubated with the aldehyde dehydrogenase specific dye, Aldefluor®, a distinct population of cells with ALDH1bright fluorescence and SSClow are indicated by an arrow in Figure 1A. This population in gate R2 was 0.89% of the total nucleated cells and was absent in the sample coincubated with the aldehyde dehydrogenase inhibitor and diethylaminobenzaldehyde (DEAB) as shown in Figure 1B.
Table 1. Mean Frequency of the ALDH1bright and ALDH1dim Cells with SSClow with CD34, CD90, CD117, and CD133 Expression
0.13% ± 0.001
0.04% ± 0.001
0.17% ± 0.001
0.28% ± 0.004
0.43% ± 0.005
0.09% ± 0.002
0.19% ± 0.05
0.30% ± 0.01
Figure 2A shows a dot plot of CD34 expression amongst the gated ALDH1bright/SSClow cells from a lineage depleted sample. Three distinct populations of cells with different levels of CD34 expression as shown in gates R3, R4, and R5 were seen. The cells with CD34negative (100–101 log), CD34dim (101–102 log), and CD34bright (102–103 log) expression of the ALDH1bright/SSClow population were 0.15, 0.33, and 0.24%, respectively. The mean and SD of ALDH1bright/SSClow cells with CD34bright expression in 44 nonlineage depleted HPC-A samples was 0.13% ± 0.12, 95% CI = 0.08–0.17, P < 0.0001.
As shown in Figure 2B, 0.21% of the ALDH1bright/SSClow cells in this lineage depleted sample had CD90dim (log 101–102) expression (gate R4). In this dot plot, 0.51% of the ALDH1bright/SSClow cells had CD90negative (log100–101 log) expression (gate R3). The mean frequency of ALDH1bright/SSClow/CD90dim cells amongst the 44 nonlineage depleted samples was 0.04% ± 0.03, 95% CI = 0.02–0.05, P < 0.0001.
As shown in dot plot 2C, 0.23% of the ALDH1bright/SSClow cells had CD117dim-bright expression and 0.51% of the cells were CD117negative-dim. The mean frequency of the CD117dim-bright cells amongst the 44 nonlineage depleted samples was 0.17% ± 0.14, 95% CI = 0.11–0.22, P < 0.0001.
In dot plot 2D, three distinct populations of cells with CD133 expression shown in gates R3, R4, and R5 were seen. The cells with CD133negative, CD133dim, and CD133bright expression of the ALDH1bright/SSClow population were 0.17, 0.39, and 0.18%, respectively. The mean frequency of CD133bright cells amongst the ALDH1bright/SSClow nonlineage depleted cells was 0.28% ± 0.37 95% CI = 0.13–0.42, P < 0.0005.
Double Marker Expression in ALDH1bright/SSClow/Linnegative Populations
In Figure 3, dot plots of HPC-A samples depleted of lineage positive cells by magnetic separation and gated for ALDH1bright/SSClow expression are shown.
As shown in Figure 3A (gate R3), 0.13% of the cells had CD34bright and CD90mid expression. In contrast, 0.11% of the cells shown in gate R5 were CD34bright with CD90negative expression.
Dot plot 3B shows that 0.25% of the ALDH1bright/SSClow/Linnegative cells had CD34bright and CD117dim-bright expression. In gate R4, 0.30% of the cells had CD34dim and CD117dim expression. In gate R5, 0.12% of the cells had CD34negative and CD117dim expression.
As shown in Figure 3C, 0.25% of the ALDH1bright/SSClow/Linnegative cells had CD34dim-bright and CD133dim expression. 0.38% of the cells had CD34dim and CD133negative expression (gate R4), and 0.31% of the cells were negative for both CD34 and CD133 expression.
Electronic Cell Volume and Marker Expression
In Table 2, the mean electronic volume of ALDH1bright/SSClow cells expressing CD90, CD34, CD133, and CD117 from 44 HPC-A samples is shown. The ALDH1bright/CD90bright cells had the smallest mean electronic volume of 264.9 μm3 (±33.6, 95% CI = 248–281, P < 0.0001) when compared with ALDH1bright/SSClow cells with CD34bright expression (270.2 μm3, ± 32.9, 95% CI = 257–282, P < 0.0001), CD133 (275.3 μm3, ± 28.8, 95% CI = 264–286, P < 0.0001), and CD117 (284.2 μm3, ± 31.9, 95% CI = 272–295, P < 0.0001). When compared with the ALDH1bright/CD117bright cells, the mean volume of the ALDH1bright/CD90positive, ALDH1bright/CD34positive, and ALDH1bright/CD133positive cells were 7.3 (±34.5, 95% CI = 247–272, P = 0.05), 5.2 (±33.4, 95% CI = 257–272, P = 0.01), and 3.2% (±29.2, 95% CI = 264–272, P = 0.02) smaller, respectively.
Table 2. The Mean Electronic Volume (μm3) of ALDH1bright and Dim Cells Expressing CD90, CD34, CD133, and CD117
264.9 ± 33.6
270.2 ± 32.9
275.3 ± 28.8
284.2 ± 31.9
223.4 ± 24.7
228.6 ± 28.7
230.1 ± 30.7
234.5 ± 28.4
The ALDH1dim/CD90positive cells had the smallest mean electronic volume of 223.4 μm3 (±24.7, 95% CI = 211–235, P < 0.0001) when compared with ALDH1dim/SSClow cells with positive expression for CD133 (228.6 μm3, ±28.7, 95% CI = 217–239, P < 0.0001), CD34 (230.1 μm3, ±30.7, 95% CI = 218–241, P < 0.0001), and CD117 (234.5 μm3, ±28.4, 95% CI = 223–245, P < 0.0001). When compared with the ALDH1dim/CD117positive cells, the mean volume of the ALDH1dim/CD90positive cells was 4.9% (±24.7, 95% CI = 211–223, P = 0.01) smaller.
ALDH1 is highly expressed in hematopoietic stem/progenitor cells, intestinal crypt cells, and in breast tumor cells. The expression of ALDH1 has been most extensively studied within the context of early hematopoiesis. In a study evaluating umbilical cord blood cells, Storms et al. (11) demonstrated that the ALDH1bright/CD34positive population had a high frequency of short and long term bone marrow repopulating cells (SRCs) in SCID mice and was highly enriched for cells that initiated primary and secondary long term cultures (LTCs). In contrast, the ALDH1bright/CD34negative cells contained no SRC, LTCs, or clonogenic myeloid cells and exhibited minimal growth in short term NK cultures. In murine bone marrow, ALDH1bright/CD34negative cells provided long term repopulation but did not contain clonogenic myeloid progenitors or primitive multilineage colony forming cells (12). In human cord blood, the ALDH1bright/SSClow cells include virtually all of the stem cells and can generate multipotent cell colonies in vitro and provide long term-reconstitution of bone marrow in NOD/SCID mice. The CD34positive/ALDH1dim/SSClow population had very limited colony-forming ability in vitro and failed to home effectively in mice and only provided short-term reconstitution in NOD-SCID mice (5). ALDH1dim cells from human cord blood can initiate short-term progenitors in mice xenograft models, whereas the ALDH1bright cells can initiate long-term cultures and grow as xenografts (10). Hess et al. (7) reported that in human cord blood, ALDH1bright/CD34negative cells might represent a primitive progenitor population, whereas Mirabelli et al. (13) reported that in human bone marrow ALDH1bright/CD34negative, cells were committed towards erythropoiesis. Dourville et al. (14) sorted peripheral blood cells from acute myeloid leukemia patients based on ALDH1 staining. He reported that ALDH1high cells with high side scatter were leukemic stem cells and on transplantation in NOD/SCID mice differentiated in to cells of myeloid lineage. On the other hand, normal cells with ALDH1high expression provided complete reconstitution of the hematopoietic system in NOD/SCID mice.
In this study, we evaluated ALDH1 activity in normal human stem/progenitor cells obtained by apheresis and to our knowledge the present report is one of the first on accurate measurement of electronic cell volume of ALDH1bright cells with different marker expression from HPC-A samples.
In this study, the ALDH1bright/SSClow cells had three subpopulations of cells with CD34 and CD133 expression. In contrast, the ALDH1bright/SSClow cells had either positive or negative expression of CD90 and CD117. The percentage of CD90 positive cells was the lowest (0.04%) amongst the ALDH1bright/SSClow cells, while CD133 positive cells were more frequent (0.28%). Furthermore, some of the ALDH1bright/SSClow cells did not express CD34, CD90, CD117, or CD133 markers.
Published studies have shown that the ALDH1bright/CD133positive cells contain both short and long term repopulating human HSCs and in contrast to ALDH1bright/CD133negative, cells have faster engraftment and repopulation capacity (7). Our data confirms published studies showing that most ALDH1bright/SSClow cells display phenotypic markers of primitive hematopoietic cells, that is, CD34, CD117, and CD133 (7, 11, 13). To our knowledge, no prior studies have evaluated the coexpression of ALDH1bright cells with CD90 expression.
In an earlier published study, we reported on the electronic volume of HPC-A cells with different stem cell marker expression. We reported that the CD34positive and CD90positive cells with CD45dim expression had the smallest volume in HPC-A samples. In this study, we found that the CD90positive cells amongst the ALDH1bright/SSClow population had the smallest mean electronic volume of 264.9 μm3 when compared with ALDH1bright/SSClow cells with CD34positive, CD133positive, and CD117positive expression. When compared with the ALDH1bright/CD117positive cells, the ALDH1bright/CD90positive cells were 7.3% smaller.
Similarly, the ALDH1dim/CD90positive cells had the smallest mean electronic volume when compared with the ALDH1dim/SSClow cells with positive expression of CD34, CD133, and CD117 stem cell markers. The ALDH1dim/CD90positive cells were 4.9% smaller when compared with the ALDH1dim/CD117positive cells.
Several earlier studies have reported on the presence of stem cells with small size in bone marrow, embryonic tissues, and cord blood (15–18). As most of these studies used either light or electron microscopy of fixed cells or forward scatter in flow cytometry, they lacked the accuracy for measurement of cell volume. As shown in our earlier study (19, 20), use of Coulter electronic volume (21) after calibration with NIST certified beads can be a reliable method for determining the volume and diameter of stem cells. The mean volume of the ALDH1bright/SSClow expressing the different stem cells markers, that is, CD90, CD34, CD133, and CD117, was 265, 270, 275, and 284 μm3 which correspond to a cell diameter of 7.97, 8.02, 8.07, and 8.16 μm. The ALDH1dim cells expressing the stem cell markers were smaller than the ALDH1bright cells.
Although prior reports have disregarded the role of ALDH1dim cells as long term repopulating cells, phenotypic expression of early stem cell markers in both the ALDHbright/SCClow and ALDHdim/SCClow cell population in our patient samples bring in to question the exact nature and reconstitutive capability of these cells. Additional studies are clearly necessary to identify characteristics which may further define the clonogenic capabilities of these cell populations. The use of electronic volume in conjunction with stem cell marker expression of the ALDH1bright and ALDH1dim cells might provide a novel and distinct method for the isolation of stem and progenitor cells and can serve as a promising alternative to routine stem cell markers for isolation of these cells. Future studies will further evaluate these cells in short and long term clonogenic assays and as xenografts in mice.