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a Hematopoietic Stem Cell Laboratory,
b FACS Laboratory, London Research Institute, Cancer Research UK, London, United Kingdom;
c Cancer Research UK Department of Medical Oncology, St. Bartholomew’s Hospital, West Smithfield, London, United Kingdom
Key Words. Human CD34+ cells • Acute myelogenous leukemia • Selection technologies • NOD/SCID model
Correspondence: Dominique Bonnet, Ph.D., Hematopoietic Stem Cell Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom. Telephone: 020-726-93281; Fax: 020-726-93581; e-mail: dominique.bonnet{at}cancer.org.uk
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Elevated levels of ALDH have been demonstrated in murine and human progenitor cells compared with other hematopoietic cells. Because these early studies used Western blotting and intra-cellular antibody staining, they were limited to the assessment of nonviable cells [5]. More recently, a method has been developed for the assessment of ALDH activity in viable cells and has been made commercially available in a kit format. This noncytotoxic method uses a cell-permeable fluorescent substrate to identify cells with high ALDH activity [6]. Substrate converted by ALDH is a charged molecule and is unable to leave the cell as freely as the unconverted substrate. In this way, converted ALDH substrate accumulates in cells with a high ALDH activity. This approach has allowed the analysis of viable murine and human ALDH+ progenitors by flow cytometry. Human cord blood hematopoietic cells with high ALDH activity are highly enriched for primitive CD34+ cells and depleted for lineage-positive (Lin+) cells (CD3, CD14, CD20, and CD56), indicating that they do indeed represent a primitive hematopoietic cell population [7].
Acute myeloid leukemia (AML) is characterized by a relentless accumulation of immature, abnormal hematopoietic cells in the bone marrow and peripheral blood. It has been postulated that AML is a disease maintained by leukemic stem cells and, hence, may be organized in a similar way to normal hematopoiesis. Indeed, only a subset of AML cells is capable of forming colonies in vitro, and an even smaller fraction can maintain colony production for 6 weeks while on feeder layers [8]. Definitive proof that a small population of putative leukemic stem cells produces the AML blasts comes from 6-week primary and secondary engraftment experiments in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice [9]. It has been postulated that the hierarchical organization of AML explains the pattern of remission and subsequent relapse that is typical of the response to cytotoxic chemotherapy. This has led to the suggestion that although most AML blasts are killed by cytotoxic therapy, leukemic stem cells may be spared and might be able to propagate the disease at a later time.
Two leukemia cell lines exist that have cyclophosphamide-sensitive and -resistant clones. In both cell lines reported in the literature (L1210 and BNML), the cyclophosphamide-resistant clone exhibits a higher ALDH activity as detected by Western blot and antibody labeling [10, 11]. Furthermore, constitutive expression of human ALDH1 or ALDH3 in human hematopoietic cells increases their resistance to cytotoxic agents [3, 12]. Accordingly, when expression of ALDH1 is blocked by the expression of antisense ALDH mRNA, cell sensitivity to 4-hydroperoxycyclophosphamide is greatly increased in vitro [13]. In this study, we have investigated the incidence and significance of cell subsets with high ALDH activity from different patients with AML. First, we confirmed the use of the ALDH substrate kit to identify cord blood CD34+ stem/progenitors cells and expanded on their phenotypic analysis. Via multicolor flow cytometry, we then assessed the incidence, morphology, phenotype, and NOD/SCID engraftment ability of ALDH+ cells in AML samples. AML samples had either no ALDH+ cells at all, an extremely rare nonmalignant stem/progenitor cell population, or a less rare, leukemic stem cell population. Accordingly, when injected into mice, ALDH+ cells demonstrated either normal, multilineage engraftment or malignant, unilineage AML growth. Hence, in addition to identifying nonmalignant stem cells within some AML samples, a high ALDH activity also identifies some patients’ leukemic stem cells. The incidence of normal or leukemic stem cells with an extremely high ALDH activity may have important implications for resistance to chemotherapy. Furthermore, the identification of leukemic stem cells on the basis of ALDH activity offers a new technique for their isolation that relies on stem cell function rather than surface phenotype.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice
All animal experiments were performed in compliance with Home Office and institutional guidelines. NOD/SCID mice were originally obtained from Dr. Leonard Schultz (Jackson Laboratory, Bar Harbor, ME) and bred at Charles Rivers Laboratories, London. They were kept in microisolators and fed sterile food and acidified water. Mice aged 8–12 weeks were irradiated at 375 rads (137Cesium source) up to 24 hours before intravenous injection of cells.
Cell Labeling
Cells were labeled with Aldefluor reagent (Becton, Dickinson [BD] Biosciences, Oxford, U.K., http://bdbiosciences.com) as described by the manufacturer. Cells were then stained with phycoerythrin (PE)–conjugated, PE-cyanin-5 (PE-CY5)–conjugated, PE-CY7–conjugated, or allophycocyanin-conjugated anti-CD34, anti-CD7, anti-CD38 (all BD Biosciences), and anti-AC133 (CD133) (Miltenyi Biotec, Cologne, Germany, http://www.miltenyibiotec.com) antibodies for 30 minutes at 4°C. Cells were washed and resuspended in phosphate-buffered saline (PBS) with 2% fetal calf serum and 4,6-diamidino-2-phenylindoiole (DAPI). Cells were then analyzed on a BD LSR flow cytometer. Aldefluor reagent was excited at 488 nm. Gates were set up to exclude nonviable cells and debris. The negative fraction was determined using appropriate isotype controls.
Assessment of Engraftment Potential
Samples were injected into the tail vein of sublethally irradiated 8- to 12-week-old mice. Six weeks after transplantation, mice were euthanized by cervical dislocation. The femurs, tibias, and pelvis were dissected and flushed with PBS. Red blood cells were lysed via ammonium chloride. Cells were stained with human-specific fluorescein isothiocyanate–conjugated anti-CD19, PE-conjugated anti-CD33, and PE-Cy5–conjugated anti-CD45 antibodies (all from BD Biosciences). Dead cells and debris were excluded via DAPI staining. A BD LSR flow cytometer was used for analysis. More than 100,000 DAPI-negative events were collected for each sample. Engraftment of AML was said to be present if a single population of CD45+ CD33+CD19– cells was present without accompanying CD45+ CD33–CD19+ cells.
Fluorescent In Situ Hybridization
Briefly, FACSorted human cells were swollen in hypotonic (0.075 M) KCl solution and fixed in Carnoy’s fixative (3:1 methanol: acetic acid) before dropping onto clean glass slides. Nuclei were aged overnight before pepsin digestion, dehydration, and application of fluorescent probes. Nuclei were incubated with probes overnight at 37°C before analysis at x1,000 magnification on an Axioplan-2 microscope (Carl Ziess, Jena, Germany, http://www.zeiss.com) equipped with Axiovision software.
Statistics
The Student’s paired t-test for significance of no difference was used throughout this report.
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RESULTS |
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High ALDH Activity Identifies Cord Blood Cells with Immature Cell Morphology
During preliminary experiments, it was noted that the signal-to-noise ratio of ALDH staining could be improved considerably if, after labeling, cells were washed twice in a large volume of buffer to reduce residual background labeling. This refinement gave us more clearly ALDH-positive and -negative populations. Consequently, we were able to visualize a subpopulation of cells that were very bright for the ALDH substrate (Fig. 1A
). These cells had the medium side and forward scatter that is characteristic of stem/progenitor cells (superimposed in Fig. 1B). These low-side-scatter, highly ALDH-positive cells accounted for 0.82% ± 0.39% of mononucleated cells in cord blood (range, 0.35%–1.29%; n = 17).
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). These ALDH+ cells were separate and distinct from the ALDH– cells and therefore easily identified.
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; Table 1). Although CD38 coexpression analysis revealed that most of the rare Lin–/CD34–/ALDH+ cells coexpressed CD38 in large amounts (86.3% ± 4.1%), indicating that most of this subset are probably committed cells [14], rare Lin–/CD34–/ALDH+/CD38– cells do exist. These Lin–/CD34–/ ALDH+/CD38– cells represent an extremely rare cell population, which accounts for 0.19% of Lin– cells and approximately one mononuclear cell in six million. Lin–/CD34–/ALDH+/CD38– cells coexpressed low levels of CD133, but the level was the same as the whole Lin–/CD34–/ALDH+ cell fraction (p = .73).
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Interestingly, there seemed to be an association between the primitive CD34+/CD38– cell phenotype and ALDH+ cells (91.3% ± 2.5% ALDH+). Relatively more mature cells with a CD34+/ CD38+ cell phenotype seemed to possess a slightly lower proportion of ALDH+ cells (78.3% ± 6.3%; example in Fig. 2C and summary of data in Table 1). This difference in percentage of ALDH+ cells between the CD38+ and CD38– subsets of cord blood CD34+ cells was statistically significant (p = .001; n = 7). Accordingly, the primitive CD133+ subset of CD34+ cells was more enriched for ALDH+ cells than the relatively mature CD133– fraction of CD34+ cells (p = .001, n = 7; data in Table 1).
Because ALDH+/Lin– cells have already been assessed in NOD/SCID mice [15], we merely confirmed the NOD/SCID engraftment of ALDH+ cells in our laboratory (nine of nine mice injected gave 6-week, multilineage engraftment).
Three Different Patterns of ALDH Activity Are Detectable in AML Samples
Having confirmed the ability of the kit to identify CD34+stemcells, we then progressed to the analysis of malignant hematopoietic cells with a high ALDH activity. In contrast to the remarkable consistency of cord blood ALDH labeling, the staining of AML samples gave more varied profiles. When cells with a high ALDH activity were detected in AML samples, two highly different patterns of high ALDH activity were observed. In one pattern, ALDH+ cells were similar to cord blood ALDH+ cells; they were extremely rare and possessed typical stem/progenitor cell scatter characteristics (rare pattern). In the other pattern, ALDH+ cells were more frequent and often possessed a higher side scatter than normal stem/ progenitor cells (numerous pattern). In approximately one fourth of AML samples examined (5 of 19), no subset of cells with a high ALDH activity was observed (defined as negative pattern).
The rare pattern occurred in 37% (7 of 19) of samples tested (Fig. 3). To confirm the specificity of our ALDH labeling, we incubated an aliquot of the stain with an inhibitor specific to ALDH (diethylaminobenzaldehyde [DEAB], supplied with kit). The mean (± SD) percentage frequency of ALDH+ cells in these six AML samples was 0.14% ± 0.14% (range, 0.02%–0.35%). The frequency of cells in the same gate, but applied to the inhibitor control, was 0.01% ± 0.03%. This difference in ALDH+ cell frequency in the presence of the inhibitor was statistically significant (p = .03). Similar to cord blood, most of these rare ALDH+ cells were CD34+ (88.7% ± 9.2%), confirming their primitive nature. This was even true when the AML itself was CD34–. On a CD34 versus ALDH dotplot, these cells appeared to be completely distinct and separate to the main AML cell population (Fig. 3, first row).
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, second row). In this pattern, ALDH+ cells were often of a higher side scatter than normal stem/progenitor cells. The mean percentage frequency of ALDH+ cells in this pattern was 19.6% ± 18.4% of total live cells. The frequency of cells in the DEAB inhibitor control was 0.03% ± 0.03%. This difference in ALDH+ cell frequency in the presence of the inhibitor was also statistically significant (p = .03). In contrast to the coexpression profile of the rare pattern, in the numerous pattern, a lower proportion of ALDH+ cells coexpressed the CD34 antigen (38.0% ± 33.7% CD34+). This difference in CD34 expression between the two ALDH staining patterns was statistically significant (p = .003).
NOD/SCID Repopulating Activity of Patterns 1 and 2 Reveals a Normal Versus Leukemic Stem Cell Potential, Respectively
To investigate the leukemic or nonleukemic nature of ALDH+ cells in AML samples, we injected cells sorted on the basis of ALDH into sublethally irradiated NOD/SCID mice. Murine marrows were analyzed 6 weeks after transplant for the presence of human myeloid (CD45+/CD33+) and lymphoid (CD45+/CD19+) cells (Table 2).
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, second row) and patient 4 (data not shown) produced only CD33+ myeloid cells (four of four). This generation of myeloid cells without any accompanying CD19+ lymphoid cells suggests to us that in the numerous pattern, ALDH+ cells are enriched for leukemic stem cells.
Despite the low numbers of cells injected (up to 40,000), normal multilineage engraftment was observed in the two representative rare-pattern samples analyzed (patients 2 and 8; example of patient 2 in Fig. 3, first row). Interestingly, when 107 total cells (equivalent to 13,000 ALDH+ cells) from patient 2 were injected into NOD/SCID mice, leukemia was propagated (three of three). However, when the rare ALDH+ cells present in this sample were isolated and injected into NOD/SCID mice (21,000 cells per mouse), normal multilineage engraftment was observed, suggesting a nonleukemic nature. Hence, in this particular leukemia, it seems that the AML cell population inhibits normal hematopoietic development, and ALDH activity provides a tool to examine this phenomenon.
Evaluation of the Leukemic Status of ALDH+ Cells via Fluorescence In Situ Hybridization Analysis
Wherever possible, ALDH+ cells were FACSorted from the main AML population and examined for genetic abnormalities that were characteristic of the particular AML. ALDH+ cells were sorted from patient 6 (numerous pattern) and were almost exclusively (95%) +21 (Fig. 4A), confirming their leukemic nature. Although ALDH– cells from the same patient (6) contained a significant proportion of +21 leukemic cells (65%), the remainder were normal hematopoietic cells that possessed the usual two copies of chromosome 21 (Fig. 4B). Most (91%) ALDH+ cells from a numerous-pattern sample (patient 15; Fig. 3, example plot in middle row) were lacking one copy of the q arm of chromosome 5, indicating a leukemic origin (Fig. 4F). ALDH– cells from patient 15 also featured many leukemic cells (58%), but the remaining cells were nonleukemic (data not shown).
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), whereas ALDH– cells contained a higher proportion of leukemic cells (81% of cells had three copies of chromosome 3; Fig. 4D). Almost all (96%) ALDH+ cells from patient 8 lacked the characteristic translocation of this patient (t [15:17], Fig. 4E).
Numerous-Pattern ALDH+ Cells Are Enriched for Cells with a Primitive Phenotype
To further define the significance of leukemic cells with a high ALDH activity, we analyzed ALDH in conjunction with CD34, CD133, CD38, and CD7. This simultaneous assessment of five parameters revealed a relationship between CD38 and ALDH within CD34+ cells that was similar to our cord blood profile. Although in all experiments analyzed (n = 5), primitive CD34+/ CD38– cells displayed a higher ALDH activity than their more differentiated CD34+/CD38+ counterparts, because of the wide variation between AML samples, this association did not reach statistical significance (45.1% ± 40.7% ALDH+ within CD34+/ CD38– cells versus 25.1% ± 34.8% of CD34+/CD38+ cells; p = .18) (Fig. 5) [9].
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Also in contrast to the cord blood situation, CD7 expression was not completely restricted to CD34–/ALDH– cells (70.3% ± 33.3%; Fig. 5). AML samples were not depleted for Lin+ cells, and hence these CD7+/CD34–/ALDH– cells possessed scatter characteristics typical of mature lymphocytes. However, the remaining 30% of CD7+ cells that coexpressed CD34 and ALDH were probably part of the AML clone, because they often had abnormal scatter characteristics (data not shown).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Combining our refined Aldefluor protocol with our use of >99% pure Lin– fractions and a live/dead assay (DAPI staining) allowed an accurate analysis of Lin– cells for ALDH activity. We confirm that a large proportion of Lin– cells is ALDH+ but can now report that this represents almost three times the proportion of Lin– cells that was previously quoted [15]. Furthermore, the extent to which the Lin–/ALDH+ and Lin–/CD34+ populations overlapped has not been fully appreciated. Although it has been reported that a high proportion of Lin–/ALDH+ cells are CD34+, the proportion of Lin–/CD34+ cells that are ALDH+ was previously underestimated [17]. Although we report that most Lin–/ CD34+ cells are also positive for ALDH and, hence, cord blood Lin–/ALDH+ and Lin–/CD34+ are almost overlapping populations, a high ALDH activity did identify other candidate stem cell populations (further discussed below).
Multiparameter analysis of Lin– populations has allowed us to confirm that most cells within phenotypically defined stem cell subsets are enriched for ALDH+ cells. Conversely, cell subsets known to contain a higher proportion of maturing progenitors contained a lower proportion of ALDH+ cells. Interestingly, there was a significant proportion of CD34+ cells with a very high ALDH activity that did not possess the CD34+/CD38– phenotype. This suggests that analysis of ALDH activity may provide an opportunity to isolate previously unidentified CD34+ stem cells.
A high ALDH activity also identifies a small population of Lin–/CD34– cord blood cells. It has been reported that the NOD/SCID repopulating subset within Lin–/CD34– cells is negative for CD38, and although most of our Lin–/CD34–/ ALDH+ cells are CD38+, we can observe a small population of Lin–/CD34–/ALDH+/CD38– cells [14]. In addition, CD7 is reportedly expressed on a population of Lin–/CD34– natural killer cell progenitors that do not have a multilineage NOD/ SCID repopulating ability [18, 19]. Interestingly, our Lin–/ CD34–/ALDH+/CD38– cells are CD7–. We believe this to be a candidate stem cell population and are currently investigating their NOD/SCID engraftment.
Once we were familiar with the Aldefluor kit’s ability to identify human hematopoietic stem/progenitor cells, we progressed to examine samples from patients with AML. Phenotyping and functional analysis of ALDH+ cells in samples from patients with AML has allowed us to suggest that in roughly one third of cases, cells with a high ALDH activity actually represent nonleukemic stem cells. Although the presence of nonleukemic stem/progenitors in AML samples has been previously demonstrated [20], to our knowledge, this is the first description of a nontoxic, functional method for their discrimination and isolation from the main AML cell population [21, 22].
It could be argued that the proportion of blast cells in each AML peripheral blood sample would affect the ALDH pattern observed. If all rare-pattern and negative-pattern AML samples possessed a low leukemic blast percentage, then this could explain the apparent lack of leukemic ALDH+ cells. However, comparison of the ALDH pattern we observed with each patient’s leukemic blast percentage (Table 3) has revealed that many of the rare-pattern and negative-pattern AML samples had a high leukemic blast cell percentage. Because we analyzed a million live cells during each acquisition, the lowest number of leukemic blasts we analyzed was approximately 80,000 in one sample (patient 9). Consequently, we have assessed the ability of the leukemic blasts in these samples to convert the Aldefluor reagent and can conclude that the AML cells are nonreactive.
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The isolation of leukemic and nonleukemic stem cells from the same patient should allow further investigations into the interaction between the two populations. As mentioned previously, even in this preliminary study we have been able to observe an apparent inhibition of normal stem cell development by the leukemic cell population (NOD/SCID engraftment patterns of patient 2).
Our data demonstrate that CD7+ T cells do not react with the Aldefluor reagent. Therefore, T-cell contamination of leukemic stem cells isolated via ALDH activity is likely to be minimal and restricted to CD34+ progenitors rather than mature T cells.
The coincidence of a high ALDH activity with the leukemic stem cell has important implications for resistance to chemotherapy. Successful chemotherapy is dependent on the relative advantage of the nonleukemic stem cell population over the leukemic stem cells. If the ALDH activity we detected is a major chemoresistance factor, one would expect the leukemia cases with a detectable leukemic ALDH+ population to have a worse prognosis than cases without any detectable ALDH+ cells. During this study we did not detect any association between the ALDH pattern detected and the patient’s karyotype-defined prognosis or French-American-British morphological classification type. Furthermore, we cannot find any prognostic link in the literature; however, analysis of more patients and other isoforms of ALDH may well reveal an association between prognosis and ALDH activity.
According to the manufacturers, the Aldefluor kit is active against the ALDH-1 isoform but not the ALDH-3 isoform. Both ALDH1 and ALDH3 are reportedly involved in chemoresistance [3, 11, 13]. Although we have investigated ALDH-1 expression, we have not assessed the levels of ALDH-3. It is possible that certain leukemias may express detectable levels of cellular ALDH-3. It will be interesting to develop reagents that are specific to particular forms of ALDH and assess the incidence of ALDH-3+ cells in leukemia. Various other tumor cell types possess elevated levels of ALDH, and it should be possible to investigate the incidence/ significance of ALDH+ cells in other malignant cell types.
It has been reported that certain ALDH isoforms are involved in the metabolism of retinoic acid from retinal [4]. It is known that promyelocytic leukemia (M3) responds to retinoic acid–mediated differentiation, and this is an effective therapy for this disease [23, 24]. During our analysis of ALDH staining in AML, we investigated three patients with the promyelocytic form of the disease. Each of the three possible situations was observed in these patients. One patient possessed an extremely rare, multilineage-engrafting population, another seemed to have a large leukemic ALDH+ cell population, and the other did not contain any detectable ALDH+ cells at all. Hence, we could not find an association between promyelocytic leukemia and ALDH activity. This is not surprising considering the observation that although inhibition of ALDH affects the oxidation of retinal to retinoic acid, this inhibition does not affect differentiation of HL-60 cells in the presence of retinal [25]. Our results support the notion that other mechanisms are also responsible for the conversion of retinol to retinoic acid in AML-M3.
In summary, a high ALDH-1 activity identifies primitive subsets of Lin–/CD34+ cells as well as candidate Lin–/CD34– stem cells in cord blood. In addition, in approximately one third of patients with AML, a high ALDH activity identifies the leukemic stem cell, and in a similar proportion of patients, nonleukemic hematopoietic stem cells are highlighted.
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ACKNOWLEDGMENTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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REFERENCES |
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