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发表于 2009-3-5 10:50 |只看该作者 |倒序浏览 |打印

a Hematopoietic Stem Cell Laboratory,. O& s# J' d& w& I1 R

b FACS Laboratory, London Research Institute, Cancer Research UK, London, United Kingdom;8 @9 ^* ]5 V2 L! S% U- d
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c Cancer Research UK Department of Medical Oncology, St. Bartholomew’s Hospital, West Smithfield, London, United Kingdom% \' E5 \0 F: y! I

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@cancer.org.uk3 d2 U. m6 E- d2 J3 \& n1 Q
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ABSTRACT

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme that is responsible for the oxidation of intracellular aldehydes. More than 17 human ALDH genes have been identified, and the ALDH superfamily is highly conserved across a variety of species . This enzyme is thought to have an important role in oxidation of alcohol and vitamin A and in cyclophosphamide chemoresistance .! Z, x% S9 A; T" `0 c! o
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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 . 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 . 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 .
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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 . 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 . 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 . Furthermore, constitutive expression of human ALDH1 or ALDH3 in human hematopoietic cells increases their resistance to cytotoxic agents . Accordingly, when expression of ALDH1 is blocked by the expression of antisense ALDH mRNA, cell sensitivity to 4-hydroperoxycyclophosphamide is greatly increased in vitro . 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

For consistent results, Aldefluor-stained cells must be analyzed within 2 hours of labeling. However, cells retain their ability to convert the ALDH substrate for at least 24 hours after collection. We typically stored our cord blood samples overnight before Aldefluor labeling and analysis without any detectable effect on the ALDH profile (data not shown). For consistency, all AML samples compared in this study (see below) were prepared in the same fashion and for the same time before Aldefluor labeling and analysis.

High ALDH Activity Identifies Cord Blood Cells with Immature Cell Morphology
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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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Figure 1. High aldehyde dehydrogenase (ALDH) activity identifies cells with stem/progenitor cell morphology. (A): Example given of cord blood mononuclear cell ALDH staining exhibits a bright, low/ medium side-scatter population that is extremely bright for ALDH substrate when compared with monocytes (M), lymphocytes (L), or debris. Dead cells were excluded via 4, 6-diamidino-2-phenylindoiole negativity. (B): When superimposed over the whole mononuclear cell population (gray), ALDH  cells (black) seem to have the scatter characteristics of stem/progenitor cells.
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Most Lin– Cells Are CD34 , ALDH  Cells

Once we had investigated the presence of cells with a high ALDH activity in mononuclear cells, we progressed to examine Lin–cells (n = 9). Cord blood mononuclear cells were StemSep depleted for cells positive for lineage antigens (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and CD235a). When representative negative fractions (n = 4) were analyzed, the mean (± SD) purity of Lin– cells was 99.3% ± 0.35%. Of these Lin– cells, 71.1% ± 9.1% possessed a high ALDH activity (Fig. 2A). These ALDH  cells were separate and distinct from the ALDH– cells and therefore easily identified.4 H3 X# q6 y7 I! }
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Figure 2. Phenotyping of cord blood lineage-negative (Lin–) cells. (A): CD34 versus aldehyde dehydrogenase (ALDH) pseudo-color dotplot of a representative cord blood, Lin– ALDH/CD34/CD38/ CD133 stain. Density of events is represented by color in 25% intervals from red to yellow to green to blue. Dead cells were excluded via 4,6-diamidino-2-phenylindoiole staining. (B): CD34/CD38 dot-plot generated from the same data file with CD34 /CD38–, CD34 / CD38 , CD34–/CD38 , and CD34–/CD38– populations gated for further analysis. (C): A CD34 versus ALDH dotplot that displays the populations defined by CD34 and CD38 in (B). Provided in the background for comparison is the whole Lin– population (gray). In addition, CD34 /CD38– cells (blue), CD34 /CD38  cells (green), Lin–/CD34–/ALDH /CD38  cells (turquoise, upper left quadrant), and Lin–/CD34–/ALDH /CD38– stem cells (red dots, upper left quadrant) are displayed. (D): CD34 versus CD133 dotplot of a representative cord blood, Lin– ALDH/CD34/CD38/CD133/CD7 stain with ALDH  (red) and ALDH– (blue) populations indicated. (E): CD133/ CD7 dotplot of Lin– cells with CD133 , CD133–, and CD7  cell populations identified. (F): CD34 versus ALDH dotplot that displays the populations defined by CD133 and CD7 in (E). Provided are CD133 / CD34  cells (green), CD133–/CD34  cells (red), and Lin–/CD34–/ ALDH–/CD7  cells (blue dots, lower left quadrant).; Z  b8 [: q. J) \7 E6 K: |) `4 a

Simultaneous analysis of ALDH, AC133 (CD133), CD34, and CD38 (n = 8) revealed some interesting staining patterns. Although the Lin–/CD34  cell population virtually overlaps with the Lin–/ALDH  cell population, this is not a complete correlation. Indeed, 93.3% ± 3.4% of Lin–/CD34  cells are ALDH , and 94.3% ± 2.5% of Lin–/ALDH  cells are positive for the CD34 antigen. Interestingly, this leaves a small proportion of Lin– cells that were positive for ALDH but negative for CD34 (3.4% ± 1.9%), and a slightly larger proportion (5.1% ± 2.6%) were Lin–/CD34 / ALDH– cells (Fig. 2A; 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 , 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).  G' K- r, y$ S: Y' n3 d9 O8 G6 x0 X

Table 1. Summary of lineage-negative cord blood analysis

Previous work has indicated that the Lin–/CD34– fraction of cord blood cells contains committed CD7  lymphoid progenitors, and we wondered if these were ALDH . Our experiments reveal that most CD7 expression is restricted to Lin–/CD34–/ALDH– cells and, accordingly, we could not detect any CD7 expression in Lin–/CD34–/ALDH /CD38– cells., X8 T; Y( Q& I- h  U

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).
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Because ALDH /Lin– cells have already been assessed in NOD/SCID mice , we merely confirmed the NOD/SCID engraftment of ALDH  cells in our laboratory (nine of nine mice injected gave 6-week, multilineage engraftment).
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Three Different Patterns of ALDH Activity Are Detectable in AML Samples! m0 M1 ?1 d, V% u
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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).2 x0 A. T+ l) F# y8 W

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 , 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).

Figure 3. Three patterns of aldehyde dehydrogenase (ALDH) activity are observed in acute myeloid leukemia (AML) patient samples. Representative analysis from each of the three patterns of ALDH activity observed in AML samples. In all analyses, dead cells were excluded via 4,6-diamidino-2-phenylindoiole staining. Provided for comparison is the appropriate inhibitor control for each ALDH stain diethylaminobenzaldehyde (DEAB). Each row was generated from one data file. In the first row, ALDH  cells appear as a small but distinct subset of the main AML cell population (rare pattern; patient 2, 91% leukemic blasts). Regardless of the CD34 status of the AML, these rare ALDH  cells were almost exclusively CD34 , confirming their primitive nature. The second row is an example from the more frequent ALDH pattern (numerous pattern; patient 15, 55% leukemic blasts). In this pattern, the CD34 status of ALDH  cells was more varied than the rare pattern. ALDH  cells were not distinct to the main AML population; rather they appeared as continuation of the main body of cells. The AML analysis presented in the third row did not exhibit any detectable ALDH activity and is defined as the negative pattern (example given is patient 16, 50% blasts).* h& _; m; j5 Z1 \

The numerous pattern of ALDH labeling was also observed in 37% (7 of 19) of AML samples tested (Fig. 3, 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).0 M( T, W; i. m! U0 \! ^7 m  Q! `4 p

NOD/SCID Repopulating Activity of Patterns 1 and 2 Reveals a Normal Versus Leukemic Stem Cell Potential, Respectively, z- e8 ?, `% `4 P$ F
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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).( j3 i, A6 e, w+ T3 e

Table 2. Summary of NOD/SCID engraftment data* B, @/ _; q1 m7 q4 `
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When injected into NOD/SCID mice, ALDH  cells from patient 15 (Fig. 3, 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.; S6 d# D4 }  n8 D0 g. f- p3 Y
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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.0 G6 j/ P" I0 T6 n- f

Evaluation of the Leukemic Status of ALDH  Cells via Fluorescence In Situ Hybridization Analysis. g- a/ D! h9 t/ O" X

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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Figure 4. Aldehyde dehydrogenase–positive (ALDH ) cells present at a high frequency possess characteristic acute myeloid leukemia (AML) genetic abnormalities. Cells were FACSorted before fluorescence in situ hybridization (FISH) labeling of cellular DNA. (A): Single-color labeling of chromosome 21 (green). Most ALDH  cells possessed three copies of chromosome 21, confirming the leukemic nature of this numerous-pattern population (patient 6). (B): Analysis of ALDH– cells from the same patient as (A). Although leukemic cells were present, this was at a lower frequency than ALDH  cells. (C): Montage of images from the analysis of rare-pattern patient 12 ALDH  cells, which possess the normal two copies of chromosome 3. (D): ALDH– from the same patient as in (C). Most cells possess the abnormality that is characteristic of this AML: three copies of chromosome 3. (E): Example of dual-color, dual-fusion labeling of the t  translocation. ALDH  cells from rare-pattern patient 8 possessed two red and two green spots, indicating a lack of the AML translocation. (F): Labeling of chromosome 5p (green) and 5q (red). Most ALDH /CD34 /– cells exhibited one red and two green FISH spots, indicating a deletion of the q arm of chromosome 5 (numerous pattern, patient 15).
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Because of the rarity of ALDH  cells in the rare pattern, enough cells for fluorescence in situ hybridization (FISH) analysis could be prepared from only two patients. ALDH  cells from rare-pattern patient 12 were almost exclusively normal (88% of cells possessed two copies of chromosome 3; Fig. 4C), 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 , Fig. 4E).) C) A1 E* t: Y: }6 ~0 L( J* {

Numerous-Pattern ALDH  Cells Are Enriched for Cells with a Primitive Phenotype
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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) .$ Q2 `- z/ B% |5 O7 ]8 r

Figure 5. Phenotyping of leukemic (numerous-pattern) aldehyde dehydrogenase–positive (ALDH ) cells. (A): Psuedo-color dotplot of a representative ALDH/CD34/CD133/CD7/CD38 stain on a leukemic sample (patient 6) that exhibited a numerous-pattern ALDH staining profile. Density of events is represented by color in 25% intervals from red to yellow to green to blue. Dead cells were excluded via 4,6-diamidino-2-phenylindoiole staining. Cells were either CD34 /ALDH , CD34–/ALDH , CD34–/ALDH–, or CD34 /ALDH–. (B): CD34 versus CD38 dotplot with CD34 /CD38–, CD34 /CD38 , CD34–/CD38 , and CD34–/CD38– populations indicated. (C): A CD34 versus ALDH dotplot that displays the populations defined by CD34 and CD38 in (B). Provided in the background for comparison is the whole lineage-negative (Lin–) population (gray). Displayed are CD34 /CD38– cells (blue), CD34 /CD38  cells (green), CD34–/ CD38  cells (turquoise), and CD34–/CD38– cells (red). (D): CD133/ CD7 dotplot of leukemic cells with CD133 , CD133–, and CD7  cell populations identified. (E): CD34 versus ALDH dotplot that displays the populations defined by CD133 and CD7 in (D). CD133 /CD34  cells (green) and CD133–/CD34  cells (red) are indicated. CD7 expression was mostly restricted to the Lin–/CD34–/ALDH– cell population (blue).
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In every experiment we analyzed, the ALDH activity was higher in CD34 /CD133  cells than CD34 /CD133– cells. However, this difference in ALDH activity was not as pronounced as in cord blood and did not reach statistical significance (55.4% ± 47.1% ALDH  cells within CD34 /CD133  cells compared with 43.4% ± 44.7% of CD34 /CD133– cells). These abnormal staining profiles within numerous-pattern AML samples support our suggestion that numerous-pattern ALDH  cells are leukemic in origin.
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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).! l/ \# ?) M! i4 @/ G$ y0 L- D; v

DISCUSSION

D.P. and D.T. contributed equally to this work. This work was supported by Cancer Research UK and Association for International Cancer Research grant No. GA3160 to D.B. The authors thank Derek Davies, Gary Warnes, and Ayad Eddaoudi of the FACS Lab at Cancer Research UK for their invaluable technical expertise. Furthermore, this study would not have been possible without Julie Bee, Clare Millum, and Ella Smallcombe of our Biological Research Unit. We are also grateful to Ian Dimmick of Becton Dickinson for our first Aldefluor kit.
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