Substrate For Aldehyde Dehydrogenase Research Articles

Trapped in Cells: A Selective Accumulation Approach for Type-I Photodynamic Ablation of Cancer Stem-like Cells.

Aldehyde dehydrogenase (ALDH) is an enzyme responsible for converting aldehyde functional groups into carboxylate metabolites. Elevated ALDH activity is a characteristic feature of cancer stem-like cells (CSCs). As a novel approach to target the CSC trait of overexpressing ALDH, we aimed to utilize ALDH activity for the selective accumulation of a photosensitizer in ALDHHigh CSCs. A novel ALDH substrate photosensitizer, SCHO, with thionylated coumarin and N-ethyl-4-(aminomethyl)benzaldehyde was developed to achieve this goal. Our study demonstrated the efficient metabolism of the aldehyde unit of SCHO into carboxylate, leading to its accumulation in ALDHHigh MDA-MB-231 cells. Importantly, we established the selectivity of SCHO as an ALDHHigh cell photosensitizer as it is not a substrate for ABC transporters. SCHO-based photodynamic therapy triggers apoptosis and pyroptosis in MDA-MB-231 cells and further reduces the characteristics of CSCs. Our study presents a novel strategy to target CSCs by exploiting their cellular metabolism to enhance photosensitizer accumulation, highlighting the potential of photodynamic therapy as a powerful tool for eliminating ALDHHigh CSCs.

Development of a Retinal-Based Probe for the Profiling of Retinaldehyde Dehydrogenases in Cancer Cells.

Retinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of great utility to visualize and quantify aldehyde dehydrogenase (ALDH) activity in health and disease. Here, we present the discovery of a first-in-class chemical probe based on retinal, the endogenous substrate of retinal ALDHs. We unveil the utility of this probe in quantitating ALDH isozyme activity in a panel of cancer cells via both fluorescence and chemical proteomic approaches. We demonstrate that our probe is superior to the widely used ALDEFLUOR assay to explain the ability of breast cancer (stem) cells to produce all-trans retinoic acid. Furthermore, our probe revealed the cellular selectivity profile of an advanced ALDH1A1 inhibitor, thereby prompting us to investigate the nature of its cytotoxicity. Our results showcase the application of substrate-based probes in interrogating pathologically relevant enzyme activities. They also highlight the general power of chemical proteomics in driving the discovery of new biological insights and its utility to guide drug discovery efforts.

Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31- /CD45- /CD34+ /CD146- cells (adventitial stromal/stem cells [ASCs]) and CD31- /CD45- /CD34- /CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbr ASC (most primitive); (b) ALDHdim ASC; (c) ALDHbr PC; (d) ALDHdim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289.

Abstract 2508: A red-shifted fluorescent substrate for aldehyde dehydrogenase, AldeRed 588-A, for labeling viable ALDH-positive cells

Abstract Normal and cancer stem cells can be isolated based upon the enzymatic activity of aldehyde dehydrogenase I (ALDH1), a detoxifying enzyme responsible for oxidation of hazardous aldehyde byproducts. ALDH1 has been used to isolate cancer stem cells from various human malignancies including bladder, breast, cervical, colon, head and neck, liver, lung, pancreas, prostate and ovary. Currently, the ALDEFLUOR assay, is the only commercially available reagent for ALDH detection in live cells. The substrate used in this assay primarily emits in the green region of the electromagnetic spectrum (512nm). For researchers with valuable cell and transgenic animal models in which the target gene of interest has been tagged with eGFP, ALDEFLUOR therefore cannot be used. Selection of cells positive for aldehyde dehydrogenase (ALDH) activity from a green fluorescent background is thus difficult with existing reagents. We now describe a red-shifted fluorescent substrate for ALDH, AldeRed 588-A, that provides additional flexibility for utilizing ALDH as a marker for stem cell and cancer stem cell isolation. The activity of AldeRed 588-A was compared with the ALDEFLUOR reagent and demonstrated similar ability to fractionate ALDHpos cells in a number of cell lines tested. Citation Format: Nick Asbrock, Vi Chu, Martin Pomper. A red-shifted fluorescent substrate for aldehyde dehydrogenase, AldeRed 588-A, for labeling viable ALDH-positive cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2508.

Abstract 1404: A red-shifted fluorescent substrate for aldehyde dehydrogenase, AldeRed 588-A, for labeling viable ALDH-positive cells

Abstract Normal and cancer stem cells can be isolated based upon the enzymatic activity of aldehyde dehydrogenase I (ALDH1), a detoxifying enzyme responsible for oxidation of hazardous aldehyde byproducts. ALDH1 has been used to isolate cancer stem cells of various human malignancies including bladder, breast, cervical, colon, head and neck, liver, lung, pancreas, prostate and ovary. Currently, the ALDEFLUORTM assay which uses a fluorescent substrate is the only commercially available reagent for ALDH detection. The substrate used in this assay primarily emits in the green region of the electromagnetic spectrum (512nm). For researchers with valuable cell and transgenic animal models in which the target gene of interest has been tagged with eGFP, ALDEFLUOR therefore cannot be used. Selection of cells positive for aldehyde dehydrogenase (ALDH) activity from a green fluorescent background is thus difficult with existing reagents. We now describe a red-shifted fluorescent substrate for ALDH, AldeRed 588-A, that provides additional flexibility for utilizing ALDH as a marker for stem cell and cancer stem cell isolation. The activity of AldRed 588-A was compared with the ALDEFLUOR reagent and demonstrated similar ability to fractionate ALDHpos cells in a number of cell lines tested. Citation Format: Vi Chu, Konstantin Taganov, D Ramesh, Nick Asbrock, Martin Pomper. A red-shifted fluorescent substrate for aldehyde dehydrogenase, AldeRed 588-A, for labeling viable ALDH-positive cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1404. doi:10.1158/1538-7445.AM2015-1404

A red-shifted fluorescent substrate for aldehyde dehydrogenase.

Selection of cells positive for aldehyde dehydrogenase (ALDH) activity from a green fluorescent background is difficult with existing reagents. Here we report a red-shifted fluorescent substrate for ALDH, AldeRed 588-A, for labeling viable ALDHpos cells. We demonstrate that AldeRed 588-A successfully isolates ALDHhi human hematopoietic stem cells from heterogeneous cord blood mononuclear cells. AldeRed 588-A can be used for multi-color applications to fractionate ALDHpos cells in the presence of green fluorophores including the ALDEFLUOR™ reagent and cells expressing eGFP. AldeRed 588-A stains ALDHpos murine pancreatic centroacinar and terminal duct cells, as visualized by fluorescent microscopy. AldeRed588-A provides a useful tool to select stem cells or study ALDH within a green fluorescent background.

P56: Aldehyde dehydrogenase plays an important role in nitrite-mediated vasorelaxation during hypoxic conditions

Background Several experimental studies have reported that the conversion of nitrite to nitric oxide (NO) may occur by a family of (hemo) proteins that exhibit ‘nitrite reductase’ activity, including globins, xanthine oxidoreductase and endothelial NO synthase. Moreover, recent studies have demonstrated aldehyde dehydrogenase (ALDH) to be an important source of nitrite-derived NO in rat blood vessels and heart, respectively. However to date, no prior research has been undertaken to investigate the effects of ALDH inhibition on nitrite-mediated vasodilatation during normoxic and hypoxic conditions. Aim To determine the role of ALDH in nitrite-mediated vasorelaxation, a dose response curve to sodium nitrite was investigated in isolated rat thoracic arota. Briefly, the vessels were subjected to normoxic or hypoxic conditions and a dose response curve to sodium nitrite was then constructed in the presence and absence of ALDH inhibitor, cyanamide, or ALDH substrate, propionaldehyde. Results No significant difference was observed between the degree of nitrite-induced relaxation in the presence and absence of cyanamide under normoxic conditions. In contrast, hypoxia enhanced nitrite-induced relaxation, but following ALDH inhibition with cyanamide, there was a significant rightward shift of the concentration response curves to sodium nitrite. Similar results were also obtained using the ALDH substrate, propionaldehyde. Conclusion These results suggest that ALDH plays an important role in the nitrite-mediated vasorelaxation in isolated rat thoracic aorta during hypoxic conditions. Disclosure Supported by a research grant from British Heart Foundation.

Acrolein detection based on alcohol dehydrogenase inhibition

This paper presents the effect of acrolein on three dehydrogenases and proposes a fast spectrometric method for acrolein analysis. We have found that alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AlDH) are inhibited by low acrolein concentrations (0.2 mM) while inhibition of glutamate dehydrogenase (GDH) is not observed even at higher acrolein concentrations (1 mM). Acrolein is a suicide substrate for AlDH and ADH inhibition by acrolein is competitive. Cysteine (L-Cys) and glutathione (GSH) react with acrolein and thus reduce its expected inhibitory effect. ADH was chosen to develop a spectrophotometric method for acrolein analysis based on enzyme inhibition. The calibration curve is linear between 0.2 and 1.0 mM acrolein.

Isolation and Characterization of Progenitor-Like Cells from Human Renal Proximal Tubules

The tubules of the kidney display a remarkable capacity for self-renewal on damage. Whether this regeneration is mediated by dedifferentiating surviving cells or, as recently suggested, by stem cells has not been unequivocally settled. Herein, we demonstrate that aldehyde dehydrogenase (ALDH) activity may be used for isolation of cells with progenitor characteristics from adult human renal cortical tissue. Gene expression profiling of the isolated ALDH(high) and ALDH(low) cell fractions followed by immunohistochemical interrogation of renal tissues enabled us to delineate a tentative progenitor cell population scattered through the proximal tubules (PTs). These cells expressed CD24 and CD133, previously described markers for renal progenitors of Bowman's capsule. Furthermore, we show that the PT cells, and the glomerular progenitors, are positive for KRT7, KRT19, BCL2, and vimentin. In addition, tubular epithelium regenerating on acute tubular necrosis displayed long stretches of CD133(+)/VIM(+) cells, further substantiating that these cells may represent a progenitor cell population. Furthermore, a potential association of these progenitor cells with papillary renal cell carcinoma was discovered. Taken together, our data demonstrate the presence of a previously unappreciated subset of the PT cells that may be endowed with a more robust phenotype, allowing increased resistance to acute renal injury, enabling rapid repopulation of the tubules.

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.Recently, we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent, a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Aldehyde dehydrogenase 1a1 is dispensable for stem cell function in the mouse hematopoietic and nervous systems

AbstractHigh levels of aldehyde dehydrogenase (ALDH) activity have been proposed to be a common feature of stem cells. Adult hematopoietic, neural, and cancer stem cells have all been reported to have high ALDH activity, detected using Aldefluor, a fluorogenic substrate for ALDH. This activity has been attributed to Aldh1a1, an enzyme that is expressed at high levels in stem cells and that has been suggested to regulate stem cell function. Nonetheless, Aldh1a1 function in stem cells has never been tested genetically. We observed that Aldh1a1 was preferentially expressed in mouse hematopoietic stem cells (HSCs) and expression increased with age. Hematopoietic cells from Aldh1a1-deficient mice exhibited increased sensitivity to cyclophosphamide in a non–cell-autonomous manner, consistent with its role in cyclophosphamide metabolism in the liver. However, Aldh1a1 deficiency did not affect hematopoiesis, HSC function, or the capacity to reconstitute irradiated recipients in young or old adult mice. Aldh1a1 deficiency also did not affect Aldefluor staining of hematopoietic cells. Finally, Aldh1a1 deficiency did not affect the function of stem cells from the adult central or peripheral nervous systems. Aldh1a1 is not a critical regulator of adult stem cell function or Aldefluor staining in mice.

280 ISOLATION, PRESERVATION, AND CHARACTERIZATION OF EQUINE UMBILICAL CORD BLOOD STEM CELLS

The isolation, preservation, and identification of hematopoietic and mesenchymal stem cells from fresh umbilical cord blood (UCB) has been extensively reported in humans. Although both types of stem cells may be of therapeutic interest in horses, data on equine UCB cells are scarce. In the present study, two separation methods to isolate stem and progenitor cells from equine UCB and two cryoprotectant solutions for their subsequent freezing were compared. Characterization of the isolated cells was evaluated flow cytometrically, based on the presence of the cytosolic enzyme aldehyde dehydrogenase (ALDH), which has been shown to be highly expressed in primitive hematopoietic cells in a number of species. Cord blood was collected from 15 foals immediately after birth. While the placenta was still in utero, the umbilical cord was clamped and disinfected. A sterile blood bag collection system containing citrate-phosphate-dextrose-adenine anticoagulant was used to collect the UCB by gravity. The UCB units were stored at 4�C and processed within 36 h. Percoll density gradient separation and rouleaux formation induced by hydroxyethyl starch (HES) were tested in parallel on equal volumes of each UCB unit. The enriched progenitor cell fraction was cryopreserved at 10 � 106 nucleated cells mL–1 using two cryoprotectant solutions based on plasma or RPMI 1640, and both containing 10% DMSO and DNase I (20 IU mL–1). Before and after thawing, cells were labeled using a fluorescent ALDH substrate (Aldefluor�, StemCell Technologies SARL, Grenoble, France) including a negative control. Cell viability was simultaneously evaluated by means of exclusion of propidium iodide. Cryopreservation was performed using a programmable freezer (–1�C/min–1 until –70�C, then –10�C/min–1 until –140�C) prior to storage in liquid nitrogen. Results were analyzed statistically with a nonparametric Mann-Whitney test. The concentration of the isolated UCB cells ranged from 0.3 to 4 � 106 cells mL–1 for Percoll and from 0.4 to 7.3 � 106 cells mL–1 for HES. The average viability before and after freezing was 94% and 93% for Percoll-, and 93% and 94% for HES-separated cells, respectively. No significant differences in concentration or in viability were observed between both isolation procedures and both cryoprotectant solutions. Before freezing, the proportion of Aldefluor�-positive cells after Percoll and HES isolation ranged between 0.5 and 38% and between 1 and 60.5%, respectively. No significant differences were found. In conclusion, the percentage of ALDH-positive cells as determined by flow cytometry was highly variable between foals, but was independent of the isolation procedures used. Whether the isolated cells represent true progenitor cells remains to be confirmed. Ongoing, flow cytometrical experiments showed that the isolated cells are CD29+ and CD44+, which may be indicative for their mesenchymal origin.

Clonotypic B Cells Circulate in Hodgkin's Lymphoma (HL).

Reed-Sternberg (RS) cells, the hallmark of HL, are clonal B lineage cells generally characterized by their co-expression of CD15 and CD30. RS cells also occasionally express the plasma cell (PC) cell surface antigen CD138 (syndecan-1), and we thus hypothesized that RS may represent aberrant PC differentiation. We found that RS cells from the L428 HL cell line exhibited an expression profile similar to normal PC and those from multiple myeloma (MM): high expression of the PC transcription factor Blimp-1 and low expression of the germinal center B cell markers Pax-5 and Bcl-6. We recently demonstrated that CD138+ MM PC, like their normal counterparts, have limited replicative potential; rather, self-renewing CD138neg clonotypic B cells appear to be responsible for the initiation and maintenance of MM, giving rise to the differentiated CD138+ MM PC (Matsui et al Blood 103: 2332–2336, 2004). To determine if RS cells similarly were differentiated progeny of rare HL cancer stem cells, we studied HL cell lines (L428, KM-H2) and found that each line contained a small (<5%) subpopulation of cells that did not express the RS markers CD15 and CD30 present on most of the cells. This small subpopulation instead resembled memory B cells (CD19+CD20+CD27+) and in addition possessed most of the clonogenic capacity of the HL cell lines; this suggested that RS cells, similar to the neoplastic PC in MM, are not the cells responsible for the growth and maintenance of HL. Using Aldefluor, a fluorescent aldehyde that is a substrate for aldehyde dehydrogenase (ALDH), we previously showed that ALDH is highly expressed in both normal and MM stem cells. The clonogenic subpopulation within the HL cell lines also expressed high ALDH activity, while the predominant RS cells exhibited low ALDH activity. To determine if clonotypic memory B cells were similarly present in HL patients, CD19+ cells were isolated from the marrow or blood of 5 HL patients. The bulk CD19+ cells, as well as those that were ALDHlow, isolated from the HL patients were a mixture of non-clonal naive and memory B cells. However, the ALDHhigh CD19+ cells were a highly enriched population of immunoglobulin (Ig) light chain-restricted CD27+ memory B cells that represented 0.7 to 3% of the circulating CD19+ cells, in 4/5 patients (none with evidence of marrow involvement) studied. These ALDHhigh CD19+ cells also displayed clonal Ig gene rearrangement by polymerase chain reaction (PCR) amplification. In two of these patients, CD15+CD30+ RS cells were isolated from a fresh diagnostic lymph node and contained the same clonal Ig gene rearrangement as the circulating ALDHhigh CD19+ B cells. Thus, clonotypic memory B cells can be found in both HL cell lines and patients. These data suggest that these clonotypic memory B cells circulate in relatively high numbers even in early stage patients, and may represent the HL stem cells. Our results also suggest that therapies, such as rituximab, that target these putative HL stem cells may have clinical activity in HL patients.

Identification of a Primitive Brain–Derived Neural Stem Cell Population Based on Aldehyde Dehydrogenase Activity

Stem cells are undifferentiated cells defined by their ability to self-renew and differentiate to progenitors and terminally differentiated cells. Stem cells have been isolated from almost all tissues, and an emerging idea is that they share common characteristics such as the presence of ATP-binding cassette transporter G2 and high telomerase and aldehyde dehydrogenase (ALDH) activity, raising the hypothesis of a set of universal stem cell markers. In the present study, we describe the isolation of primitive neural stem cells (NSCs) from adult and embryonic murine neurospheres and dissociated tissue, based on the expression of high levels of ALDH activity. Single-cell suspension was stained with a fluorescent ALDH substrate termed Aldefluor and then analyzed by flow cytometry. A population of cells with low side scatter (SSC(lo)) and bright ALDH (ALDH(br)) activity was isolated. SSC(lo)ALDH(br) cells are capable of self-renewal and are able to generate new neurospheres and neuroepithelial stem-like cells. Furthermore, these cells are multipotent, differentiating both in neurons and macroglia, as determined by immunocytochemistry and real-time reverse transcription-polymerase chain reaction analysis. To evaluate the engraftment potential of SSC(lo)ALDH(br) cells in vivo, we transplanted them into mouse brain. Donor-derived neurons with mature morphology were detected in the cortex and subcortical areas, demonstrating the capacity of this cell population to differentiate appropriately in vivo. The ALDH expression assay is an effective method for direct identification of NSCs, and improvement of the stem cell isolation protocol may be useful in the development of a cell-mediated therapeutic strategy for neurodegenerative diseases.

Aldehyde Dehydrogenase (ALDH) Activity in Fresh (Pre-Freezing) and Post-Thawing Leukapheresis and Cord Blood Collections.

Introduction: Primitive hematopoietic progenitor cells may be characterized by the detection of the intracellular enzymatic activity of aldehyde dehydrogenase (ALDH ). The use of a fluorescent substrate for ALDH (Aldefluor) permits the cytofluorimetric study of different stem cell populations in the functional way rather then as markers of cell surface. Aim of this study was to evaluate the ALDH activity of the stem cells in pre-freezing (fresh) leukapheresis (LKF) colllections and in cord blood (CB) units, to verify the feasibility of detecting the ALDH activity in the post-thawing units and finally to compare the results between the fresh and post thawed stem cells to be transplanted.Materials and methods: Samples containing 2 x106 total nucleated cells obtained from 5 LKF and 5 CB units immediately after collection and from the same units after thawing, were incubated with Aldeflour (StemCo Biomedical, Durham, NC, USA) at 37°C for 45 min. and successively for 15 min. with anti CD34 PE, (Beckman Coulter, Fullerton, CA, USA) and anti CD45 PerCP (BD Biosciences, San Jose, CA, USA). Viability of CD34+ cells was evaluated using 7AAD, (Beckman Coulter, Fullerton, CA, USA). The setting of thawed samples was carried out immediately after thawing, taking care to make the sample dilution very quickly, in order to avoid the detrimental action on the cells exerted by DMSO at 37°C and at room temperature. All samples were analized using a BD Biosciences FACSCalibur flow cytometer device (San Jose, CA, USA).Results: The results of the ALDH and CD34+ cell analysis and stem cell viability on fresh and thawed samples are detailed in table 1.Conclusions: Our results demonstrate the feasibility of ALDH determination even in post thawed samples on condition that the test is conducted with accuracy with particular attention at the dilution step that must be completed in a very short time. The ALDH activity tends to decline in thawed stem cells demonstrating the detrimental action on stem cell functionality of the entire cryopreservation and thawing processes. In particular the contact of the stem cell with cryoprotectant mixture (DMSO) at room temperature has a negative, time dependent, impact on stem cell quality, confirming that the reinfusion phase of the graft must be carried out as soon as possible. The introduction of a functional test to evaluate the graft quality may be helpful in transplant clinical practice.Table 1:Results of ALDH assayes on pre-freezing and post-thawing leukapheresis and cord blood collections.CD34+ (%)ALDH+ (%)CD34+/ALDH+ (%)ALDH+/CD34+ (%)Viability (%)Fresh LKF n=50.560.1264.697.899.2Fresh CB n=50.280.2376.799.898.1Thawed LKF n=50.480.0965.594.481.2Thawed CB n=50.240.1971.596.877.3

Aldehyde Dehydrogenase Expression in Primitive Human Hematopoietic Progenitor Cells with Side Population Characteristic and in Samples from Patients with Acute Myeloid Leukemia.

High activity of the detoxifying enzyme aldehyde dehydrogenase (ALDH) has been attributed to primitive hematopoietic stem cells in the murine and human system. ALDH activity can be measured by a functional assay employing BODIPY-aminoacetaldehyde (BAAA) as a fluorescent intracellular substrate. High ALDH activity within hematopoietic stem cells is associated with CD34 and AC133 surface expression.Given the co-expression of these markers in acute myeloid leukemia (AML), we sought to investigate whether ALDH-activity can be detected in early blood stem cell disorders and in primitive hematopoietic progenitor cells with the side population phenotype. Employing a commercial Aldefluor™ -kit thirteen peripheral blood and bone marrow samples from patients with AML were stained for ALDH activity. Four samples exhibited a clearly distinctive positive staining pattern when compared to the inhibitor control, i.e. 16–91% of AML blast cells were considered as ALDH positive. The staining pattern was similar to marrow samples from healthy donors or peripheral blood sample controls from G-CSF-mobilised healthy stem cell donors.In addition we investigated ALDH activity in primitive bone marrow side population (SP) cells from healthy individuals. Although the ALDH substrate BAAA is actively extruded out of cells and especially SP stem cells by an ABC-transporter-activity, we established a co-staining method for Hoechst 33342 and BAAA by inclusion of specific ABC-transporter inhibitors.Both SP cells and ALDH-positive cells appeared as a clear distinctive population with some overlap between both populations. Quantitative RT-PCR of different sorted stem cell samples confirmed that SP cells had higher ABCG2-expression than ALDH positive cells and ABCG2-expression was higher in ALDH positive cells compared to the total cell fraction or granulocytes.We conclude that ALDH is expressed in some but not all AML blast cells. This is in accordance with the cytogenetic heterogeneity of this disease and may have therapeutic implications because of the detoxifying activity of the ALDH enzyme. In addition ALDH activity can also be found on a minor population of very primitive marrow progenitor cells. Whether high ALDH activity is a feature of the leukemic stem cell requires further investigation.

Characterization of Cells with a High Aldehyde Dehydrogenase Activity from Cord Blood and Acute Myeloid Leukemia Samples

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme that is responsible for the oxidation of intracellular aldehydes. Elevated levels of ALDH have been demonstrated in murine and human progenitor cells compared with other hematopoietic cells, and this is thought to be important in chemoresistance. A method for the assessment of ALDH activity in viable cells recently has been developed and made commercially available in a kit format. In this study, we confirmed the use of the ALDH substrate kit to identify cord blood stem/progenitor cells. Via multicolor flow cytometry of cord blood ALDH+ cells, we have expanded on their phenotypic analysis. We then assessed the incidence, morphology, phenotype, and nonobese diabetic/ severe combined immunodeficiency engraftment ability of ALDH+ cells from acute myeloid leukemia (AML) samples. AML samples had no ALDH+ cells at all, an extremely rare nonmalignant stem/progenitor cell population, or a less rare, leukemic stem cell population. Hence, in addition to identifying nonmalignant stem cells within some AML samples, a high ALDH activity also identifies some patients' CD34+/ CD38- 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. Identification and isolation of leukemic cells on the basis of ALDH activity provides a tool for their isolation and further analysis.

A High ALDH Activity Identifies Both Normal and Leukemic Stem Cells.

Aldehyde Dehydrogenase (ALDH) is a cytosolic enzyme that is responsible for the oxidation of intracellular aldehydes. Elevated levels of ALDH have been demonstrated in murine and human progenitor cells when compared to other haematopoietic cells and this is thought to be important in chemoresistance. A method to assess ALDH activity in viable cells has been developed and has recently been made commercially available in a kit format. Here, we confirmed the use of the ALDH substrate kit to identify cord blood stem/progenitor cells. Cells with a high ALDH activity accounted for only 0.82%±0.39% of mononucleated cells in cord blood (range = 0.35–1.29%, n=17). When cells negative for lineage antigens (Linneg - 99.3%±0.35% pure) were analyzed, 71.1%±9.1% possessed a high ALDH activity (n=9). We can now report that Linneg/CD34+ and Linneg/ALDH+ are essentially overlapping populations. Indeed, 93.3%±3.4% of Linneg/CD34+ cells are ALDH+, and 94.3%±2.5% of Linneg/ALDH+ cells are positive for the CD34 antigen. A small proportion of Linneg cells were positive for ALDH, but negative for CD34 (3.4%±1.9%) and a larger proportion (28.4%±7.7%) were Linneg/CD34neg/ALDHneg cells. Linneg/CD34neg/ALDHneg cells were almost exclusively CD7+, indicating that this subset probably represents NK progenitors. In addition, the majority of Linneg/CD34neg/ALDH+ cells co-express CD38 indicating that they are also committed cells. The remaining candidate Linneg/CD34neg/ALDH+/CD38neg stem cells account for 0.19% of lineage negative cells and approximately 0.0006% to 0.002% of mononuclear cells.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. One pattern (pattern-1, 6/17 of AML samples tested) was very similar to the cord blood profile; ALDH+ cells were rare (0.16%±0.14%), had a low/medium side scatter and were almost exclusively CD34+ (87.1%±9.2%). In another group of patients (pattern-2, 6/17) ALDH+ cells were much more frequent 13.9%±11.8%, had a higher side scatter and were not exclusively CD34+ (41.1%±9.2%). In a similar proportion of patients (pattern-3) we could not detect any ALDH activity above the level of inhibitor controls.When FACSorted and injected into NOD/SCID mice, ALDH+ cells from pattern-2 gave rise to unilineage myeloid engraftment in 2 separate experiments (four mice), suggesting a leukemic origin. However, when ALDH+ cells from pattern-1 were injected into sublethally irradiated NOD/SCID mice, multilineage engraftment was observed (two patients, two mice).Hence, it seems that a high ALDH activity may be used to identify non-malignant stem cells within some AML samples. In addition, 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. Identification and isolation of leukemic cells on the basis of ALDH activity provides a tool for their isolation and further analysis.

Mobilized peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation.

We have developed an approach for identifying primitive mobilized peripheral blood cells (PBSC) that express high levels of aldehyde dehydrogenase (ALDH). PBSC were stained with a fluorescent ALDH substrate, termed BODIPY trade mark -aminoacetaldehyde (BAAA), and then analysed using flow cytometry. A population of cells with a low side scatter (SSC) and a high level of BAAA staining, termed the SSCloALDHbr population, was readily discriminated and comprised a mean of 3 +/- 5% of leukapheresis samples. A mean of 73 +/- 11% of the SSCloALDHbr population expressed CD34 and 56 +/- 25% of all the mobilized CD34+ cells resided within the SSCloALDHbr population. The SSCloALDHbr population was largely depleted of cells with mature phenotypes and enriched for cells with immature phenotypes. Sorted SSCloALDHbr and SSCloALDHbr CD34+ PBSC were enriched for progenitors with the ability to (1) generate colony-forming units (CFU) and long-term culture (LTC)-derived CFU, (2) expand in primary and secondary LTC, and (3) generate multiple cell lineages. In 21 cancer patients who had undergone autologous PBSC transplantation, the number of infused SSCloALDHbr cells/kg highly correlated with the time to neutrophil and platelet engraftment (P < 0.015 and P < 0.003 respectively). In summary, peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation.

Reactivity of atropaldehyde, a felbamate metabolite in human liver tissue in vitro

Antiepileptic therapy with a broad spectrum drug felbamate (FBM) has been limited due to reports of hepatotoxicity and aplastic anemia associated with its use. It was proposed that a bioactivation of FBM leading to formation of α,β-unsaturated aldehyde, atropaldehyde (ATPAL) could be responsible for toxicities associated with the parent drug. Other members of this class of compounds, acrolein and 4-hydroxynonenal (HNE), are known for their reactivity and toxicity. It has been proposed that the bioactivation of FBM to ATPAL proceeds though a more stable cyclized product, 4-hydroxy-5-phenyltetrahydro-1,3-oxazin-2-one (CCMF) whose formation has been shown recently. Aldehyde dehydrogenase (ALDH) and glutathione transferase (GST) are detoxifying enzymes and targets for reactive aldehydes. This study examined effects of ATPAL and its precursor, CCMF on ALDH, GST and cell viability in liver, the target tissue for its metabolism and toxicity. A known toxin, HNE, which is also a substrate for ALDH and GST, was used for comparison. Interspecies difference in metabolism of FBM is well documented, therefore, human tissue was deemed most relevant and used for these studies. ATPAL inhibited ALDH and GST activities and led to a loss of hepatocyte viability. Several fold greater concentrations of CCMF were necessary to demonstrate a similar degree of ALDH inhibition or cytotoxicity as observed with ATPAL. This is consistent with CCMF requiring prior conversion to the more proximate toxin, ATPAL. GSH was shown to protect against ALDH inhibition by ATPAL. In this context, ALDH and GST are detoxifying pathways and their inhibition would lead to an accumulation of reactive species from FBM metabolism and/or metabolism of other endogenous or exogenous compounds and predisposing to or causing toxicity. Therefore, mechanisms of reactive aldehydes toxicity could include direct interaction with critical cellular macromolecules or indirect interference with cellular detoxification mechanisms.