ALDHbr Cells Research Articles

Sex-related differences in efficacy of bone marrow-derived high aldehyde dehydrogenase activity cells against pulmonary fibrosis

BackgroundAlthough bone marrow-derived cells with high aldehyde dehydrogenase activity (ALDHbr) have shown therapeutic potential against various diseases in animal studies, clinical trials have failed to show concurrent findings. We aimed to clarify the optimal conditions for the efficacy of ALDHbr cells by using a murine bleomycin-induced pulmonary fibrosis model.MethodsWe intravenously transferred male or female donor C57BL/6 mice-derived ALDHbr cells into recipient C57BL/6 mice under various conditions, and used mCherry-expressing mice as a donor to trace the transferred ALDHbr cells.ResultsPulmonary fibrosis improved significantly when (1) female-derived, not male-derived, and (2) lineage (Lin)-negative, not lineage-positive, ALDHbr cells were transferred during the (3) fibrotic, not inflammatory, phase. Consistent with the RNA-sequencing results, female-derived Lin−/ALDHbr cells were more resistant to oxidative stress than male-derived cells in vitro, and transferred female-derived Lin−/ALDHbr cells were more viable than male-derived cells in the fibrotic lung. The mechanism underlying the antifibrotic effects of Lin−/ALDHbr cells was strongly associated with reduction of oxidative stress.ConclusionsOur results indicated that Lin−/ALDHbr cell therapy could ameliorate pulmonary fibrosis by reducing oxidative stress and suggested that their efficacy was mediated by sex-related differences. Thus, sex-awareness strategies may be important for clinical application of bone marrow ALDHbr cells as a therapeutic tool.

Antifibrotic effect of lung-resident progenitor cells with high aldehyde dehydrogenase activity

BackgroundAldehyde dehydrogenase (ALDH) is highly expressed in stem/progenitor cells in various tissues, and cell populations with high ALDH activity (ALDHbr) are associated with tissue repair. However, little is known about lung-resident ALDHbr. This study was performed to clarify the characteristics of lung-resident ALDHbr cells and to evaluate their possible use as a tool for cell therapy using a mouse model of bleomycin-induced pulmonary fibrosis.MethodsThe characteristics of lung-resident/nonhematopoietic (CD45−) ALDHbr cells were assessed in control C57BL/6 mice. The kinetics and the potential usage of CD45−/ALDHbr for cell therapy were investigated in bleomycin-induced pulmonary fibrosis. Localization of transferred CD45−/ALDHbr cells was determined using mCherry-expressing mice as donors. The effects of aging on ALDH expression were also assessed using aged mice.ResultsLung CD45−/ALDHbr showed higher proliferative and colony-forming potential than cell populations with low ALDH activity. The CD45−/ALDHbr cell population, and especially its CD45−/ALDHbr/PDGFRα+ subpopulation, was significantly reduced in the lung during bleomycin-induced pulmonary fibrosis. Furthermore, mRNA expression of ALDH isoforms was significantly reduced in the fibrotic lung. When transferred in vivo into bleomycin-pretreated mice, CD45−/ALDHbr cells reached the site of injury, ameliorated pulmonary fibrosis, recovered the reduced expression of ALDH mRNA, and prolonged survival, which was associated with the upregulation of the retinol-metabolizing pathway and the suppression of profibrotic cytokines. The reduction in CD45−/ALDHbr/PDGFRα+ population was more remarkable in aged mice than in young mice.ConclusionsOur results strongly suggest that the lung expression of ALDH and lung-resident CD45−/ALDHbr cells are involved in pulmonary fibrosis. The current study signified the possibility that CD45−/ALDHbr cells could find application as novel and useful cell therapy tools in pulmonary fibrosis treatment.

IFN-γ inhibit ALDHbr breast cancer cells in murine 4T1 tumor model

Abstract Background Due to the heterogeneity of breast cancer cells, cytotoxic treatments targeting proliferative cancer cells could not eradicate all cancer cells. Compelling evidence suggests that breast cancer stem cells (BCSCs) have a crucial impact in current shortcomings of cancer therapy because they are largely responsible for tumor initiation, relapse, metastasis, and chemo-resistance. Cytokines in the tumor microenvironment can regulate the self-renewal and survival of BCSCs in a variety of ways. Methods and Results We have utilized in vitro and mouse xenograft models (immunocompetent Balb/c mice and thymus-deficient nude mice (Balb/c nude)) to examine the interaction between breast cancer stem cells (BCSCs) and T cells or cytokine IFN-γ. In vitro assay, such as migration and invasion and sphere-forming, we demonstrate that CD8+ T lymphocytes regulate aldehyde dehydrogenase (ALDH) expressing in BCSCs (ALDHbr 4T1 cells) may through cytokine IFN-γ. Utilizing immunocytochemistry, western blotting and gene expression microarray of ALDHbr vs ALDHdim 4T1 cells, we identified cytokine IFN-γ decreased ALDH1A protein expression and regulated the cancer stem cell population. In various in vivo-like screening assays, depletion of either CD4+ or CD8+ T cells associated with increased proportions of BCSCs. Conclusion The collective results suggest that IFN-γ as a highly secreted cytokine may affect cancer stem cells in mouse breast cancer model.

In Vitro Identification and Characteristics of CD34- Leukemia Stem Cell in t(8;21) Acute Myeloid Leukemia

To preliminarily identify the existence of CD34- leukemia stem cell (LSC) in t(8;21) acute myeloid leukemia (AML) by in vitro test. Bone marrow samples collected from newly diagnosed t(8;21) AML patients were tested. Lin-CD34+ CD38-(abbreviation, CD34+CD38-), Lin-CD34+CD38+ (abbreviation, CD34+CD38+) and Lin-CD34-CD38-CD45dimSSClow(abbreviation, CD34-"LSC") cell fractions were gated by flow cytometry after staining with fluorescent antibodies. Cells in G0 phase were identified through Hoechst 33342 and pyronin Y staining. Aldefluor reagent was used to test aldehyde dehydrogenase (ALDH) activity. The above-mentioned 3 cell fractions were sorted, and mRNA levels of AML1-ETO and WT1 were measured by real-time quantitative PCR. The 3 tested samples displayed the same tendency in ratio of the cells in G0 phase: CD34-"LSC">CD34+ CD38->CD34+CD38+. The paired t-test of 53 patients showed that frequency of ALDHbr cells of both CD34+CD38- and CD34-"LSC" cell fractions was significantly higher than that of CD34+CD38+ (P<0.01), furthermore, the ALDHbr cell frequency was significantly higher in CD34-"LSC" than that in CD34+ CD38- (P<0.01). AML1-ETO mRNA levels of cells sorted from 3 patients were similar among the 3 cell fractions within each patient, whereas WT1 mRNA levels were significantly higher in CD34-"LSC" than that in other 2 cell fractions. CD34- LSC may exist in t(8;21) AML, and may be more primitive than CD34+ LSC. These results promote the necessity to perform in vivo xenogeneic transplantation mice.

Identification of differentially expressed genes in oral squamous cell carcinoma TCA8113 cells.

Previous studies have demonstrated that cancer cells with increased levels of aldehyde dehydrogenase ‘bright’ activity (ALDHbr) exhibit stem cell properties compared with cells exhibiting decreased ALDH activity (ALDHlow). To screen possible biomarkers of cancer stem cells in tongue squamous cell carcinoma, ALDHbr and ALDHlow cells were isolated from the tongue squamous cell carcinoma TCA8113 cell line, and suppression subtractive hybridization was performed to identify differentially expressed genes in the two subpopulations. A total of 240 positive clones were randomly selected for sequencing and were functionally characterized using bioinformatical tools. The results of the present study identified the differential expression of 104 clones, 62 of which corresponded to known genes and 42 of which corresponded to unknown genes. Cluster analysis revealed that the known genes were involved in the regulation of the cell cycle and cell differentiation. In addition, analysis of 10 signaling pathways revealed that genes were markedly altered in the ALDHbr cell subpopulation. Additional study is required to identify the function that these genes serve in the biomolecular regulatory mechanisms of cancer stem cells and to assist in explaining the biological behavior of oral squamous cell carcinoma.

Enhancement of tumor initiation and expression of KCNMA1, MORF4L2 and ASPM genes in the adenocarcinoma of lung xenograft after vorinostat treatment.

Cancer stem cells (CSCs) are usually tolerant to chemotherapy and radiotherapy and associated with tumor relapse. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACI), is currently being used in clinical trials of lung cancer. However, SAHA facilitates the formation of induced pluripotent stem cells from somatic cells. We hypothesized that SAHA would mediate the CSCs properties and subsequently confer a more malignant phenotype in lung cancer. Transfected H1299 lung cancer cells, which stably expresses a triple fused reporter gene (DsRedm-Fluc-tTKsr39) under the control of CMV promoter was used to establish a xenograft mouse model. After the treatment of SAHA, H1299 cell line and tumor xenografts were sorted by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity. We found that SAHA could suppress the growth of xenografted H1299 tumors with decreased proportion of ALDHbr lung cancer cells indicating that SAHA may target CSCs. However, SAHA significantly enhanced the tumor initiating capacity and the expression of malignant genes such as KCNMA1, MORF4L2 and ASPM in the remaining living ALDHbr cells. These findings suggested that SAHA treatment created a more drug-resistant state in residual ALDHbr cells. The in vivo imaging technique may facilitate searching and characterization of CSCs.

Development of a flow cytometry–based pulse-width assay for detection of aggregates in cellular therapeutics to be infused by catheter

Background aimsDelivery of cell-based therapies through the carotid artery with the use of an intra-arterial catheter could introduce aggregates and cause focal ischemia in the brain. We developed a pulse-width flow cytometry method for aggregate detection and quantification. The assay was designed to be used as a cell product release assay in a clinical trial seeking to treat ischemic stroke with sorted cells brightly expressing aldehyde dehydrogenase (ALDHbr cells) delivered through intra-arterial catheters. MethodsThe forward light scatter pulse-width axis of a flow cytometer was calibrated for particle diameter measurements through the use of traceable standard microspheres and linear regression. As a positive control, Concanavalin A–aggregated cells were counted manually and sorted onto slides to compare with pulse width–determined values. Known numbers of aggregates were spiked into purified singlet cells for quantification. A clinical standard for aggregate count and diameter was determined. The assay was used to qualify catheters with the use of ALDHbr cells. ResultsThe pulse-width axis was highly linear for microsphere diameter (r2 > 0.99), which allowed for size calibration. Microscopically determined counts and diameters corresponded to pulse width-determined values. Known aggregate counts were linear with pulse width–determined aggregate counts (r2 = 0.98). The limit of detection was determined to be 0.004%. Flow of ALDHbr cells through catheters did not generate aggregates. The final method to be used as a release assay for the stroke clinical trial was tested successfully on samples from volunteer donors. ConclusionsThe pulse-width aggregate detection assay provides a reliable, reproducible, accurate and rapid means of detection, classification and quantification of aggregates in cell therapy products.

Mass Spectrometry Imaging of Sphingolipids in Preeclamptic Placentae

Mass Spectrometry Imaging of Sphingolipids in Preeclamptic Placentae

Bone formation induced by BMP-2 in human osteosarcoma cells

Our previous studies demonstrated that BMP-2 inhibits the tumorigenicity of cancer stem cells identified as cells with high aldehyde dehydrogenase activity (ALDH(br) cells) from the human osteosarcoma cell line OS99-1. We further investigated whether BMP-2 is capable of inducing bone formation in OS99-1 cells. Flow cytometry sorting was used to isolate tumorigenic ALDH(br) and non-tumorigenic ALDH(lo) cells. qRT-PCR was used to quantify the gene expression. A xenograft model was used to verify the bone formation in vivo. There was significantly higher mRNA expression of BMPR1B and BMPR2 in ALDH(lo) cells compared with that in ALDH(br) cells and the BMPR1B expression in ALDH(lo) cells was ~8-fold higher compared to that in ALDHbr cells. BMP-2 was also found to induce higher transcription of osteogenic markers Runx-2, Osterix (Osx), alkaline phosphatase (ALP) and collagen type I in ALDH(lo) cells compared to ALDH(br) cells, which were mediated by the canonical Smad signaling pathway. In vivo, BMP-2 was identified to induce bone formation in both ALDH(br) and ALDH(lo) cells. All animals receiving 1 x 10()4 ALDH(lo) cells treated with 30 µg of BMP-2 per animal showed bone formation within 1-2 weeks after injection in mice. Bone formation induced by BMP-2 in ALDH(lo) cells showed significantly more bone mineral content compared to that in ALDH(br) cells. BMP-2 induces bone formation in heterogeneous osteosarcoma cells and BMP-2 may have a promising therapeutic role for treating human osteosarcoma by inducing differentiation along an osteogenic pathway.

Doxorubicin-enriched, ALDHbr mouse breast cancer stem cells are treatable to oncolytic herpes simplex virus type 1

BackgroundThe primary objective of this study was to test whether oncolytic herpes simplex virus type 1 (HSV1) could eradicate chemoresistant cancer stem cells (CSCs).MethodsThe fluorescent aldefluor reagent-based technique was used to identify and isolate ALDHbr cells as CSCs from the 4T1 murine breast cancer cell line. The presence of ALDHbr 4T1 cells was also examined in 4T1 breast cancer transplanted in immune-competent syngeneic mice.ResultsCompared with ALDHlo cells, ALDHbr cells had a markedly higher ability to form tumor spheres in vitro and a higher tumorigenic potential in vivo. ALDHbr cells also exhibited increased doxorubicin resistance in vitro, which correlated with a selective increase in the percentage of ALDHbr cells after doxorubicin treatment and an increased expression of P-glycoprotein (P-gp), a known chemoresistance factor. In contrast, oncolytic HSV1 was able to kill ALDHbr cells in vitro and even more markedly in vivo. Furthermore, in in vivo studies, systemic administration of doxorubicin followed by intratumoral injection of oncolytic HSV1 resulted in much more significant suppression of tumor growth with increased median survival period compared with each treatment given alone (p<0.05). Though more CD8+ T lymphocytes were induced by oncolytic HSV1, no significant specific T cell response against CSCs was detected in vivo.ConclusionsThese results suggested that the use of oncolytic HSV1 following doxorubicin treatment may help eradicate residual chemoresistant CSCs in vivo.

Relationships Among Commonly Used Measures of Cord Blood Potency, Aldhbr Cell Content, and Colony Forming Cell Content in Cord Blood Units Prior to Cryopreservation: Towards an Improved Metric for Potency of Banked Cord Blood

Relationships Among Commonly Used Measures of Cord Blood Potency, Aldhbr Cell Content, and Colony Forming Cell Content in Cord Blood Units Prior to Cryopreservation: Towards an Improved Metric for Potency of Banked Cord Blood

Mechanisms of Action of Human Aldehyde Dehydrogenase Bright Cells in Therapy of Cardiovascular Diseases: Expression Analysis of Angiogenic Factors and Aldehyde Dehydrogenase Isozymes

Human stem cell populations that express high aldehyde dehydrogenase activity [ALDHbr cells] have angiogenic activity in preclinical models and have been used safely to treat patients in early clinical trials. Bone marrow ALDHbr cells are being developed for therapeutic use in ischemic cardiovascular diseases. The mechanisms by which ALDHbr cells repair ischemic tissue are unknown, but available data suggest that angiogenic factors released by ALDHbr cells are involved. Gene expression studies were performed, and bone marrow ALDHbr cells were found to express 69 of 84 angiogenic factors tested. The 25 most highly expressed genes included soluble cytokines and growth factors, cytokine receptors, extracellular matrix proteins, and cell-cell signaling receptors. ALDHdim bone marrow cells that do not express high levels of ALDH and that have no angiogenic activity in preclinical models expressed a different group of genes. CD105 and Ephrin B4 transcripts were expressed about 65-fold more highly in ALDHbr than ALDHdim cells, and expression of these proteins was demonstrated in ALDHbr cells by flow cytometry. Expression analysis probing all 19 ALDH isozymes and immunofluorescence both demonstrated that ALDH1A1, an enzyme that can generate retinoic acids from retinaldehyde, was the ALDH isozyme most highly over expressed in ALDHbr cells compared to ALDHdim cells. Transwell studies demonstrated that ALDHbr cells responded to hypoxic conditions and to factors released from human endothelial vein cells (HUVEC) with changes in expression of specific angiogenic factors. Soluble factors released from ALDHbr cells in transwell cultures under hypoxic conditions stabilized endothelial tubules formed by HUVEC. The results are consistent with the hypothesis that human bone marrow ALDHbr cells can promote angiogenesis in ischemic tissues of patients with cardiovascular disease by several mechanisms including the release of soluble mediators.

Relationships Among Commonly Used Measures of Cord Blood Potency, ALDHbr Cell Content, and Colony Forming Cell Content in Cord Blood Units Prior to Cryopreservation: Towards An Improved Metric for Potency of Banked Cord Blood,

Abstract 4054 Background:Despite adequate total nucleated cell (TNC) dosing, graft failure and engraftment delays remain a significant issue for patients undergoing cord blood transplantation (CBT). Previously, we have shown that recovery of colony forming units (CFU) after cord blood unit (CBU) thaw is superior to TNC and CD34+ content in predicting engraftment. However, the CFU assay requires weeks and is not standardized. Aldehyde dehydrogenase (ALDH), an enzyme expressed in stem and progenitor cells (ALDHbr cells), is detected by a functional, flow-based intracellular assay and can be completed within a few hours. In this study, we determined the statistical relationships among TNC, CD34+, ALDHbr cells and CFU in a large cohort of fresh CBUs prior to cryopreservation with the ultimate goal of defining the best parameter(s) to predict potency defined by successful engraftment. Methods: As part of routine banking procedures, post-processing TNC, mononuclear cell count (MNC), CD34+cells and CFU content were enumerated on fresh CBUs donated to the Carolina Cord Blood Bank (n=5268) or identified for directed donation (n=14) between 9/07–7/09. ALDHbr cell content was measured using Aldecount (Aldagen, Inc.). Correlations among these various CBU characteristics were determined. Results: Of the 5282 fresh CBUs analyzed, the median values for TNC, CD34+, MNC, ALDHbr cells and CFU content were: 11.8×108 (range, 2.9–55.5×108), 3.36 x106 (range, 0.17–98.2×106), 5.5×108(range, 1.7–20.0×108), 4.3x 106 (0–36.6×106) and 34.0×105 (range, 0.6–193.3×105), respectively. There were significant correlations (p<0.0001) among all of the cord blood (CB) parameters measured as presented in Table 1. In standardized, univariate modeling which allowed for ranked comparisons of the CB parameters using AIC values, CFU was best predicted by TNC (AIC=11,501) followed closely by ALDHbr (AIC=12,012) and, to a lesser degree, MNC (AIC=12,412) and CD34+ (AIC=12,612). In multivariate modeling of CB parameters (Table 2), results were similar to the standardized univariate analysis.Table 1:Correlation coefficients for parameters measured on fresh CB (all p<0.0001)TNCCFUMNCALDHbrCD34+TNC1.000.700.820.580.50CFU0.701.000.610.660.60MNC0.820.611.000.470.41ALDHbr0.580.660.471.000.70CD34+0.500.600.410.701.00Table 2:Standardized multivariate models of CBU parametersModels - Slope Estimate (p value)PredictorTNCCFUALDHbrCD34+TNC–0.35*0.23*NSCFU0.22*–0.26*0.23*MNC0.56*0.12*−0.05 (p=0.002)NSALDHbr0.15*0.26*–0.54*CD34+NS0.19*0.46*–Volume0.09*NS−0.03 (p=0.01)0.03 (p=0.01)*p<0.0001; NS - not significant Conclusions:In this large dataset of fresh CBUs, the parameters measured (TNC, MNC, CFU, CD34+ and ALDHbr) are well correlated and predictive of each other. Furthermore, total ALDHbr measured on fresh CB was well correlated with other CB parameters, including CFUs that also assesses viable cells and is the unit characteristic that best predicts engraftment. Given the technical difficulties associated with measuring CFUs, ALDHbr content has promise as a potential CFU surrogate as a potency assay for CBUs. ALDHbr measurements were modestly, but significantly more reliable in predicting CFU content than CD34+ or MNC content in this cohort of fresh CBU. We and others have previously shown that different types of cells survive freezing and thawing of the CBU with different efficiencies. Consequently, we are studying which CBU parameter best correlates with engraftment after thawing of CBUs or from segments attached to CBUs in order to improve assessment of potency during the process of CBU selection for CBT. Disclosures:Balber:Aldagen, Inc: Consultancy, Equity Ownership.

Intradialytic Hypertension and its Association with Endothelial Cell Dysfunction

Intradialytic hypertension is associated with adverse outcomes, yet the mechanism is uncertain. Patients with intradialytic hypertension exhibit imbalances in endothelial-derived vasoregulators nitric oxide and endothelin-1, indirectly suggesting endothelial cell dysfunction. We hypothesized that intradialytic hypertension is associated in vivo with endothelial cell dysfunction, a novel predictor of adverse cardiovascular outcomes. We performed a case-control cohort study including 25 hemodialysis (HD) subjects without (controls) and 25 with intradialytic hypertension (an increase in systolic BP pre- to postdialysis ≥10 mmHg ≥4/6 consecutive HD sessions). The primary outcome was peripheral blood endothelial progenitor cells (EPCs) assessed by aldehyde dehydrogenase activity (ALDH(br)) and cell surface marker expression (CD34(+)CD133(+)). We also assessed endothelial function by ultrasonographic measurement of brachial artery flow-mediated vasodilation (FMD) normalized for shear stress. Parametric and nonparametric t tests were used to compare EPCs, FMD, and BP. Baseline characteristics and comorbidities were similar between groups. Compared with controls, 2-week average predialysis systolic BP was lower among subjects with intradialytic hypertension (144.0 versus 155.5 mmHg), but postdialysis systolic BP was significantly higher (159.0 versus 128.1 mmHg). Endothelial cell function was impaired among subjects with intradialytic hypertension as measured by decreased median ALDH(br) cells and decreased CD34(+)CD133(+) cells (ALDH(br), 0.034% versus 0.053%; CD34(+)CD133(+), 0.033% versus 0.059%). FMD was lower among subjects with intradialytic hypertension (1.03% versus 1.67%). Intradialytic hypertension is associated with endothelial cell dysfunction. We propose that endothelial cell dysfunction may partially explain the higher event rates observed in these patients.

Abstract 293: Characterization of renal tumor-initiating cells in human renal carcinoma cell lines

Abstract High aldehyde dehydrogenase activity (ALDH) has been used as a putative marker for the tumor- initiating subpopulation in many cancer types. We previously showed that tumor-initiating cells in human osteosarcoma OS99-1 cell line can be identified using ALDH. In the current study we sought to determine whether ALDH can be also used as a marker to isolate tumor-initiating populations from human renal carcinoma cell lines. By using a xenograft model in which cells from human renal carcinoma cell line ACHN and Caki-2 were grown in NOD/SCID mice, we identified a subset of renal cells that were highly tumorigenic based on the high aldehyde dehydrogenase activity. Isolated cancer cells with relatively high aldehyde dehydrogenase activity (ALDHbr cells) revealed a greater colony-forming efficiency, higher proliferative output, the ability to grow in non-adhesive spheroids, the capability to undergo adipogenic and osteogenic differentiation, and increased expression of stem cell marker genes Oct3/4A, Nanog and Pax-2. In vivo experiments showed that ALDHbr cells were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDHlo cells), generating new tumors with as few as 25 cells. The isolation of renal tumor-initiating cells by their high ALDH activity may lead to a new insight into the study of how renal cell carcinoma may take place, and may lead to effective prognostic and therapeutic strategies for the treatment of patients with kidney cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 293. doi:10.1158/1538-7445.AM2011-293

BMP-2 inhibits the tumorigenicity of cancer stem cells in human osteosarcoma OS99-1 cell line

Previously, based on high ALDH activity, we showed that cancer stem cells (CSCs) could be identified as ALDHbr cells from an aggressive human osteosarcoma OS99-1 cell line. In this study, we evaluate the impact of BMP-2 on CSCs.Three types of BMP receptors were expressed in freshly sorted ALDHbr cells. In vitro, growth of the sorted ALDHbr cells was inhibited by BMP-2. Using RT-PCR analysis, BMP-2 was found to down-regulate the expression of embryonic stem cell markers Oct3/4, Nanog, and Sox-2, and up-regulate the transcription of osteogenic markers Runx-2 and Collagen Type I. In vivo, all animals receiving ALDHbr cells treated with BMP-2 did not form significant tumors, while untreated ALDHbr cells developed large tumor masses in NOD/SCID mice. Immunostaining confirmed few Ki-67 positive cells were present in the sections of tumor containing ALDHbr cells treated with BMP-2. These results suggest that BMP-2 suppresses tumor growth by reducing the gene expression of tumorigenic factors and inducing the differentiation of CSCs in osteosarcoma. BMP-2 or BMP-2-mimetic drugs, if properly delivered to tumor and combined with traditional therapies, may therefore provide a new therapeutic option for treatment of osteosarcoma.

Evaluation of mobilized peripheral stem cells according to CD34 and aldehyde dehydrogenase expression and effect of SSClo ALDHbr cells on hematopoietic recovery

We evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte-colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m²) once daily, intravenously on day 1). We also investigated the relationship between the number of SSC(lo) CD45(dim) CD34(hi) cells, SSC(lo) ALDH(br) cells and engraftment. Thirty patients (20 males and 10 females), who were candidates for autologous peripheral blood stem cell transplantation, were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n = 14), Hodgkin's lymphoma (n = 7), non-Hodgkin's lymphoma (n = 2), acute myloid leukemia (n = 2), chronic lymphocytic leukemia (n = 1) and germ cell testis tumor (n = 1). Numbers of SSC(lo) CD45(dim) CD34(hi) cells and SSC(lo) ALDH(br) cells were highly correlated in both peripheral blood and apheresis products (P < 0.001). We could not find a relationship between the transplanted SSC(lo) CD45(dim) CD34(hi) cell dose or SSC(lo) ALDH(br) cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC(lo) CD45(dim) CD34(hi) cells were 5.40 × 10⁶/kg for neutrophil recovery and 7.22 x 10⁶/kg for platelet recovery. The optimal thresholds for SSC(lo) ALDH(br) cells were 6.53 x 10⁶/kg for neutrophil recovery and 8.72 x 10⁶/kg platelet recovery. According to our data, numbers of SSC(lo) ALDH(br) cells are in very good agreement with numbers of SSC(lo) CD45(dim) CD34(hi) cells and can be a predictor of stem cell mobilization.

Aldehyde Dehydrogenase (ALDH) Vs CD34 for Predicting Engraftment After Autologous Hematopoietic Progenitor Cell (autoHPC) Transplant

AbstractAbstract 4441 Introduction:When the cell surface marker CD34 became available for enumerating HPC there was controversy as to whether it was useful for predicting time to engraftment after autologous peripheral blood HPC transplant (autoHPCT). It was demonstrated that a threshold dose of 2.0–2.5 × 106 CD34+ cells/kg in the product, measured before cryopreservation, predicted reliable engraftment within a reasonable period of time. CD34 is expressed on the surface of HPC ranging from primitive to committed progenitor cells. ALDH is highly expressed in primitive cells such as HPC, endothelial progenitor cells and mesenchymal stromal cells. ALDHbr cells are by definition viable, while measurement of viable CD34+ cells requires an additional assay. We tested the correlation of CD34 and ALDH activity in fresh HPC products and then examined the usefulness of these two assays for predicting time to WBC and platelet engraftment after autoHPCT. Materials and Methods:We identified 42 consecutive HPC apheresis products used for autoHPCT for which data on CD34+ numbers and viability as well as ALDHbr cell numbers were available. Data from 5 of these product were not used for predicting engraftment due to lack of engraftment data. Viability was assesed by flow cytometry on pre-cryopreservation (fresh) and post-thaw samples by looking at propidium iodide or 7-AAD uptake within the CD34+ population identified using the ISHAGE method. ALDH activity was measured by flow cytometry on pre-freeze samples using the manufacturers prescribed method. HPC were cryopreserved using a controlled rate freezer and 10% DMSO, then stored in liquid nitrogen. Data on time to WBC and platelet engraftment were determined by chart review. The patents received a median of 3.78 × 106 CD34+ cells/kg (range 2.43–8.59 × 106 CD34+ cells/kg) for autoHPCT. Regression analyses were performed to see if viable CD34 in the fresh product, and in the thawed product, correlated with ALDH and whether any of these measures correlate with time to WBC (ANC >500/mm3) or platelet engraftment (>20,000/mm3). Results:The correlation coefficients for ALDHbr vs viable CD34+ cells pre-cryopreservation (r=0.97; n=42) and for ALDHbr cells vs post-thaw CD34+ cells (r=0.96; n=42) were both significant. The percent of variation for ALDHbr cells to WBC engraftment (r2 = 2.2%; n=37) and for CD34+ cells both pre-freezing and post-thaw to WBC engraftment (r2 =1.9%; n=37) were not significant. The r2 for ALDHbr cells and platelet engraftment was 0.9%, the r2 for CD34+ cells and platelet engraftment was 1.1% pre-freeze (n=37) and was 0.9% post-thaw (n=37) were also not significant. Conclusion:Enumeration of HPC by ALDH correlates well with CD34, suggesting that the two assays are equivalent when analyzing autologous peripheral blood. Neither the number of ALDHbr cells or CD34+ cells in fresh products, nor the number of viable CD34+ cells in the thawed products, correlates with time to WBC and platelet engraftment. Since our patients received at least 2.4 × 106 CD34+ cells/kg, our observations support previous data showing no correlation between cell dose and time to engraftment if more than the HPC threshold dose for engraftment is given for transplant. Disclosures:No relevant conflicts of interest to declare.

Abstract 5155: BMP-2 inhibits the tumorigenicity of human osteosarcoma tumor-initiating cells

Abstract Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-β superfamily, were originally identified as molecules that induce bone and cartilage formation and are now considered as multifunctional cytokines. More recently, there have been a number of studies showing the inhibitory effects of BMP-2 on tumorigenesis in many cancers. However, very little has been investigated to date about the role of BMP-2 in human osteosarcoma. In the present study we show that BMP-2 inhibits the tumorigenicity of an aggressive human osteosarcoma cell line, OS99-1. Based on the stem cell theory of cancer, a subpopulation of cells showing characteristics of stem cells was identified using high aldehyde dehydrogenase (ALDH) activity (ALDHbr cells) from xenografts. These stem-like cells were found to be highly tumorigenic and were classified as tumor initiating cells. In vitro, the growth of the sorted tumorigenic ALDHbr cells was significantly inhibited by the addition of 300 ng/ml of BMP-2 in the presence of 0.5% fetal bovine serum (FBS) for 48h, as evaluated by cell proliferation assay. In addition, using RT-PCR analysis, we found BMP-2 down-regulated the expressions of embryonic stem cell markers Oct3/4, Nanog, and Sox-2. Conversely there was an increase in the transcription of osteogenic markers Runx-2 and Collagen Type I in the sorted tumorigenic ALDHbr cells treated with the same BMP-2 solution for 48h compared with the same volume of vehicle. In vivo, tumor formation in animals was compared to a control receiving ALDHbr cells maintained under identical conditions without BMP-2. All animals receiving untreated ALDHbr cells developed large tumor masses that showed characteristic osteosarcoma features, including nuclear atypia, extensive neovascularization, and high mitotic activity. Conversely, ALDHbr cells treated with 30μg/animal of BMP-2 did not form significant tumors. Further immunostaining confirmed most Ki-67 positive cells were shown in the tumor sections from mice injected with ALDHbr cells without BMP-2, whereas few Ki-67 positive cells were present in the sections of tumor from ALDHbr cells treated with BMP-2. Based on these results, it appears that BMP-2 may suppress tumor growth by inducing the differentiation of tumor -initiating cells. BMP-2 or BMP-2-mimetic drugs, if properly delivered to tumor and combined with traditional therapies, may therefore provide a new therapeutic option. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5155.

ALDH br Hematopoietic Progenitors Promote Short-Term Engraftment In Experimental Models For Cord Blood Transplantation

We previously developed methods to purify human hematopoietic progenitors based on their high expression of aldehyde dehydrogenase (ALDHbr cells), which enrich both short- and long-term SCID-repopulating cells. One clinical trial now suggests that augmenting a conventional cord blood transplant (CBT) with ALDHbr cells accelerates both neutrophil and platelet engraftment. We now describe two experimental models for this clinical regimen, performed in different strains of NOD/SCID mice. Each study included three cohorts: (1) mice transplanted with 4,000 purified ALDHbr cells; (2) mice transplanted with total CB at a dose that contained 4,000 ALDHbr cells; and, (3) mice that first received the same dose of unmanipulated CB and, after 4hours, the ALDHbr cells.