Aldehyde Dehydrogenase High Cells Research Articles

Abstract 1761: Examination of therapeutic potential of epigenetic modulation by dual HDAC-LSD1 inhibition in murine models of triple negative breast cancer (TNBC)

Abstract This study examines the effects of the epigenetic modulator 4SC-202, a dual histone deacetylase (HDAC) /lysine-specific histone demethylase 1A (LSD1) small molecule inhibitor, on tumor growth and metastasis in a mouse model of breast cancer. 4SC-202 differs from clinically available HDAC inhibitors such as Vorinostat (SAHA) by specifically targeting class I HDACs including HDAC1, HDAC2, and HDAC3 and the histone demethylase LSD1. The hypothesis to be examined is that epigenetic modulation via dual inhibition of HDAC1 and LSD1 improves disease control in breast cancer. In vitro data indicates that 4SC-202 is significantly cytotoxic and cytostatic to the highly metastatic murine Triple Negative Breast Cancer (TNBC) murine model 4T1 and the human TNBC cell line MDA-MB-231 and reduces cancer cell migration. In vivo studies conducted in the syngeneic 4T1 model which closely mimics human TNBC in terms of sites of metastasis, show increased necrosis in 4T1 tumors, reduced tumor burden in vivo as compared to Vorinostat treatment, and significantly reduced metastasis. Furthermore, multiple studies in different cell types indicate that the mechanism of action of 4SC-202 may involve effects on cancer stem cells, which are able to self-renew and form metastatic lesions. It has been shown that a marker of cancer stem cells (CSC's) in breast cancer is high aldehyde dehydrogenase (ALDH) activity and that ALDH high cells mediate metastasis. In the 4T1 syngeneic mouse model of breast cancer, inhibition of ALDH activity suppresses stem-like cell properties. Preliminary data suggests 4SC-202 substantially reduces the ALDH high 4T1 cancer stem population. Transcriptome bulk RNA -sequencing analysis from 4T1 tumors reveal changes in metastatic related pathways in 4SC-202 treated tumors, including significant changes to the expression levels of genes implicated in focal adhesion and ECM-receptor interaction pathways. Furthermore, 4SC-202 appears to alter expression of cytokines which serve as chemokines, potentially leading to increased expression of tumor infiltrating lymphocytes. In summary, treatment of a highly metastatic murine model of TNBC with 4SC-202 significantly reduces metastasis, and warrants further preclinical studies in conjunction with standard of care chemotherapy treatment. Citation Format: Shanta M. Messerli, Mariah M. Hoffman, Jessica S. Zylla, Etienne Gnimpieba, Keith W. Miskimins. Examination of therapeutic potential of epigenetic modulation by dual HDAC-LSD1 inhibition in murine models of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1761.

CD44+/EPCAM+ cells detect a subpopulation of ALDHhigh cells in human non-small cell lung cancer: A chance for targeting cancer stem cells?

Objectives: Several studies demonstrated that aldehyde dehydrogenase (ALDH) and CD44 are the most considered cancer stem cells (CSC) markers. However, a comparison between ALDH high cells and CD44+ cells have been previously described with no significant correlation. Indeed, the aim of the present research is to identify a superficial marker able to match with ALDH high cells population in freshly isolated human lung cancer cells.Materials and Methods: This cross-sectional study analyzed the expression of ALDHhigh/low cells and the positivity for CD44 and epithelium cell adhesion molecule (EPCAM) antigens in surgical lung cancer tissues. The main approach was a cytofluorimetric analysis of ALDH expression and positivity for CD44/EPCAM on primary cell population obtained from 23 patients harboring NSCLC.Results: There was a highly positive correlation between the expressions of ALDHhigh and CD44+/EPCAM+ cells, with a Pearson’s correlation coefficient equal to 0.69 (95% CI 0.39–0.86; P = 0.0002), and Spearman’s correlation coefficient equal to 0.52 (P = 0.0124). The average paired difference between the expression of ALDHhigh and CD44+/EPCAM+ cells was very close to 0, being 0.1% (SD 2.5%); there was no difference between these subpopulations in terms of means (95% CI = –1.0; 1.2%, P = 0.8464). These results highlight a strong similarity between ALDHhigh and CD44+/EPCAM+ cells.Conclusions: Our study is the first attempt which identifies a high correlation between the ALDHhigh and the CD44+/EPCAM+ cells, thus suggesting the possibility to use this superficial marker for future target treatments against lung cancer stem cells.

Abstract B007: Identification of prostate cancer stem cell antigens for T-cell immunotherapy by HLA ligandome analysis

Abstract Introduction: Localized prostate cancer (PCa) can be successfully treated by androgen deprivation, radiotherapy and surgery, however these may not be sufficient to eradicate cancer stem cells (CSCs). CSCs are more resistant to such treatments than the bulk of the tumor; and can contribute to disease relapse. PCa patients who relapse have a poor prognosis. We hypothesize that CSCs could be killed by T-cells in an antigen specific way, thus preventing the possibility of relapse. In this study we identified novel PCa CSC antigens by HLA ligandome analysis and isolated antigen specific CD8+ T-cells. Methods: We identified CSCs using aldehyde dehydrogenase (ALDH) activity as a CSC marker. ALDH high and low cells from the DU145 PCa cell line and from prostate adenocarcinoma primary tissue were characterized in vitro; DU145 CSCs and non-CSCs were additionally characterized in vivo. We isolated peptide-HLA complexes from DU145 cells by immunoprecipitation and analyzed the eluted peptides by mass spectrometry. We identified CSC antigens based on the gene expression in ALDH high and low DU145 cells (measured by qPCR). To select antigens for further analysis we performed homology modelling of the HLA-peptide interface using COOT software and the YASARA server for energy minimization. The interface interactions were quantified using PISA software. We additionally confirmed antigen expression in the primary cells by fluorescence microscopy and PCR. Tetramers were produced to isolate T-cells which recognized a selection of these antigens. Results: The ligandome analysis identified over 1900 peptides. We selected antigens with low gene expression in healthy tissues (www.GTexportal.org) and high predicted binding to DU145 HLA alleles (http://tools.immuneepitope.org/mhci/). ALDH high DU145 cells were more tumorigenic in vivo than ALDH low cells. ALDH high DU145 and primary prostate cancer cells grew larger colonies and spheres in vitro. We identified 11 CSC antigens by qPCR; 6 upregulated in ALDH high DU145 cells (e.g., TACSTD2) and 5 abundant in both ALDH high and ALDH low DU145 cells (e.g., XPO1). Relevant 9-mer epitopes from three antigens* induced CD8+ T-cell responses in vitro. Antigen-specific CD8+ T-cells were identified by tetramer staining at a frequency of approx. 15 per 100000 cells. These cells are currently being expanded to use in CTL assays. Conclusion: We have identified CSC antigens which could lead to specific targeting by T-cells and prevention of PCa relapse. Further epitopes restricted to the more frequent HLA alleles could additionally be predicted in silico from the novel antigens we identified. We are also investigating prediction of viral epitopes highly aligned with the self-peptides (determined in silico) to boost the immune response against CSCs. *Subject of an ongoing patent application. Citation Format: Amy S. Codd, Saly Al-Taei, Serina Tokita, Emi Mizushima, Pierre J. Rizkallah, Tom Whalley, Barbara Szomolay, Kristin Ladell, James E. McLaren, Sian Llewellyn-Lacey, David A. Price, Takayuki Kanaseki, Toshihiko Torigoe, Stephen Man, Zsuzsanna Tabi. Identification of prostate cancer stem cell antigens for T-cell immunotherapy by HLA ligandome analysis [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B007.

The sonic hedgehog signaling pathway maintains the cancer stem cell self-renewal of anaplastic thyroid cancer by inducing snail expression.

Cancer stem cells (CSCs) have been recently identified in thyroid neoplasm. Anaplastic thyroid cancer (ATC) contains a higher percentage of CSCs than well-differentiated thyroid cancer. The signaling pathways and the transcription factors that regulate thyroid CSC self-renewal remain poorly understood. The objective of this study is to use two ATC cell lines (KAT-18 and SW1736) as a model to study the role of the sonic hedgehog (Shh) pathway in maintaining thyroid CSC self-renewal and to understand its underlying molecular mechanisms. The expression and activity of aldehyde dehydrogenase (ALDH), a marker for thyroid CSCs, was analyzed by Western blot and ALDEFLUOR assay, respectively. The effect of three Shh pathway inhibitors (cyclopamine, HhAntag, GANT61), Shh, Gli1, Snail knockdown, and Gli1 overexpression on thyroid CSC self-renewal was analyzed by ALDEFLUOR assay and thyrosphere formation. The sensitivity of transfected KAT-18 cells to radiation was evaluated by a colony survival assay. Western blot analysis revealed that ALDH protein levels in five thyroid cancer cell lines (WRO82, a follicular thyroid cancer cell line; BCPAP and TPC1, two papillary thyroid cancer cell lines; KAT-18 and SW1736, two ATC cell lines) correlated with the percentage of the ALDH(High) cells as well as Gli1 and Snail expression. The Shh pathway inhibitors, Shh and Gli1 knockdown, in KAT-18 cells decreased thyroid CSC self-renewal and increased radiation sensitivity. In contrast, Gli1 overexpression led to increased thyrosphere formation, an increased percentage of ALDH(High) cells, and increased radiation resistance in KAT-18 cells. Inhibition of the Shh pathway by three specific inhibitors led to decreased Snail expression and a decreased number of ALDH(High) cells in KAT-18 and SW1736. Snail gene knockdown decreased the number of ALDH(High) cells in KAT-18 and SW1736 cells. The Shh pathway promotes the CSC self-renewal in ATC cell lines by Gli1-induced Snail expression.

High aldehyde dehydrogenase activity identifies cancer stem cells in human cervical cancer

High aldehyde dehydrogenase (ALDH) activity characterizes a subpopulation of cells with cancer stem cell (CSC) properties in several malignancies. To clarify whether ALDH can be used as a marker of cervical cancer stem cells (CCSCs), ALDH high and ALDH low cells were sorted from 4 cervical cancer cell lines and 5 primary tumor xenografts and examined for CSC characteristics. Here, we demonstrate that cervical cancer cells with high ALDH activity fulfill the functional criteria for CSCs: (1) ALDH high cells, unlike ALDH low cells, are highly tumorigenic in vivo; (2) ALDH high cells can give rise to both ALDH high and ALDH low cells in vitro and in vivo, thereby establishing a cellular hierarchy; and (3) ALDH high cells have enhanced self-renewal and differentiation potentials. Additionally, ALDH high cervical cancer cells are more resistant to cisplatin treatment than ALDH low cells. Finally, expression of the stem cell self-renewal-associated transcription factors OCT4, NANOG, KLF4 and BMI1 is elevated in ALDH high cervical cancer cells. Taken together, our data indicated that high ALDH activity may represent both a functional marker for CCSCs and a target for novel cervical cancer therapies.

Abstract 4899: Surface marker screening revealed the marker distinguishes gastric cancer stem cells in CD44 negative population.

Abstract Background: Gastric cancer (GC) is the second leading cause of cancer-related deaths in the world. Reportedly, cancer tissue is maintained by a small fraction of cancer cells called cancer stem cells (CSCs). CSCs are considered to initiate therapy resistance, recurrence and metastasis. Although several reports have shown that GC is maintained by CSCs, detailed mechanism of gastric CSCs (GCSCs) maintenance is unclear. It has been shown that high aldehyde dehydrogenase (ALDH) activity is one of the markers for GCSCs by several groups including us. To elucidate the molecular mechanism which regulate GCSCs, we performed screening to identify the novel marker which is able to distinguish GSCSs with high specificity. Materials and methods: We performed flowcytometric screening of 8 common CSC markers (CD44, CD133, EpCAM, CD90, CD13, CD117 and ALDH) for 6 GC cell lines (MKN45, AGS, GSU, NUGC3, MKN1 and MKN7). Fresh surgically removed GC specimens were obtained with informed patient consent. To obtain xenograft, tissues were dissociated with mechanical and enzymatic digestion, filtered to remove clumps, and injected subcutaneously to flank of NOD/SCID mice. Cells were stain with ALDEFLUOR assay kit for 45 minutes at 37°C detection of ALDH activity. Result: Among 8 common CSC makers tested, we decided to focus on ALDH activity because the cell populations with high ALDH activities (ALDHhigh cells) were detected in the most of the cell lines and the fresh tumor tested. ALDHhigh cells showed higher ability of sphere formation, tumorigenicity in NOD/SCID mice, and chemoresistance. Notch1, a critical regulator in maintenance of normal gastric stem/progenitor cell, was highly expressed in ALDHhigh cells. We identified several cell surface markers in the course of searching for markers selectively overexpressed in ALDHhigh cells of GC xenograft. By staining GC cells with several markers including CD44, GCSCs in CD44- fraction became distinguishable. Expression of the several surface markers was highly increased after treatment of chemotherapeutic agents. Our data suggest that the cell populations distinguished by CD44 are heterogenous in terms of chemoresistance and tumorigenicity. Conlcusion: High ALDH activity is a marker for GCSCs and analysis of ALDH high cells revealed that fresh gastric cancer samples contain CD44- gastric cancer stem cells. Citation Format: Shimpei Nishikawa, Hideshi Ishii, Masamitsu Konno, Naotsugu Haraguchi, Yoshihiro Kano, Atsushi Hamabe, Shinichiro Hasegawa, Hisataka Ogawa, Takahito Fukusumi, Katsuya Ohta, Toshihiro Kudo, Daisuke Sakai, Shuji Takiguchi, Taroh Satoh, Masaki Mori, Yuichiro Doki. Surface marker screening revealed the marker distinguishes gastric cancer stem cells in CD44 negative population. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4899. doi:10.1158/1538-7445.AM2013-4899

Aldehyde dehydrogenasehigh gastric cancer stem cells are resistant to chemotherapy

Cancer stem cells (CSCs) are known to influence chemoresistance, survival, relapse and metastasis. Aldehyde dehydrogenase (ALDH) functions as an epithelial CSC marker. In the present study, we investigated the involvement of ALDH in gastric CSC maintenance, chemoresistance and survival. Following screening for eight candidate markers (CD13, CD26, CD44, CD90, CD117, CD133, EpCAM and ALDH), five gastric cancer cell lines were found to contain small subpopulations of high ALDH activity (ALDH(high) cells). We also examined the involvement of ALDH(high) cell populations in human primary tumor samples. Immunodeficient NOD/SCID mice were inoculated with tumor tissues obtained from surgical specimens. ALDH(high) cells were found to persist in the xenotransplanted primary tumor samples. in the immunodeficient mice, ALDH(high) cells exhibited a greater sphere‑forming ability in vitro and tumorigenic potential in vivo, compared with subpopulations of low ALDH activity (ALDH(low) cells). Cell cultures treated with 5-fluoro-uracil and cisplatin exhibited higher numbers of ALDH(high) cells. Notch1 and Sonic hedgehog (Shh) expression was also found to increase in ALDH(high) cells compared with ALDH(low) cells. Therefore, it can be concluded that ALDH generates chemoresistance in gastric cancer cells through Notch1 and Shh signaling, suggesting novel treatment targets.

Aldehyde dehydrogenase activity in cancer stem cells from canine mammary carcinoma cell lines

Increasing evidence suggests that diverse solid tumours arise from a small population of cells known as cancer stem cells or tumour-initiating cells. Cancer stem cells in several solid tumours are enriched for aldehyde dehydrogenase (ALDH) activity. High levels of ALDH activity (ALDH(high)) were detected in four cell lines derived from canine mammary carcinomas. ALDH(high) cells were enriched in a CD44(+)CD24(-) population having self-renewal capacity. Xenotransplantation into immunodeficient mice demonstrated that 1×10(4) ALDH(high) cells were sufficient for tumour formation in all injected mice, whereas 1×10(4) ALDH(low) cells failed to initiate any tumours. ALDH(high)-derived tumours contained both ALDH(+) and ALDH(-) cells, indicating that these cells had cancer stem cell-like properties.

Abstract 5193: Wnt signaling in colitis derived colon cancer initiating cells promotes the tumorigenicity in colitis to cancer transition

Abstract Introduction: Wnt (Wingless) signaling in colon stem cells is involved in the development and maintenance of crypt architecture. In addition, activating mutations in the Wnt signaling pathway initiate and perpetuate colon cancer initiating cells (CCIC). Recently, we have isolated and propagated CCICs from colitis. Here, we hypothesize that Wnt signaling in the colitis derived CCICs potentiates colitis-associated tumor initiation. Methods: In order to test the above hypothesis, we have designed a dual fusion Wnt reporter construct (TTLG). The construct comprises of luminescent firefly luciferase and enhanced green fluorescent protein (eGFP), which are preceded by the TCF/LEF binding site. TTLG was transduced into colitis sphere isolates that were derived from Aldehyde dehydrogenase (ALDH) high cells. Immunohistochemistry validated the staining for activated β-Catenin in TTLG GFP high vs eGFP low colitis sphere isolates Tumorigenicity was evaluated by injecting eGFP high and eGFP low cells (1000 each) in the flanks of NOD-SCID mice. Secondary tumors were generated from 10% eGFP bright vs dimmest cells (1000, 100 and 10 each) obtained from eGFP high xenograft derived cells. In parallel, in vitro limiting dilution assays (LDA) were performed. Results: The eGFP high sphere cell population had nuclear localized Active Beta Catenin staining, indicating active Wnt signaling (95%). Conversely, little to no (<5%) Active Beta Catenin (ABC) staining was observed in the nucleus of eGFP low sphere cells. In vitro studies demonstrated that the clonogenic potential of eGFP high colitis sphere cells was significantly higher than that of eGFP low cells. This was further confirmed by in vivo studies, where the tumors generated from eGFP high cells grew more rapidly and larger when compared to eGFP low cells. The ability of the eGFP low cells to induce the tumor relates to the fact that eGFP low cells are a subset of the ALDH high population. Furthermore, secondary tumors that were generated from the 10% eGFP bright cells derived from eGFP high xenograft were initiated within few days after injection. At the time of harvest, the tumors formed from 10% eGFP dimmest cells were less than ¼ the size of the 10% eGFP bright cells derived tumor. The clonogenic potential of these 10% eGFP bright cells was about 50-100 fold higher than the 10% eGFP dimmest cells. Conclusions: The Wnt high ALDH high cells have higher clonogenic potential when compared to Wnt low ALDH high cells. Also, the tumors generated from Wnt high ALDH high colitis cells demonstrated decreased tumor latency and increased tumorigenicity compared to Wnt low ALDH high colitis cells. Collectively, our observations indicate that Wnt high CCICs from colitis promote colitis-associated tumorigenicity. 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 5193. doi:10.1158/1538-7445.AM2011-5193

Aldehyde dehydrogenase (ALDH) – a promising new candidate for use in preclinical and clinical selection of pluripotent very small embryonic-like stem cells (VSEL SCs) of high long-term repopulating hematopoietic potential

In the field of stem cells (SCs) biology there is great need for a universal marker that can effectively identify the rare populations of SCs in order to characterize and isolate them for research and therapeutic purposes. In line with this idea, the measurement of aldehyde dehydrogenase (ALDH) activity shows promising potential as a common marker for the identification of SCs. Recently discovered very small embryonic-like stem cells (VSEL SCs) have a pluripotent nature and high long-term repopulating hematopoietic potential. The aim of the present study was to determine whether VSEL SCs isolated from umbilical cord blood (CB) and different murine organs express high ALDH activity. To address this issue, 12 CB units were analyzed by employing flow cytometry to detect the following populations: (i) CXCR4(+)/Lin(-)/CD45(-)/ALDH(high), (ii) CD34(+)/Lin(-)/CD45(-)/ALDH(high) and (iii) CD133(+)/Lin(-)/CD45(-)/ALDH(high). Additionally, selected organs from 16 5-week-old female inbred C57BL/6J mice and 16 7-month-old female C57BL/6J mice were analyzed for detection of the following populations: (i) Sca-1(+)/Lin(-)/CD45(-) and (ii) Sca-1(+)/Lin(-)/CD45(+). All these populations were assessed for ALDH activity. We found that CB contains VSEL SCs with high ALDH activity. We also observed that ALDH(high) cells constitute a modest percentage of VSEL SCs present in selected murine organs. We demonstrated that CB and adult murine organs possess a subpopulation of ALDH(high) VSEL SCs. Above all, the observed high level of ALDH activity can be considered a functional marker of organ-derived pluripotent SCs and allows for simple, efficient isolation of cells with primitive features for their utility in targeted cell therapies.

Immunohistochemical staining of cancer stem cell markers in hepatocellular carcinoma

Background Cancer stem cells (CSCs) are thought to be a critical subpopulation in tumor development, progression, metastasis and recurrence, and the identification of these cells is an initial step in understanding their role in oncogenesis and in seeking valuable markers for diagnosis or development of targeting therapeutics. Aims To identify CSCs in hepatocellular carcinoma (HCC) specimens and define their tissue specificity. Methods Immunohistochemical staining of CSC markers: CD44, CD90, CD133 and aldehyde dehydrogenase (ALDH) was performed in 25 HCC specimens, 4 hepatoblastomas, 8 peri-malignant tissues, and 19 cases of viral hepatitis. Results The positivity of CD44 staining in HCC specimens was significantly lower than in viral hepatitis specimens. The positive rate of CD133 in HCC was similar to viral hepatitis specimens. CD133 + cells were largely localized to ALDH-positive cells in HCC as revealed by confocal microscopy. In contrast, the co-expression of both markers was visualized within vessels or in the portal areas in viral hepatitis. Moreover, among 7 liver specimens adjacent to HCC tissue, 3–6 samples were positive for CD44, CD90, CD133 and ALDH, especially in dysplastic cells. One of 4 hepatoblastoma cases was positive for all these markers; whereas, the other three specimens were negative for all these CSC markers. Conclusions In HCC and dysplastic tissues, clusters of CD133 +/ALDH high cells were identified. The use of cancer stem cell markers to screen tissues with chronic liver diseases provides limited guidance in the identification of malignant cells.

ALDH high adenoid cystic carcinoma cells display cancer stem cell properties and are responsible for mediating metastasis

The cancer stem cell (CSC) theory has been proposed to explain the tumor heterogeneity and carcinogenesis process. Recent studies indicate that aldehyde dehydrogenase (ALDH) activity represents a promising CSC marker. Here, we aimed to determine whether human adenoid cystic carcinoma (AdCC) also follows CSC model by exploring the CSC properties of AdCC cells expressing high level of ALDH activity. Utilizing in-vivo series transplantation assays, we found ALDH high AdCC cells were capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. Utilizing in-vitro assay, we found only ALDH high AdCC cells have tumorsphere-forming ability in anchorage-independent cultures. Finally, we showed ALDH high AdCC cells possess highly invasive capability and are responsible for mediating metastasis. These findings suggest the existence of a developmental hierarchy within human AdCC and further elucidation of the unique survival mechanism of AdCC derived CSC population may provide novel therapeutic strategies to treat AdCC.

898. Human Hematopoietic Repopulating Stem and Progenitor Cells Can Be Efficiently Selected Based on High Aldehyde Dehydrogenase Activity

Top of pageAbstract Human hematopoietic stem cells (HSC) are commonly purified by the expression of cell surface markers, such as CD34. Since cell phenotype can be altered by cell cycle progression or ex vivo culture, purification based on conserved stem cell function may represent a more reliable way to isolate stem cell populations. Human hematopoietic progenitor cells express increased levels of cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for the resistance of HSC to alkylating agents such as cyclophosphamide. We purified primitive HSC from human umbilical cord blood (UCB) by lineage depletion (Lin-) followed by the selection of cells with high ALDH activity using the fluorescent substrate Aldefluor™. Side scatter low, ALDH high (ALDHhi) cells comprised 22.6 ± 3.0% of the Lin- population (n=9), and represented 0.1 ± 0.01% of total MNC from UCB (n=5). In comparison to ALDHloLin- cells, the ALDHhiLin- population demonstrated significantly (p < 0.05) enriched expression of the pan-leukocyte marker CD45 (98.6 ± 0.7%), the primitive cell surface markers CD34 (91.0 ± 2.9%), CD133 (70.9 ± 4.2%), CD117 (83.1 ± 2.7%), and platelet-derived endothelial cell adhesion molecule (PECAM-1) or CD31 (97.9 ± 0.8%). ALDHhiLin- cells were also highly enriched for primitive HSC markers CD34 +CD38− (25.5 ± 3.7% vs 5.1 ± 1.3%, p < 0.005), and CD34 + CD133+ (72.7 ± 4.0 vs 12.0 ± 3.1, p < 0.00005), cell phenotypes associated with enhanced repopulating function in vivo. Human hematopoietic progenitor function was enriched in the ALDHhiLin- population (1 CFU in 4.4 cells, n=5), while the ALDHloLin- population had little in vitro colony forming ability at cell densities up to 5 × 104 cells. Multilineage human hematopoietic repopulation was consistently observed in the bone marrow, spleen and peripheral blood of immune deficient mice transplanted with ALDHhiLin- cells, whereas ALDHloLin- cells produced no detectable human engraftment. Direct comparison of repopulation using the NOD/SCID (n=37) and NOD/SCID beta-2 microglobulin (B2M) null (n=41) models demonstrated that 10-fold greater numbers of ALDHhiLin- cells were needed to engraft the NOD/SCID mouse as compared to the more permissive NOD/SCID B2M null mouse, suggesting that the ALDHhiLin- population contained committed progenitors as well as primitive SCID repopulating cells. Engrafting ALDHhiLin- cells in the mouse bone marrow differentiated into cells that express lineage markers for mature myeloid (CD33), mature B-lymphoid (CD19) and primitive repopulating (CD34 + CD38−) cells. We are currently evaluating the ALDHhiLin- population as a novel target for retroviral and lentiviral gene therapy applications. Cell fractionation based on lineage depletion and ALDH activity provides a reproducible method to purify human stem and progenitor cells based primarily on stem cell function rather than hematopoietic cell surface markers.