Stem Cell-associated Antigens Research Articles

Immunophenotypically Aberrant Stem Cells in Myelodysplastic Syndromes: A Biomarker for Leukemic Progression

Introduction: Myelodysplastic syndromes (MDS) are generally referred to as a spectrum of myeloid malignancies that are notorious for their heterogeneity. Subsequently, the question arises as to what is the best method to dissect the heterogeneity of these syndromes, which may range from a normal life expectancy and MDS-unrelated dead to rapid progression to acute myeloid leukemia (AML). Most effort is still being put into the development of morphological classification and prognostic models like the classification of the World Health Organization (WHO) and the revised International Prognostic Scoring System (IPSS-R). However, the need for a more refined classification based on new insights into MDS pathophysiology is emerging.Methods: Hitherto we propose a new approach to predict leukemic progression among MDS patients using hematopoietic stem cell immunoprofiles. In this study, leukemic stem cell associated antigens - CD7, CD11b, CD22, CD33, CD44, CD45RA, CD56, CD123, CD366, CD371 - were analyzed on CD34+CD38- hematopoietic stem cells (HSCs) derived of 130 patients and controls at time of diagnosis.Results: Aberrant expression of these antigens was found in 33% (24/72) and in 20% (1/5) of the MDS and chronic myelomonocytic leukemia (CMML) patients, respectively. Even in patients diagnosed with idiopathic cytopenia of undetermined significance (ICUS) based on inexplicable cytopenia but less than 10% dysplasia, expression of aberrant antigens was found in 50% (4/8). In contrast to MDS, CMML, and ICUS patients, aberrant antigen expression was absent on HSCs of healthy, age-matched controls (0/9) and pathological controls (0/36), i.e. patients with non-myeloid clonal disorders. In MDS patients, the presence of aberrant immunophenotypic HSCs (AIP-HSCs) at diagnosis was negatively associated with the leukemia free and overall survival time (Figure 1a-b, p<.001), and increased the likelihood of leukemic progression and death by 99.5% (HR=0.005, 95%CI: 0-10.0) and 87.2% (HR=0.128, 95%CI: 0.04-0.41). The robust performance of flow cytometric detection of AIP-HSCs as diagnostic test to identify MDS patients prone to AML was illustrated by a high sensitivity of 88% and specificity of 69% at a follow-up time of six months. Although a slide trend toward increased AIP-HSCs prevalence for higher risk categories, MDS patients with AIP-HSCs were found across almost all WHO 2016 and IPSS-R categories (Figure 2a-b). Interestingly, multivariate regression analyses, including AIP-HSC percentage as well as the IPSS-R variables cytogenetic abnormalities, bone marrow blasts, hemoglobin, absolute neutrophil count, and platelets, revealed AIP-HSC percentage to be the only determinant with a significant effect on leukemia free survival. In predicting overall survival, apart from AIP-HSCs, cytogenetic abnormalities rendered statistical significant value.Conclusion: Flow cytometric detection of AIP-HSCs can be used as a biomarker to predict which MDS patients will finally develop AML. As a result, the presence of AIP-HSCs may improve currently used classification models and substitute the 20% blast cut-off to discriminate between MDS and AML. Addition of molecular features will be needed to validate the malignant nature of these AIP-HSCs. In the near feature, we anticipate that flow cytometric detection of AIP-HSCs will not only contribute to the dissection of MDS heterogeneity and an increased understanding of MDS stem cell biology, but also to therapy decision making, disease monitoring, and the clinical outcome of MDS patients. [Display omitted] DisclosuresOssenkoppele:J&J: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Roche: Honoraria; Karyopharm: Consultancy, Research Funding; Novartis: Research Funding.

CD133 mRNA-Loaded Dendritic Cell Vaccination Abrogates Glioma Stem Cell Propagation in Humanized Glioblastoma Mouse Model

Cancer stem cells are initiating cells of cancer and propagate its growth through self-renewal and differentiation of its daughter cells. CD133 is a cell surface antigen that is present on glioma stem cells and has been used to prospectively isolate glioma stem cells. We hypothesized that a major histocompatibility complex (MHC)-independent and long-lasting immune response against CD133 could be generated by transfecting CD133 mRNA into dendritic cells and vaccinating animals with experimental gliomas. To test this hypothesis, we developed a novel humanized mouse model using CD34-positive hematopoietic stem cells. We confirmed the robust simultaneous activation of CD8- and CD4-positive T cells by dendritic cell vaccination with modified CD133 mRNA leading to a potent and long-lived immune response, with subsequent abrogation of CD133-positive glioma stem cell propagation and tumor growth. This study for the first time demonstrates in both a humanized mouse model and in a syngeneic mouse model of glioblastoma that targeting a glioma stem cell-associated antigen is an effective strategy to target and kill glioma stem cells. This novel and simple humanized mouse model for immunotherapy is a significant advance in our ability to test human-specific immunotherapies for glioblastoma.

Treatment of ovarian cancer by targeting the tumor stem cell-associated carbohydrate antigen, Sialyl-Thomsen-nouveau.

Recurrent ovarian cancer (OvCa) is thought to result in part from the inability to eliminate rare quiescent cancer stem cells (CSCs) that survive cytotoxic chemotherapy and drive tumor resurgence. The Sialyl-Thomsen-nouveau antigen (STn) is a carbohydrate moiety present on protein markers of CSCs in pancreatic, colon, and gastric malignancies. We have demonstrated that human OvCa cell lines contain varying levels of cells that independently express either STn or the ovarian CSC marker CD133. Here we determine co-expression of STn and CD133 in a subset of human OvCa cell lines. Analyses of colony and sphere forming capacity and of response to standard-of-care cytotoxic therapy suggest a subset of OvCa STn+ cells display some CSC features. The effect of the anti-STn antibody-drug conjugates (ADCs) S3F-CL-MMAE and 2G12-2B2-CL-MMAE on OvCa cell viability in vitro and in vivo was also assessed. Treatment with S3F-CL-MMAE reduced the viability of two of three OvCa cell lines in vitro and exposure to either S3F-CL-MMAE or 2G12-2B2-CL-MMAE reduced OVCAR3-derived xenograft volume in vivo, depleting STn+ tumor cells. In summary, STn+ cells demonstrate some stem-like properties and specific therapeutic targeting of STn in ovarian tumors may be an effective clinical strategy to eliminate both STn+ CSC and STn+ non-CSC populations.

Differentiation-Associated MicroRNA Alterations in Mouse Heart-Derived Sca-1(+)CD31(-) and Sca-1(+)CD31(+) Cells.

Cardiac resident stem/progenitor cells (CSC/CPCs) are critical to the cellular and functional integrity of the heart because they maintain myocardial cell homeostasis. Several populations of CSC/CPCs have been identified based on expression of different stem cell-associated antigens. Sca-1+ cells in the cardiac tissue may be the most common CSC/CPCs. However, they are a heterogeneous cell population and, in transplants, clinicians might transplant more endothelial cells, cardiomyocytes, or other cells than stem cells. The purposes of this study were to (1) isolate CSC/CPCs with Lin−CD45−Sca-1+CD31− and Lin−CD45−Sca-1+CD31+ surface antigens using flow-activated cell sorting; (2) investigate their differentiation potential; and (3) determine the molecular basis for differences in stemness characteristics between cell subtypes. The results indicated that mouse heart-derived Sca-1+CD31− cells were multipotent and retained the ability to differentiate into different cardiac cell lineages, but Sca-1+CD31+ cells did not. Integrated analysis of microRNA and mRNA expression indicated that 20 microRNAs and 49 mRNAs were inversely associated with Sca-1+CD31− and Sca-1+CD31+ subtype stemness characteristics. In particular, mmu-miR-322-5p had more targeted and inversely associated genes and transcription factors and might have higher potential for CSC/CPCs differentiation.

Multiparameter Flow Cytometry in Differential Diagnostics of Unclear Cytopenias, CLL-1 As an Additional Aberrant Marker on Myeloid Stem Cells

Flow cytometric analysis of the surface antigen profile of bone marrow (BM) cells has been shown to increase diagnostic sensitivity when combined with BM morphology and conventional cytogenetics (CG) and is currently recommended (Porwit et al Leukemia 2014). We wanted to test the differential diagnostic value of multiparameter flow cytometry (MFC) in a tertiary referral hospital with a wider array of colors to detect distinct aberrancies in antigen expression using simultaneously altogether 10 antibody conjugates.From I/2013 to VII/2015, BM samples from 88 pts with unclear cytopenia or suspected MDS were analyzed in parallel by cytomorphology (CM), CG and MFC. Anemia and/or thrombocytopenia were typical findings at presentation, median age 69 years (range: 29-89), M/F ratio 1.44:1. They were followed until end-VII/2015, for a median time of 15 months (1-30). The 35 MDS or CMML diagnoses were based on the WHO 2008 classification, distribution: 1 RA, 2 RT, 2 RARS, 10 RCMD, 2 RAEB-1, 5 RAEB-2, 2 MDS-U, 3 del(5q), 7 CMML-1 and 1 CMML-2. The two other groups: pts in whom MDS could not be confirmed during the follow-up (MDS suspicion) and those who were diagnosed as various non-clonal cytopenias.MFC was performed with 10-color flow in 4-5 tubes following ELN recommendations (Westers et al Leukemia 2012) in myeloid progenitor cells (myPC), granulocytes and monocytes. As a modification to the ELN protocol, the novel AML stem cell-associated antigen, C-type Lectin-like molecule-1 (CLL-1, also termed ClecA12, van Rhenen et al Blood 2007) together with CD38 was studied on myPCs. Our analysis included the robust four parameter flow cytometric score (FCM score) originally reported by Della Porta and ELN (Haematologica 2012). In their study, a FCM score ≥2 was significantly associated with MDS.The value of the recently published strategy to identify patients with a deletion 5q on the basis of MFC (Oelschlaegel et al Haematologica 2015) was also evaluated for patients recruited since I/2014 (n = 50). Parameters included in the 5q- score: 1) CD45 MFI ratio (Ly/myPC) ≤7.0 - score 10, 2) myPC >2% - score 3, 3) SSC-ratio (Gr/Ly) <6 -score 2, 4) CD71 on Gr ≤20% - score 1.5, 5) Sex, female - score 1.5.As MFC controls we had BM samples from six healthy subjects.Clinical, cytogenetic and flow cytometric characteristics of the study cohort:In myPCs, defined as CD34+, CD117+ cells, the most common aberrant lymphoid markers expressed were CD7, CD5 and CD56 (5, 4 and 2 of 35 MDS/CMML patients, respectively). Abnormal CLL-1 expression was seen in four of 35 MDS/CMML patients (in 3 pts it was expressed on CD38neg/dim myPCs and in one it was negative on all myPCs). In five patients, percentage of myPCs expressing CLL-1 was higher compared with normal controls: two of those pts had MDS, two non-clonal cytopenias and one was in the MDS suspicion group. A 5-parameter-del(5q)-score was studied in 50 patients, 20 with MDS/CMML and 30 with non-clonal cytopenia. Four patients had 5-parameter-del(5q)-score ≥15,0, all in the MDS group. Of those, two had deletion 5q, one t(3;3)(q21;q26) and monosomy 7, one had a complex karyotype.In MDS diagnostics, 10-color flow cytometry allows better characterization of small blast populations and analysis of a wider selection of antigens in fewer tubes compared with conventional 4-5 color flow. It also provides a rough scoring system to help to screen potential MDS patients among unclear cytopenias. We found CD7, CD5 and the novel AML stem cell-associated antigen CLL-1 to be the most commonly aberrantly expressed antigens on myPC in MDS/CMML patients studied. This study also confirmed the value of the Ogata score as a screening tool in differential diagnostics of unclear cytopenias. With the del(5q)-score, we were able to identify both two patients with deletion 5q in 50 patients evaluated so far. [Display omitted] DisclosuresEbeling:Celgene: Consultancy.

Dual Face of Immune Microenvironment in Myeloma

An important property of human multiple myeloma (MM) is the growth of tumor cells and their precursors in the bone marrow microenvironment. This specialized microenvironment includes a distinct component of immune cells that includes cells of innate as well as adaptive immune system. Growing body of evidence points to a central role for cross-talk between this immune microenvironment and tumor cells in regulating the growth of tumor cells in myeloma. These interactions play a dual role-with some interactions promoting tumor growth while others suppress growth of tumor cells. Cross talk between T and B cells has been implicated in normal B/plasma cell differentiation and may also play a role in pathogenesis of myeloma, explaining increased risk of plasma cell malignancies in certain settings such as Gaucher disease. The bone marrow of MM patients is often infiltrated with innate cells such as dendritic cells (DCs) and cross-talk between myeloid/plasmacytoid DCs or macrophages may promote growth and survival of MM cells. This cross-talk may also have fundamental implications for inducing genomic instability in tumor cells, such as via the induction of cytidine deaminases in tumor cells. Genomic instability is an early feature of human MM and many of the mutations found in MM tumor cells are also present in the precursor states. The immune system has the capacity to recognize these precursor lesions and even in the setting of clinical MM, the tumor bed contains T cells that can be recruited to kill tumor cells. Specific targets of tumor immunity may differ between preclinical and clinical malignancy. Immunity to certain stem cell-associated antigens are in particular associated with reduced risk of progression to clinical MM. With advanced disease, tumor infiltrating T cells encounter several immune inhibitory checkpoints which leads to suppression of function of tumor-infiltrating T cells. In addition to T cells, cells of the innate immune system such as natural killer and natural killer T cells may also be attractive targets of protective tumor immunity in MM. Immunologic approaches may also be combined with other strategies such as other immunomodulatory drugs (IMiDs) already in clinical use in MM. Recent advances in mouse modeling provide new opportunities to better understand how to harness the immune system against human MM. Such approaches have the advantage of the potential for durable control of tumor cells via immunologic memory. Such approaches are now entering the clinic as attractive targets to restore anti-tumor immunity, both in the context of therapy or prevention of MM. Disclosures Dhodapkar: Celgene: Research Funding. Off Label Use: checkpoint blockade drugs, imids.

Abstract 3886: Clec12a: A new AML stem cell-associated antigen

Abstract Introduction: Acute myeloid leukemias (AML), a heterogeneous and complex group of diseases, is a clonal malignant disorder derived from a small number of leukemic stem cells (LSCs), which are sustained by self-renewing and responsible for the propagation of leukemic blasts (LBs). Monoclonal antibodies have emerged as effective targeted therapies for the treatment of human malignancies and their mechanisms of action are able to deliver the therapeutic effects with minimal toxicity. Aim: The challenge is the identification of cell surface antigens which could be preferentially expressed on AML LSC compared with normal hematopoietics stem cells and that could be helpful to target therapies. Materials and methods: On 16 AML patients (pts) at diagnosis (peripheral blood of 12 pts and bone marrow of 4 pts) myeloblast leukemic cells were identified using a FACSCanto flow cytometer (Becton Dickinson), based on low expression of CD45 and low side scatter (SSC) properties (CD45 low/SSC low). On these subpopulation we used an antibody panel against cell surface antigens, for the detection of immature markers and potential leukemia-associated antigens: CD34, CD38, CD90, CLEC12A (C-type lectin domain family 12 member A), CD44, CD99, TIM-3 (T cell immunoglobulin mucin-3), CD32, CD133, CD74, CD47, CD58, CD25, CD22, CD96. Results:. The proportion of LBs was positively correlated with CD45 low/SSC low (median 51,8 %). This gated population of LBs were positive for CD34 (median 44,65%), CD38 (median 18,75%), CD90 (median 0,90%), CLEC12A (median 93,7%), CD44 (median 99,9%), CD99 (median 96,6%), TIM-3 (median 84,4%), CD32 (median 14%), CD123 (29,15%), CD133 (median (8,95%), CD58 (median 97,5%), CD47 (median 99,9%), CD74 (median 3,2%), CD25 (median 0,6%), CD96 (median 87%), CD22 (median 0,55%). Conclusion: The expression of CD34 and CD38 antigens is heterogeneous in LBs. In particular we found that the 50% of patients were CD34+ and 50% were CD38+. CLEC12A, CD44, CD99, TIM-3, CD58, CD47 and CD96 were highly expresses in LCSs. CD90, CD32, CD123, CD133,CD74, CD25 and CD22 were low. Interestingly we identified that the expression of CLEC12A distinguished two different populations: the CLEC12Ahigh cells correlated with the blast cells CD45low/SSClow; on the other hand, CLEC12Alow cells could be compared with CD45high/SSChigh population, representing normal hematopoietic cells. In conclusion, this marker is a good candidate to target therapies against leukemic stem cells. However further studies with an higher number of patients must be carried out to confirm that CLEC12A is an appropriate antigen for a new monoclonal antibody-based therapy. Acknowledgements: European LeukemiaNet, AIL, AIRC, PRIN 2010-2011, FP7 NGS-PTL project. Citation Format: Viviana Guadagnuolo, Enrica Imbrogno, Andrea Ghelli Luserna di Rorà, Antonella Padella, Giorgia Simonetti, Emanuela Ottaviani, Cristina Papayannidis, Ilaria Iacobucci, Anna Ferrari, Simona Soverini, Giovanni Martinelli. Clec12a: A new AML stem cell-associated antigen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2014-3886

Stage-specific embryonic antigen-4 identifies human dental pulp stem cells

Embryonic stem cell-associated antigens are expressed in a variety of adult stem cells as well as embryonic stem cells. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 can be used to isolate dental pulp (DP) stem cells. DP cells showed plastic adherence, specific surface antigen expression, and multipotent differentiation potential, similar to mesenchymal stem cells (MSC). SSEA-4+ cells were found in cultured DP cells in vitro as well as in DP tissue in vivo. Flow cytometric analysis demonstrated that 45.5% of the DP cells were SSEA-4+. When the DP cells were cultured in the presence of all-trans-retinoic acid, marked downregulation of SSEA-3 and SSEA-4 and the upregulation of SSEA-1 were observed. SSEA-4+ DP cells showed a greater telomere length and a higher growth rate compared to ungated and SSEA-4− cells. A clonal assay demonstrated that 65.5% of the SSEA-4+ DP cells had osteogenic potential, and the SSEA-4+ clonal DP cells showed multilineage differentiation potential toward osteoblasts, chondrocytes, and neurons in vitro. In addition, the SSEA-4+ DP cells had the capacity to form ectopic bone in vivo. Thus, our results suggest that SSEA-4 is a specific cell surface antigen that can be used to identify DP stem cells.

Expression of the hMICL in acute myeloid leukemia—a highly reliable disease marker at diagnosis and during follow‐up

Stable flow cytometric markers for malignant myeloid cells are highly warranted. Based on data from stem cell research, we hypothesized that the human inhibitory C-type lectin like receptor (hMICL) might represent a novel diagnostic and prognostic vehicle in a standard flow cytometry (FCM) setting. Standard four-color FCM was employed to uncover the expression patterns of hMICL in bone marrow in a test set of 55 retrospectively collected diagnostic acute myeloid leukemia (AML) samples and in a set of 36 prospectively collected diagnostic AML samples. Ninety-two percent of the AML patients stained positive for hMICL and in the otherwise poorly characterized CD34 negative patient group hMICL staining revealed a very homogenous expression profile in the blast cell compartment with a mean of 88% hMICL positive cells. Moreover, hMICL displayed significantly higher expression in AML as compared with normal donors as measured by median fluorescence intensity (MFI) ratios (P = 0.01). There was no difference in hMICL MFI ratios between the CD34 positive and the CD34 negative subgroups (P = 0.89). Importantly, there was no difference in MFI ratios between paired diagnostic and relapse samples (P = 0.76) in 23 cases studied, indicating stable expression of hMICL during the course of the disease. In contrast to the other stem cell associated antigens analyzed (CD34, CD96, CD117, and CD133), hMICL was expressed on myeloid blast cells only, revealing hMICL as a diagnostic marker in AML. These data identify hMICL as a myeloid leukemia-associated antigen and establishes its applicability for diagnosis and follow-up of AML patients in a standard FCM setting.

How to Overcome Myeloma Stem Cell Resistance to Therapy - Targeting the Stem Cell Niche

AbstractAbstract SCI-6The concept of cancer stem cells has attracted again intense research interest, as the drug resistance attributed to this infrequent subpopulation of tumor cells could explain how patients can relapse even after prolonged complete clinical, biochemical, radiologic or even molecular remissions. In multiple myeloma (MM), several aspects of the cancer stem cell concept remain to be elucidated, including the potential heterogeneity of this cell subpopulation or whether CD138+ expression is incompatible or not with a MM stem cell. As these questions are being resolved, emerging data highlight that the drug resistance of MM cells with clonogenic/stem cell-like features is heavily influenced by interactions with non-malignant accessory cells of the local microenvironment, including bone marrow stromal cells (BMSCs). Indeed, transcriptional signatures of “stemness”, as identified in normal stem cells or cancer stem cells from other neoplasias, are detected in the bulk population of MM cell lines and are upregulated after MM cell interaction with BMSCs. MM cell lines and primary tumor cells contain subpopulations with clonogenic potential, such as the side population (SP) cells. SP cells, detected by low intracellular accumulation of Hoechst 33342 dye (in contrast to the tumor's “main population” (MP), are considered an enriched source of tumor-initiating cells in diverse neoplasias and were detected among CD138− CD20+ CD27+ clonogenic cells in primary MM samples. Interaction with BMSCs increases the viability of SP cells and their percentage within the MM cell population. While interaction with BMSCs or other accessory cells of the microenvironmental niche suppresses the anti-MM activity of glucocorticoids, conventional chemotherapeutics and certain investigational agents, other agents (e.g. immunomodulatory thalidomide derivatives (IMIDs), such as lenalidomide) have increased activity against MM SP cells in the context of this tumor-microenvironment interaction. These observations suggest that MM cells with stem cell-like features exhibit functional plasticity depending on which specific microenvironmental niche they interact with. The Hedgehog, Wnt and Notch pathways, as well as regulators of chromatin remodeling, e.g. histone demethylases, have emerged as putative links between drug resistance, “cancer stemness” and how these functional outcomes are modulated by the local microenvironment in MM. These pathways, as well as embryonic stem cell-associated antigens (e.g. SOX2), represent intriguing targets for investigational therapies. However, clinical translation of such treatments has notable challenges, as conventional criteria for response assessment may not accurately reflect the treatment's impact on clonogenic tumor cells. Progression-free survival is considered a more appropriate endpoint for cancer stem-cell targeting agents, its assessment, however, may be confounded without concomitant treatment that suppresses the bulk of the tumor. Consequently, candidate cancer stem cell-targeting agents may have to be evaluated in combination with regimens (including lenalidomide-bortezomib-Dex (RVD) or other combinations built around the therapeutic “backbone” of proteasome inhibition and IMIDs) which potently target the bulk MM cell population and induce high rates of complete/near complete responses. Further improvements of this dual targeting of clonogenic and bulk tumor cells may be facilitated by recently developed high-throughput platforms (e.g. compartment-specific bioluminescence imaging, CS-BLI) which screen, in the presence vs. absence of stroma or other accessory cells, large numbers of anti-tumor agents and combinations thereof against the bulk tumor cell population or its clonogenic compartments. These new platforms will inform the rational design of regimens that will hopefully improve the long-term outcome of MM patients by suppressing a clonogenic/stem cell-like tumor compartment and circumventing microenvironment-dependent drug resistance. Disclosures:Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Immunohistochemical and Flow Cytometric Analysis of Long-Term Label-Retaining Cells in the Adult Heart

Cardiac-resident stem/progenitor cells have been identified based on expression of stem cell-associated antigens. However, no single surface marker allows to identify a definite cardiac stem/progenitor cell entity. Hence, functional stem cell markers have been extensively searched for. In homeostatic systems, stem cells divide infrequently and therefore retain DNA labels such as 5-bromo-2'-deoxyuridine, which are diluted with division. We used this method to analyze long-term label-retaining cells in the mouse heart after 14 days of 5-bromo-2'-deoxyuridine administration. Labeled cells were detected using immunohistochemical and flow-cytometric methods after varying chasing periods up to 12 months. Using mathematical models, the observed label dilution could consistently be described in the context of a 2-population model, whereby a population of rapidly dividing cells accounted for an accelerated early decline, and a population of slowly dividing cells accounted for decelerated dilution on longer time scales. Label-retaining cells were preferentially localized in the atria and apical region and stained negative for markers of the major cell lineages present in the heart. Most cells with long-term label-retention expressed stem cell antigen-1 (Sca-1). Sca-1(+)CD31(-) cells formed cell aggregates in culture, out of which lineage-negative (Lin(-))Sca-1(+)CD31(-) cells emerged, which could be cultured for many passages. These cells formed cardiospheres and showed differentiation potential into mesenchymal cell lineages. When cultured in cardiomyogenic differentiation medium, they expressed cardiac-specific genes. In conclusion, recognition of slow-cycling cells provides functional evidence of stem/progenitor cells in the heart. Lin(-)Sca-1(+)CD31(-) cardiac-derived progenitors have a potential for differentiation into cardiomyogenic and mesenchymal cell lineages.

Production and Characterization of Monoclonal Antibodies to Mouse Germ Cells

In mammals, primordial germ cells (PGCs) are generated in the extra-embryonic epiblast, and thereafter migrate into the developing gonads. Following the development of the gonads to the testes or ovaries, germ cells mature into sperms or eggs. In the present study, we report production and characterization of monoclonal antibodies (MAb) that recognize PGCs. Extracts from E12.5 mouse embryonic gonads were immunized as an antigen, and hybridomas were generated using the rat medial iliac lymph node method. The hybridoma supernatants were screened by immunohistochemical analyses of E12.5 mouse embryonic sections. The antibody, referred to herein as MAb 5B5, provided strong signals on PGCs. Moreover, immunofluorescence analyses using a variety of the tissue sections of mouse embryos revealed that MAb 5B5 also recognizes the ventricular zone of the cerebral cortex and the neural canal in the spinal cord in which neural specific stem cells are present in abundance. Based on these findings, MAb 5B5 might recognize stem cell-associated antigens.

Isolation of multipotent stem cells in human periodontal ligament using stage-specific embryonic antigen-4

The periodontal ligament (PDL) comprises adult stem cells, which are responsible for periodontal tissue regeneration. In the present study, we investigated the specific profile of the stem cells in the human PDL. Microscopic analysis demonstrated that PDL cells showed a fibroblastic appearance, forming flat and loose aggregates. PDL cells expressed embryonic stem cell-associated antigens (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT4, NANOG, SOX2, and REX1, and alkaline phosphatase activity), as well as conventional mesenchymal stem cell markers. When PDL cells were cultured in the presence of all- trans-retinoic acid, the numbers of SSEA-3+ and SSEA-4+ PDL cells were significantly decreased, while that of SSEA-1+ was increased. SSEA-4+ PDL cells showed a greater telomere length and growth rate. SSEA-4+ PDL cells exhibited the potential to generate specialized cells derived from three embryonic germ layers: mesodermal (adipocytes, osteoblasts, and chondrocytes), ectodermal (neurons), and endodermal (hepatocytes) lineages. Our findings demonstrated that SSEA-4, a major antigen to distinguish human embryonic stem cells, could also be used to identify multipotent stem cells in the PDL. Hence, SSEA-4+ human PDL cells appear to be a promising source of stem cells for regenerative medicine.

Ovine Periodontal Ligament Stem Cells: Isolation, Characterization, and Differentiation Potential

Periodontal disease leads to destruction of the connective tissues responsible for restraining teeth within the jaw. To date, various conventional therapies for periodontal regeneration have shown limited and variable clinical outcomes. Recent studies have suggested that newly identified human periodontal ligament stem cells (PDLSCs) may offer an alternate and more reliable strategy for the treatment of periodontal disease using a cell-based tissue engineering approach. In the present study, we generated enriched preparations of PDLSCs derived from ovine periodontal ligament using immunomagnetic bead selection, based on expression of the mesenchymal stem cell-associated antigen CD106 (vascular cell adhesion molecule 1). These CD106+ ovine PDLSCs demonstrated the capacity to form adherent clonogenic clusters of fibroblast-like cells when plated at low densities in vitro. Ex vivo-expanded ovine PDLSCs exhibited a high proliferation rate in vitro and expressed a phenotype (CD44+, CD166+, CBFA-1+, collagen-I+, bone sialoprotein+) consistent with human-derived PDLSCs. Furthermore, cultured ovine PDLSCs expressed high transcript levels of the ligament/tendon-specific early transcription factor scleraxis. Importantly, ex vivo-expanded ovine PDLSCs demonstrated the capacity to regenerate both cementum-like mineral and periodontal ligament when transplanted into NOD/SCID mice. The results from the present study suggest that ovine PDLSCs may potentially be used as a novel cellular therapy to facilitate successful and more predictable regeneration of periodontal tissue using an ovine preclinical model of periodontal disease as a prelude to human clinical studies.

Retroviral Mediated Transfer of MLL Fusion Genes into Human CD34+ Cord Blood Cells Supports the Establishment of Long-Term Myeloid Cultures with Growth Rates, Immunophenotypic and Transcriptional Features Distinct from Those of AML1-ETO Transduced Cultures.

Translocations involving the MLL gene, primarily t(9;11) and t(10;11) together with rearrangements affecting the core binding factor (CBF) genes, t(8;21) and inv(16) comprise the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). Although all of these rearrangements generate chimeric transcription factors (MLL-AF9, MLL-AF10, AML1-ETO and CBFβ-MYH11) clinicopathologic features and transcriptional profiles clearly distinguish MLL-rearranged from CBF-rearranged AML. To understand how these distinct subgroups of AML arise, we have developed a model for studying the effects of MLL and CBF fusion proteins on the growth, survival and differentiation human myeloid progenitors in vitro. Using retroviral mediated gene transfer, we transduced CD34 selected normal human cord blood (CB) cells with vector (MIEG3) alone or with vectors expressing MLL-AF9, MLL-AF10 or AML1-ETO fusion genes. Whereas CB transduced with MIEG3 proliferated in liquid culture for 6 to 8 weeks, MLL-AF9, MLL-AF10 and AML1-ETO transduced cells have continued to proliferate continuously in culture for more than 16 weeks without any sign of crisis. At any point after transduction, CB expressing MLL-AF9 or MLL-AF10 exhibited a faster rate of growth as compared with AML1-ETO or MIEG3 transduced CB. This difference in growth rate was associated with a reduced frequency of spontaneous apoptosis by annexin staining in the MLL cultures, as compared with the AML1-ETO cultures, but no difference in the fraction of cells in S-phase. MLL-AF9 and MLL-AF10 transduced CB also exhibited evidence of early myeloid maturation arrest based on morphology and surface antigen expression. However, while AML1-ETO transduced cells continue to express CD34 throughout their time in culture, MLL cultures lose expression of this stem cell-associated antigen and acquire expression of c-Kit and CD33, neither of which is expressed in AML1-ETO cultures. Also, unlike AML1-ETO transduced cells, CB transduced with with MLL fusions retain serial clonogenicity for 3 or more rounds of plating in methylcellulose assays. We used quantitative realtime RT-PCR to measure expression of 3 genes that are differentially expressed in patients with MLL or CBF gene fusions based on published microarray data. While expression of SPARC increased over time in MIEG3 cultures or remained stable in CB transduced with AML1-ETO, it decreased to nearly undetectable levels in MLL-AF9 transduced cultures. In contrast, expression of both BMI-1 and HOXA9 increased in the MLL-AF9 cultures and decreased in the MIEG3 and AML1-ETO cultures. The transcriptional changes in our long-term cultures mirror the gene expression differences that have been observed in AML associated with MLL or CBF fusions and suggest that this will be a useful model to study how chimeric transcription factors contribute to myeloid leukemogenesis. Interestingly, CB transduced with a mutated MLL-AF10 (MA10ΔLZ) lacking the leucine zipper domain required for transformation of primary murine myeloid progenitors did not differ, in terms of growth or differentation, from MIEG3 transduced cells. This suggests that the effects of MLL-AF9 and MLL-AF10 on normal CB may reflect early events in myeloid leukemogenesis. The in vivo leukemogenic potential of MLL fusion transduced CB is currently being evaluated in NOD/SCID mice.

Enrichment and transplantation of spermatogonial stem cells.

Spermatogenesis is a complex, highly organized process originated from stem cell spermatogonia. Because there are very few stem cells and they can only be defined by their function, the identification and isolation of these cells has been very difficult. By using a spermatogonial transplantation assay system, we have identified alpha6-and beta1-integrin expression on stem cells, and cells isolated with these antigens were significantly enriched in stem cells. This is the first demonstration of spermatogonial stem cell-associated antigens. Analysis of two infertile mouse models, Steel/SteelDickie (Sl/Sld) and experimental cryptorchidism, showed that the number of stem cells is reduced in Sl/Sld testis. Whereas cryptorchid testes are greatly enriched for stem cells, and one in 200 cells is a stem cell. These techniques will provide an important starting point for further purification and characterization of spermatogonial stem cells.

Beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells.

Although spermatogenesis is essential for reproduction, little is known about spermatogonial stem cells. These cells provide the basis for spermatogenesis throughout adult life by undergoing self-renewal and by providing progeny that differentiate into spermatozoa. A major impediment to our understanding of the biology of these stem cells is the inability to distinguish them from spermatogonia that are committed to differentiation. We made use of the known association of stem cells with basement membranes and our spermatogonial transplantation assay system to identify specific molecular markers on the stem cell surface. Selection of mouse testis cells with anti-beta1- or anti-alpha6-integrin antibody, but not anti-c-kit antibody, produced cell populations with a significantly enhanced ability to colonize recipient testes and generate donor cell-derived spermatogenesis. We demonstrate spermatogonial stem cell-associated antigens by using an assay system based on biological function. Furthermore, the presence of surface integrins on spermatogonial stem cells suggests that these cells share elements of a common molecular machinery with stem cells in other tissues.

Hematopoietic Defects in Mice Lacking the Sialomucin CD34

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34-null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34-null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.

Phenotypical characteristics of acute myelocytic leukemia associated with the t(8;21)(q22;q22) chromosomal abnormality: frequent expression of immature B-cell antigen CD19 together with stem cell antigen CD34

Twenty-three acute myelocytic leukemia (AML) patients with t(8;21) chromosomal abnormality, all classified as M2 (French-American-British [FAB] classification), were investigated. Blastic cells from all patients were positive for the stem cell-associated antigens, CD34 and HLA-DR, and the immature myeloid antigens, CD13 and CD33. The nonblastic leukemic cells expressed the more mature myeloid antigens, CD11b and CD15, with loss of the immature phenotype. The incidence of positivities for the stem cell-associated antigens, CD34 and HLA-DR, in t(8;21) AML cells was significantly higher in comparison with those in other AML showing granulocytic differentiation (M2 or M3). AML cells with t(8;21) also showed some phenotypic abnormalities. Frequent expression of CD19 was found in the blastic population of t(8;21) AML (18 of 23 cases) without other B-cell antigens and Ig gene rearrangements. CD19 expression was confirmed by immunocytochemistry and Northern blotting. The CD19+ blastic cells coexpressed both CD34 and HLA-DR. In addition, CD33+ cells among the blastic fraction in t(8;21) AML cells were fewer in number than in those of M2 or M3 AML without t(8;21). Our findings indicate that leukemic blasts of t(8;21) AML commonly express CD19 while preserving the stem cell-associated antigens, and differentiate into the granulocytic pathway with discordant maturation such as low CD33 expression.

Phenotypical Characteristics of Acute Myelocytic Leukemia Associated With the t(8;2l)(q22;q22) Chromosomal Abnormality: Frequent Expression of Immature B-Cell Antigen CD19 Together With Stem Cell Antigen CD34

Abstract Twenty-three acute myelocytic leukemia (AML) patients with t(8;21) chromosomal abnormality, all classified as M2 (French-American-British [FAB] classification), were investigated. Blastic cells from all patients were positive for the stem cell-associated antigens, CD34 and HLA-DR, and the immature myeloid antigens, CD13 and CD33. The nonblastic leukemic cells expressed the more mature myeloid antigens, CD11b and CD15, with loss of the immature phenotype. The incidence of positivities for the stem cell-associated antigens, CD34 and HLA-DR, in t(8;21) AML cells was significantly higher in comparison with those in other AML showing granulocytic differentiation (M2 or M3). AML cells with t(8;21) also showed some phenotypic abnormalities. Frequent expression of CD19 was found in the blastic population of t(8;21) AML (18 of 23 cases) without other B-cell antigens and Ig gene rearrangements. CD19 expression was confirmed by immunocytochemistry and Northern blotting. The CD19+ blastic cells coexpressed both CD34 and HLA-DR. In addition, CD33+ cells among the blastic fraction in t(8;21) AML cells were fewer in number than in those of M2 or M3 AML without t(8;21). Our findings indicate that leukemic blasts of t(8;21) AML commonly express CD19 while preserving the stem cell-associated antigens, and differentiate into the granulocytic pathway with discordant maturation such as low CD33 expression.