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Despite many years of intensive effort,there is surprisingly little consensus on the most suitable markers with which to locate and isolate stem cells from adult tissues. By comparison,the study of cancer stem cells is still in its infancy; so,unsurprisingly,there is great uncertainty as to the identity of these cells. Stem cell markers can be broadly categorized into molecular determinants of self-renewal,clonogenicity,multipotentiality,adherence to the niche,and longevity. This review assesses the utility of recognizing cancer stem cells by virtue of high expression of aldehyde dehydrogenases (ALDHs),probably significant determinants of cell survival through their ability to detoxify many potentially cytotoxic molecules,and contributing to drug resistance. Antibodies are available against the ALDH enzyme family,but the vast majority of studies have used cell sorting techniques to enrich for cells expressing these enzymes. Live cells expressing high ALDH activity are usually identified by the ALDEFLUOR kit and sorted by fluorescence activated cell sorting (FACS). For many human tumours,but notably breast cancer,cell selection based upon ALDH activity appears to be a useful marker for enriching for cells with tumour-initiating activity (presumed cancer stem cells) in immunodeficient mice,and indeed the frequency of so-called ALDH(bri) cells in many tumours can be an independent prognostic indicator. View Publication

It is unknown how dendritic cells (DCs) become specialized as mucosal DCs and maintain intestinal homeostasis. We report that a subset of bone marrow cells freshly isolated from C57BL/6 mice express the retinoic acid (RA)-synthesizing enzyme aldehyde dehydrogenase family 1,subfamily A2 (ALDH1a2) and are capable of providing RA to DC precursors in the bone marrow microenvironment. RA induced bone marrow-derived DCs to express CCR9 and ALDH1a2 and conferred upon them mucosal DC functions,including induction of Foxp3(+) regulatory T cells,IgA-secreting B cells,and gut-homing molecules. This response of DCs to RA was dependent on a narrow time window and stringent dose effect. RA promoted bone marrow-derived DC production of bioactive TGF-β by inhibiting suppressor of cytokine signaling 3 expression and thereby enhancing STAT3 activation. These RA effects were evident in vivo,in that mucosal DCs from vitamin A-deficient mice had reduced mucosal DC function,namely failure to induce Foxp3(+) regulatory T cells. Furthermore,MyD88 signaling enhanced RA-educated DC ALDH1a2 expression and was required for optimal TGF-β production. These data indicate that RA plays a critical role in the generation of mucosal DCs from bone marrow and in their functional activity. View Publication

BACKGROUND: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming,which uses a defined set of transcription factors,iPS cells represent important sources of patient-specific cells for clinical applications. However,before these cells can be used in therapeutic designs,it is essential to understand their genetic stability. METHODOLOGY/PRINCIPAL FINDINGS: Here,we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation,iPS cells activate checkpoint signaling,evidenced by phosphorylation of ATM,NBS1,CHEK2,and TP53,localization of ATM to the double strand breaks (DSB),and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle,displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore,both cell types remove DSB within six hours of γ-irradiation,form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally,we report elevated expression of genes involved in DNA damage signaling,checkpoint function,and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage,dramatic changes in cell cycle structure,including a high percentage of cells in the S phase,increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest,were observed in stem cells generated by induced pluripotency. View Publication

Neuronal precursor cells (NPCs) are temporally regulated and have the ability to proliferate and differentiate into mature neurons,oligodendrocytes,and astrocytes in the presence of growth factors (GFs). In the present study,the role of the Jak pathway in brain development was investigated in NPCs derived from neurosphere cultures using Jak2 and Jak3 small interfering RNAs and specific inhibitors. Jak2 inhibition profoundly decreased NPC proliferation,preventing further differentiation into neurons and glial cells. However,Jak3 inhibition induced neuronal differentiation accompanied by neurite growth. This phenomenon was due to the Jak3 inhibition-mediated induction of neurogenin (Ngn)2 and NeuroD in NPCs. Jak3 inhibition induced NPCs to differentiate into scattered neurons and increased the expression of Tuj1,microtubule associated protein 2 (MAP2),Olig2,and neuroglial protein (NG)2,but decreased glial fibrillary acidic protein (GFAP) expression,with predominant neurogenesis/polydendrogenesis compared with astrogliogenesis. Therefore,Jak2 may be important for NPC proliferation and maintenance,whereas knocking-down of Jak3 signaling is essential for NPC differentiation into neurons and oligodendrocytes but does not lead to astrocyte differentiation. These results suggest that NPC proliferation and differentiation are differentially regulated by the Jak pathway. View Publication

Information on natural killer (NK)-cell receptor-ligand interactions involved in the response to human cytomegalovirus (HCMV) is limited and essentially based on the study of infected fibroblasts. Experimental conditions were set up to characterize the NK response to HCMV-infected myeloid dendritic cells (DCs). Monocyte-derived DCs (moDCs) infected by the TB40/E HCMV strain down-regulated the expression of human leukocyte antigen class I molecules and specifically activated autologous NK-cell populations. NKG2D ligands appeared virtually undetectable in infected moDCs,reflecting the efficiency of immune evasion mechanisms,and explained the lack of antagonistic effects of NKG2D-specific monoclonal antibody. By contrast,DNAM-1 and DNAM-1 ligands (DNAM-1L)-specific monoclonal antibodies inhibited the NK response at 48 hours after infection,although the impact of HCMV-dependent down-regulation of DNAM-1L in infected moDCs was perceived at later stages. moDCs constitutively expressed ligands for NKp46 and NKp30 natural cytotoxicity receptors,which were partially reduced on HCMV infection; yet,only NKp46 appeared involved in the NK response. In contrast to previous reports in fibroblasts,human leukocyte antigen-E expression was not preserved in HCMV-infected moDCs,which triggered CD94/NKG2A(+) NK-cell activation. The results provide an insight on key receptor-ligand interactions involved in the NK-cell response against HCMV-infected moDCs,stressing the importance of the dynamics of viral immune evasion mechanisms. View Publication

The resistance of glioma cells to a number of antitumor agents and the highly invasive nature of glioma cells that escape the primary tumor mass are key impediments to the eradication of tumors in glioma patients. In this study,we evaluated the therapeutic efficacy of a novel PI3-kinase/mTOR inhibitor,PI-103,in established glioma lines and primary CD133(+) glioma-initiating cells and explored the potential of combining PI-103 with stem cell-delivered secretable tumor necrosis factor apoptosis-inducing ligand (S-TRAIL) both in vitro and in orthotopic mouse models of gliomas. We show that PI-103 inhibits proliferation and invasion,causes G(0)-G(1) arrest in cell cycle,and results in significant attenuation of orthotopic tumor growth in vivo. Establishing cocultures of neural stem cells (NSC) and glioma cells,we show that PI-103 augments the response of glioma cells to stem cell-delivered S-TRAIL. Using bimodal optical imaging,we show that when different regimens of systemic PI-103 delivery are combined with NSC-derived S-TRAIL,a significant reduction in tumor volumes is observed compared with PI-103 treatment alone. To our knowledge,this is the first study that reveals the antitumor effect of PI-103 in intracranial gliomas. Our findings offer a preclinical rationale for application of mechanism-based systemically delivered antiproliferative agents and novel stem cell-based proapoptotic therapies to improve treatment of malignant gliomas. View Publication

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1,ALDH3A1,and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity,and that this activity is associated with ALDH1A1 expression. Isolated ALDH(+) lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH(-) counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH(+) cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH(+) lung cancer cells,commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together,these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential,that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis,and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH(+) component,implicating Notch signaling in lung cancer stem cell maintenance. View Publication

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7),the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions,and currently,there are no effective forms of therapy. Nevertheless,some advances in patient therapy are being made,and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized,gene-corrected,patient-specific cell transfer,we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly,human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis. View Publication

BACKGROUND: The radiation-induced bystander effect" (RIBE) was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR). RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC) are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However� View Publication

AIM Human embryonic stem cells (hESCs) represent a novel cell source to treat diseases such as heart failure and for use in drug screening. In this study,we aim to promote efficient generation of cardiomyocytes from hESCs by combining the current optimal techniques of controlled growth of undifferentiated cells and specific induction for cardiac differentiation. We also aim to examine whether these methods are scalable and whether the differentiated cells can be cryopreserved. METHODS & RESULTS hESCs were maintained without conditioned medium or feeders and were sequentially treated with activin A and bone morphogenetic protein-4 in a serum-free medium. This led to differentiation into cell populations containing high percentages of cardiomyocytes. The differentiated cells expressed appropriate cardiomyocyte markers and maintained contractility in culture,and the majority of the cells displayed working chamber (atrial and ventricular) type electrophysiological properties. In addition,the cell growth and differentiation process was adaptable to large culture formats. Moreover,the cardiomyocytes survived following cryopreservation,and viable cardiac grafts were detected after transplantation of cryopreserved cells into rat hearts following myocardial infarctions. CONCLUSION These results demonstrate that cardiomyocytes of high quality can be efficiently generated and cryopreserved using hESCs maintained in serum-free medium,a step forward towards the application of these cells to human clinical use or drug discovery. View Publication

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this,time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis,including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique,in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work,and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality,fast chromosomal analysis of human ES and iPS cells. View Publication

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation,overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities,which indicates the presence of other mechanisms for Evi-1 activation. In this study,we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL-mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population,in which Evi-1 particularly contributes to the propagation of MLL-ENL-immortalized cells. Furthermore,gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell-like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell-derived MLL leukemic cells. View Publication