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Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology,gene therapy,and clinical transplantation. Here,we demonstrate efficient ex vivo expansion of HSCs measured by long-term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133-sorted cells using a soluble form of Delta1. After a 3-week culture on immobilized Delta1 supplemented with stem cell factor,thrombopoietin,Flt-3 ligand,interleukin (IL)-3,and IL-6/soluble IL-6 receptor chimeric protein (FP6) in a serum- and stromal cell-free condition,we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self-renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/gammac(null) mice,which showed myeloid,B,T,and natural killer cells as well as CD133(+)CD34(+) cells,and hematopoietic reconstitution in the secondary recipients. Interestingly,the CD133-sorted cells contained approximately 4.5 times more SRCs than the CD34-sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation. View Publication

Progranulin (pgrn; granulin-epithelin precursor,PC-cell-derived growth factor,or acrogranin) is a multifunctional secreted glycoprotein implicated in tumorigenesis,development,inflammation,and repair. It is highly expressed in macrophage and monocyte-derived dendritic cells. Here we investigate its regulation in myeloid cells. All-trans retinoic acid (ATRA) increased pgrn mRNA levels in myelomonocytic cells (CD34(+) progenitors; monoblastic U-937; monocytic THP-1; progranulocytic HL-60; macrophage RAW 264.7) but not in nonmyeloid cells tested. Interleukin-4 impaired basal expression of pgrn in U-937. Differentiation agents DMSO,and,in U-937 only,phorbol ester [phorbol 12-myristate,13-acetate (PMA)] elevated pgrn mRNA expression late in differentiation,suggestive of roles for pgrn in more mature terminally differentiated granulocyte/monocytes rather than during growth or differentiation. The response of pgrn mRNA to ATRA differs in U-937 and HL-60 lineages. In U-937,ATRA and chemical differentiation agents greatly increased pgrn mRNA stability,whereas,in HL-60,ATRA accelerated pgrn mRNA turnover. The initial upregulation of pgrn mRNA after stimulation with ATRA was independent of de novo protein synthesis in U-937 but not HL-60. Chemical blockade of nuclear factor-kappaB (NF-kappaB) activation impaired ATRA-stimulated pgrn expression in HL-60 but not U-937,whereas in U-937 it blocked PMA-induced pgrn mRNA expression,suggestive of cell-specific roles for NF-kappaB in determining pgrn mRNA levels. We propose that: 1) ATRA regulates pgrn mRNA levels in myelomonocytic cells; 2) ATRA acts in a cell-specific manner involving the differential control of mRNA stability and differential requirement for NF-kappaB signaling; and 3) elevated pgrn mRNA expression is characteristic of more mature cells and does not stimulate differentiation. View Publication

Tryptophan (Trp) catabolism mediated by indoleamine 2,3-dioxygenase (IDO) plays a central role in the regulation of T-cell-mediated immune responses. In this study,we also demonstrate that natural killer (NK)-cell function can be influenced by IDO. Indeed,l-kynurenine,a Trp-derived catabolite resulting from IDO activity,was found to prevent the cytokine-mediated up-regulation of the expression and function of specific triggering receptors responsible for the induction of NK-cell-mediated killing. The effect of l-kynurenine appears to be restricted to NKp46 and NKG2D,while it does not affect other surface receptors such as NKp30 or CD16. As a consequence,l-kynurenine-treated NK cells display impaired ability to kill target cells recognized via NKp46 and NKG2D. Instead,they maintain the ability to kill targets,such as dendritic cells (DCs),that are mainly recognized via the NKp30 receptor. The effect of l-kynurenine,which is effective at both the transcriptional and the protein level,can be reverted,since NK cells were found to recover their functional competence after washing. View Publication

The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However,investigators report studies of MSC using different methods of isolation and expansion,and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes,which hinders progress in the field. To begin to address this issue,the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First,MSC must be plastic-adherent when maintained in standard culture conditions. Second,MSC must express CD105,CD73 and CD90,and lack expression of CD45,CD34,CD14 or CD11b,CD79alpha or CD19 and HLA-DR surface molecules. Third,MSC must differentiate to osteoblasts,adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds,we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators. View Publication

Expansion of hematopoietic stem cells could be used clinically to shorten the prolonged aplastic phase after umbilical cord blood (UCB) transplantation. In this report,we investigated rapid severe combined immunodeficient (SCID) repopulating activity (rSRA) 2 weeks after transplantation of CD34(+) UCB cells cultured with serum on MS5 stromal cells and in serum- and stroma-free cultures. Various subpopulations obtained after culture were studied for rSRA. CD34(+) expansion cultures resulted in vast expansion of CD45(+) and CD34(+) cells. Independent of the culture method,only the CD34(+)33(+)38(-) fraction of the cultured cells contained rSRA. Subsequently,we subfractionated the CD34(+)38(-) fraction using stem cell markers CD45RA and CD90. In vitro differentiation cultures showed CD34(+) expansion in both CD45RA(-) and CD90(+) cultures,whereas little increase in CD34(+) cells was observed in both CD45RA(+) and CD90(-) cultures. By four-color flow cytometry,we could demonstrate that CD34(+)38(-)45RA(-) and CD34(+)38(-)90(+) cell populations were largely overlapping. Both populations were able to reconstitute SCID/nonobese diabetic mice at 2 weeks,indicating that these cells contained rSRA activity. In contrast,CD34(+)38(-)45RA(+) or CD34(+)38(-)90(-) cells contributed only marginally to rSRA. Similar results were obtained when cells were injected intrafemorally,suggesting that the lack of reconstitution was not due to homing defects. In conclusion,we show that after in vitro expansion,rSRA is mediated by CD34(+)38(-)90(+)45RA(-) cells. All other cell fractions have limited reconstitutive potential,mainly because the cells have lost stem cell activity rather than because of homing defects. These findings can be used clinically to assess the rSRA of cultured stem cells. View Publication

Human rhinovirus (HRV)-induced respiratory infections are associated with elevated levels of IFN-gamma-inducible protein 10 (IP-10),which is an enhancer of T lymphocyte chemotaxis and correlates with symptom severity and T lymphocyte number. Increased IP-10 expression is exhibited by airway epithelial cells following ex vivo HRV challenge and requires intracellular viral replication; however,there are conflicting reports regarding the necessity of type I IFN receptor ligation for IP-10 expression. Furthermore,the involvement of resident airway immune cells,predominantly bronchoalveolar macrophages,in contributing to HRV-stimulated IP-10 elaboration remains unclear. In this regard,our findings demonstrate that ex vivo exposure of human peripheral blood monocytes and bronchoalveolar macrophages (monocytic cells) to native or replication-defective HRV serotype 16 (HRV16) resulted in similarly robust levels of IP-10 release,which occurred in a time- and dose-dependent manner. Furthermore,HRV16 induced a significant increase in type I IFN (IFN-alpha) release and STAT1 phosphorylation in monocytes. Neutralization of the type I IFN receptor and inhibition of JAK or p38 kinase activity strongly attenuated HRV16-stimulated STAT1 phosphorylation and IP-10 release. Thus,this work supports a model,wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity. Our findings demonstrating robust activation of monocytic cells in response to native and/or replication-defective HRV16 challenge represent the first evidence indicating a mechanistic disparity in the activation of macrophages when compared with epithelial cells and suggest that macrophages likely contribute to cytokine elaboration following HRV challenge in vivo. View Publication

In previous reports,we have demonstrated that only direct cell-cell contact with stromal cells,such as the murine stromal cell line AFT024,was able to alter the cell division kinetics and self-renewing capacity of hematopoietic progenitor cells (HPC). Because beta(1)-integrins were shown to be crucial for the interaction of HPC with the bone marrow microenvironment,we have studied the role of beta(1)-integrins in the regulation of self-renewing cell divisions. For this purpose,we used primary human mesenchymal stromal (MS) cells as in vitro surrogate niche and monitored the division history and subsequent functional fate of individually plated CD34(+)133(+) cells in the absence or presence of an anti-beta(1)-integrin blocking antibody by time-lapse microscopy and subsequent long-term culture-initiating cell (LTC-IC) assays. beta(1)-Integrin-mediated contact with MS cells significantly increased the proportion of asymmetrically dividing cells and led to a substantial increase of LTC-IC. Provided that beta(1)-integrin-mediated contact was available within the first 72 hours,human MS cells were able to recruit HPC into cell cycle and accelerate their division kinetics without loss of stem cell function. Activation of beta(1)-integrins by ligands alone (e.g.,fibronectin and vascular cell adhesion molecule-1) was not sufficient to alter the cell division symmetry and promote self-renewal of HPC,thus indicating an indirect effect. These results have provided evidence that primary human MS cells are able to induce self-renewing divisions of HPC by a beta(1)-integrin-dependent mechanism. View Publication

Various growth factors are known to stimulate both early and late stages of human hematopoietic cell development in semisolid assay systems,but their role as microenvironmental regulators is poorly understood. To address this problem,we developed a novel coculture system in which highly purified primitive human hematopoietic cells were seeded onto an irradiated feeder layer of cells from a murine marrow-derived stromal cell line (M2-10B4) previously engineered by retroviral-mediated gene transfer to produce specific human factors. Effects on cells at very early,intermediate,and late stages of hematopoiesis were then evaluated by assessing the number of clonogenic cell precursors (long-term culture initiating cells [LTC-IC]),clonogenic cells,and mature granulocyte and macrophage progeny present in the cultures after 5 weeks. In the absence of any feeders,cells at all stages of hematopoiesis decreased to very low levels. In contrast,maintenance of LTC-IC was found to be supported by control murine stromal cells as effectively as by standard human marrow adherent layers. The presence of granulocyte colony-stimulating factor (G-CSF) and interleukin-3-producing M2-10B4 cells in combination was able to further enhance the maintenance and early differentiation of these cells without a decline in their proliferative potential as measured by the clonogenic output per LTC-IC. However,this effect was lost if granulocyte-macrophage CSF (GM-CSF)-producing feeders were also present. On the other hand,in the presence of GM-CSF-producing feeders,the output of mature granulocytes and macrophages increased 20-fold. These findings show that it is possible to selectively improve the maintenance of very primitive human hematopoietic cells in vitro or their output of mature progeny by appropriate manipulation of the long-term marrow culture system. Further exploitation of this approach should facilitate investigation of the mechanisms operative within the human marrow microenvironment in vivo and the design of protocols for in vitro manipulation of human marrow for future therapeutic applications. View Publication

Hematopoietic stem cells (HSCs) have the capacity to self-renew and the potential to differentiate into all of the mature blood cell types. The ability to prospectively identify and isolate HSCs has been the subject of extensive investigation since the first transplantation studies implying their existence almost 50 years ago. Despite significant advances in enrichment protocols,the continuous in vitro propagation of human HSCs has not yet been achieved. This chapter describes current procedures used to phenotypically and functionally characterize candidate human HSCs and initial efforts to derive permanent human HSC lines. View Publication

Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers,including myeloid malignancies. We investigated the effects of an oncogenic Kras(G12D) allele on phosphorylated signaling molecules in primary c-kit(+) lin(-/low) hematopoietic stem/progenitor cells. Comparison of wild-type and Kras(G12D) c-kit(+) lin(-/low) cells shows that K-Ras(G12D) expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5,ERK,and S6 under basal and stimulated conditions. Whereas Kras(G12D) cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor,we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell-targeted interventions. View Publication

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified,biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes,a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts,including G6b,G6f,LRRC32,LAT2,and the G protein-coupled receptor SUCNR1,encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins,and flow cytometric analysis confirmed the expression of G6b,G6f,and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b,G6f,and LRRC32 are restricted to the platelet lineage,whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest,because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists. View Publication

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friend's murine erythroleukemia (MEL) cells,addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance. To identify leukemia stem cells (LSC),we used the Culture Repopulating Ability (CRA) assay we developed to quantify in vitro stem cells capable of short-term reconstitution (STR). Hypoxia strongly inhibited the overall growth of MEL cell population,which,despite its clonality,comprised progenitors characterized by markedly different hypoxia-resistance. These included hypoxia-sensitive colony-forming cells and hypoxia-resistant STR-type LSC,capable of repopulating secondary liquid cultures of CRA assays,confirming what was previously shown for normal hematopoiesis. STR-type LSC were found capable not only of surviving in hypoxia but also of being mostly in cycle,in contrast with the fact that almost all hypoxia-surviving cells were growth-arrested and with what we previously found for HSC. However,quiescent LSC were also detected,capable of delayed culture repopulation with the same efficiency as STR-like LSC. The fact that even quiescent LSC,believed to sustain minimal residual disease in vivo,were found within the MEL cells indicates that all main components of leukemia cell populations may be present within clonal cell lines,which are therefore suitable to study the sensitivity of individual components to treatments. Disclosure of potential conflicts of interest is found at the end of this article. View Publication