技术资料

The signals that control the regenerative ability of hematopoietic stem cells (HSCs) in response to damage are unknown. Here,we demonstrate that downstream activation of the Hedgehog (Hh) signaling pathway induces cycling and expansion of primitive bone marrow hematopoietic cells under homeostatic conditions and during acute regeneration. However,this effect is at the expense of HSC function,because continued Hh activation during regeneration represses expression of specific cell cycle regulators,leading to HSC exhaustion. In vivo treatment with an inhibitor of the Hh pathway rescues these transcriptional and functional defects in HSCs. Our study establishes Hh signaling as a regulator of the HSC cell cycle machinery that balances hematopoietic homeostasis and regeneration in vivo. 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

Combined deletion of chromosomes 1p and 19q is associated with improved prognosis and responsiveness to therapy in patients with anaplastic oligodendroglioma. The deletions usually involve whole chromosome arms,suggesting a t(1;19)(q10;p10). Using stem cell medium,we cultured a few tumors. Paraffin-embedded tissue was obtained from 21 Mayo Clinic patients and 98 patients enrolled in 2 North Central Cancer Treatment Group (NCCTG) low-grade glioma trials. Interphase fusion of CEP1 and 19p12 probes detected the t(1;19). 1p/19q deletions were evaluated by fluorescence in situ hybridization. Upon culture,one oligodendroglioma contained an unbalanced 45,XX,t(1;19)(q10;p10). CEP1/19p12 fusion was observed in all metaphases and 74% of interphase nuclei. Among Mayo Clinic oligodendrogliomas,the prevalence of fusion was 81%. Among NCCTG patients,CEP1/19p12 fusion prevalence was 55%,47%,and 0% among the oligodendrogliomas,mixed oligoastrocytomas,and astrocytomas,respectively. Ninety-one percent of NCCTG gliomas with 1p/19q deletion and 12% without 1p/19q deletion had CEP1/19p12 fusion (P textless 0.001,chi(2) test). The median overall survival (OS) for all patients was 8.1 years without fusion and 11.9 years with fusion (P = 0.003). The median OS for patients with low-grade oligodendroglioma was 9.1 years without fusion and 13.0 years with fusion (P = 0.01). Similar significant median OS differences were observed for patients with combined 1p/19q deletions. The absence of alterations was associated with a significantly shorter OS for patients who received higher doses of radiotherapy. Our results strongly suggest that a t(1;19)(q10;p10) mediates the combined 1p/19q deletion in human gliomas. Like combined 1p/19q deletion,the 1;19 translocation is associated with superior OS and progression-free survival in low-grade glioma patients. View Publication

Drug resistance resulting from emergence of imatinib-resistant BCR-ABL point mutations is a significant problem in advanced-stage chronic myelogenous leukemia (CML). The BCR-ABL inhibitor,nilotinib (AMN107),is significantly more potent against BCR-ABL than imatinib,and is active against many imatinib-resistant BCR-ABL mutants. Phase 1/2 clinical trials show that nilotinib can induce remissions in patients who have previously failed imatinib,indicating that sequential therapy with these 2 agents has clinical value. However,simultaneous,rather than sequential,administration of 2 BCR-ABL kinase inhibitors is attractive for many reasons,including the theoretical possibility that this could reduce emergence of drug-resistant clones. Here,we show that exposure of a variety of BCR-ABL+ cell lines to imatinib and nilotinib results in additive or synergistic cytotoxicity,including testing of a large panel of cells expressing BCR-ABL point mutations causing resistance to imatinib in patients. Further,using a highly quantifiable bioluminescent in vivo model,drug combinations were at least additive in antileukemic activity,compared with each drug alone. These results suggest that despite binding to the same site in the same target kinase,the combination of imatinib and nilotinib is highly efficacious in these models,indicating that clinical testing of combinations of BCR-ABL kinase inhibitors is warranted. View Publication

Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice,thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely,a peripheral source of enzyme,established by transplanting ARSA-overexpressing hepatocytes from transgenic donors,failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified,enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall,our data provide a strong rationale for implementing HSPC gene therapy in MLD patients. View Publication

The nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase SIRT1 has been linked to fatty acid metabolism via suppression of peroxysome proliferator-activated receptor gamma (PPAR-gamma) and to inflammatory processes by deacetylating the transcription factor NF-kappaB. First,modulation of SIRT1 activity affects lipid accumulation in adipocytes,which has an impact on the etiology of a variety of human metabolic diseases such as obesity and insulin-resistant diabetes. Second,activation of SIRT1 suppresses inflammation via regulation of cytokine expression. Using high-throughput screening,the authors identified compounds with SIRT1 activating and inhibiting potential. The biological activity of these SIRT1-modulating compounds was confirmed in cell-based assays using mouse adipocytes,as well as human THP-1 monocytes. SIRT1 activators were found to be potent lipolytic agents,reducing the overall lipid content of fully differentiated NIH L1 adipocytes. In addition,the same compounds have anti-inflammatory properties,as became evident by the reduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). In contrast,a SIRT1 inhibitory compound showed a stimulatory activity on the differentiation of adipocytes,a feature often linked to insulin sensitization. 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

BACKGROUND: We have been interested in studying the roles of two aldehyde dehydrogenases in the biology of lung cancer. In this study,we seek to apply Aldefluor flow cytometry-based assay for the measurement of aldehyde dehydrogenase (ALDH) activity in lung cancer cell lines,which may become a new tool that will facilitate our continued research in this field. EXPERIMENTAL DESIGN: Several established lung cancer cell lines were used,including A549 cell line expressing siRNA against aldehyde dehydrogenase class-1A1 (ALDH1A1). Western blot analysis,spectrophotometry assay,and Aldefluor staining were used to measure protein or enzyme activity in these cell lines. For the purpose of measurement of ALDH activity by Aldefluor in cells with known high ALDH levels,cells were mixed 1:10 with immortalized lung epithelial cell line (Beas-2B),which is known to lack ALDH activity. To delineate dead cells,double staining using Aldefluor and propidium iodide (PI) was done. Double staining was also used to detect changes in ALDH activity in two different cell lines after treatment with 4-hydroperoxycyclophosphamide (4-HC). RESULTS: Our results show a very good correlation between Aldefluor,Western blot,and spectrophotometry assays. Mixing experiments with Beas-2B cells allowed accurate assessment of ALDH activity in A549 cells at baseline and after siRNA expression,thus establishing an approach that facilitates the measurement of very high ALDH using the Aldefluor assay. Aldefluor staining was able to detect heterogeneity in ALDH expression among as well as within the same cell lines and better assess viability after 4-HC treatment when combined with PI. CONCLUSIONS: Aldefluor assay can be adapted successfully to measure ALDH activity in lung cancer cells and may have the advantage of providing real time changes in ALDH activity in viable cells treated with siRNA or chemotherapy. View Publication

Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified,but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved,we performed Northern analyses on RNA extracted from human marrow LTC adherent layers,or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL-1 beta),IL-6,and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF,GM-CSF,and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast,transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC,and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures,respectively. Together,these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC,with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF,GM-CSF,and IL-6. Given the similarities between the LTC system and the marrow microenvironment,it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells. 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