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The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2),a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly,whereas normal HSCs were rapidly exhausted after serial transplantations,overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast,similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a genetic genomics" screen� View Publication

BACKGROUND: The dose-limiting toxicity of doxorubicin on hematopoietic stem cells reduces the maximum benefit from this powerful drug. Melatonin may play a role in reducing this toxicity. MATERIALS AND METHODS: Melatonin at 10 microM was used while challenging human peripheral blood stem cells (PBSC) with doxorubicin (0.6 microM and 1 microM),and colony formation was used to evaluate the protective effect of melatonin. RESULTS: Melatonin was protective for the myeloid and erythroid series when given during or 1 hour after,but not before,doxorubicin,as measured by colony assay. This protection was independent from its antioxidant function as measured by 2',7'-dichlodihydro-fluorescein diacetate and was selective for PBSC when compared to the MCF-7 cancer cell line. CONCLUSION: The results suggest the importance of the time sequence for melatonin administration to exert its protective effect in relation to doxorubicin treatment,as well as its protective effect on both erythroid and myeloid elements independent from its antioxidant function. View Publication

By retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs),we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here,we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction,human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development,it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary,a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells. View Publication

Interleukin-6 (IL-6) and its soluble receptor (sIL-6R) are major factors for maintenance and expansion of hematopoietic stem cells (HSCs). Sensitivity of HSCs to IL-6 has been previously studied,in part by measuring the expression of IL-6R on the membrane (mIL-6R). Several studies have described the regulation of cell surface expression of IL-6R by several cytokines,but the role of glycoprotein 130 activation has not yet been investigated. In this study,CD133(+) cells were purified from adult peripheral blood and were precultured in the absence or presence of 5-fluorouracil (5-FU) for selection of quiescent HSCs. Cells were cultured with continuous or pulsed stimulations of an IL-6-sIL-6R fusion protein (hyperinterleukin-6 [HIL-6]) to 1) detect mIL-6R by flow cytometry,2) assess mIL-6R and sIL-6R RNAs by reverse transcription-polymerase chain reaction,3) measure sIL-6R in supernatants by enzyme-linked immunosorbent assay,4) analyze cell-cycle status,and 5) perform long-term culture-initiating cell assays. The level of mIL-6R(-) cells was preserved by 5-FU incubation. HIL-6 increased steady-state mIL-6R RNA and expression rate on HSCs,independently of treatment with 5-FU. Enhanced production of sIL-6R was observed with short pulses of HIL-6 on CD133(+) 5-FU-pretreated cells. This overproduction of sIL-6R was abrogated by tumor necrosis factor-alpha protease inhibitor-1,an inhibitor of a disintegrin and metalloprotease proteases,suggesting the shedding of mIL-6R. This phenomenon was mediated through the phosphatidylinositol-3'-kinase pathway and was involved in the maintenance of primitive HSCs. In conclusion,expression and production of IL-6R are tightly regulated and stage specific. We assume that sIL-6R produced by shedding should be involved in autocrine and paracrine loops in the HSC microenvironment. View Publication

Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian,Botryllus schlosseri,vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues,gametes,or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent,self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates,but not to both,is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele,these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance. View Publication

Deletions and/or mutations of p53 are relatively rare and late events in the natural history of B-cell chronic lymphocytic leukemia (B-CLL). However,it is unknown whether p53 signaling is functional in B-CLL and if targeted nongenotoxic activation of the p53 pathway by using nutlin-3,a small molecule inhibitor of the p53/MDM2 interaction,is sufficient to kill B-CLL cells. In vitro treatment with nutlin-3 induced a significant cytotoxicity on primary CD19(+) B-CLL cells,but not on normal CD19(+) B lymphocytes,peripheral-blood mononuclear cells,or bone marrow hematopoietic progenitors. Among 29 B-CLL samples examined,only one was resistant to nutlin-3-mediated cytotoxicity. The induction of p53 by nutlin-3 in B-CLL samples was accompanied by alterations of the mitochondrial potential and activation of the caspase-dependent apoptotic pathway. Among several genes related to the p53 pathway,nutlin-3 up-regulated the steady-state mRNA levels of PCNA,CDKN1A/p21,GDF15,TNFRSF10B/TRAIL-R2,TP53I3/PIG3,and GADD45. This profile of gene activation showed a partial overlapping with that induced by the genotoxic drug fludarabine. Moreover,nutlin-3 synergized with both fludarabine and chlorambucil in inducing B-CLL apoptosis. Our data strongly suggest that nutlin-3 should be further investigated for clinical applications in the treatment of B-CLL. View Publication

Niemann-Pick type C2 (NPC2) protein has been characterized as a cholesterol-binding protein. Its loss leads to NPC2 disease,an inherited neurodegenerative disorder. When analyzing gene expression profile,we noticed high expression of both NPC2 and its receptor,mannose 6-phosphate receptor (MPR),in murine hematopoietic stem cells. NPC2 protein,in the presence of thrombopoietin (TPO),causes an increase in CFU-GEMM (colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte) and a decrease in CFU-GM (colony-forming unit-granulocyte-macrophage) colony number in colony-forming cell (CFC) assays. This effect is independent of cholesterol binding but does require the presence of MPR. With M07e cells,a TPO-dependent hematopoietic leukemia cell line,NPC2 can inhibit TPO-induced differentiation and enhance TPO-mediated anti-apoptosis effects. Strikingly,these results are not observed under the standard 20% O(2) level of the standard incubator,but rather at 7% O(2),the physiological oxygen level of bone marrow. Furthermore,NPC2 protein upregulates hypoxia inducible factor 1-alpha protein level at 7% O(2),but not at 20% O(2). Our results demonstrate that NPC2 protein plays a role in hematopoiesis at the physiologic bone marrow level of O(2). View Publication

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step,we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells,and then,expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123(low)HLA-DR- phenotype,expressed myeloperoxidase,and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules,CD1a,CD11c,CD80,CD86,DC-SIGN,and CD40; and were capable of Ag processing,triggering naive T cells in MLR,and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-alpha coexpressed a high level of CD14,and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition,because ES cells can be expanded without limit,they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance. View Publication

Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that,unlike steady-state erythropoiesis,erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM),severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In short-term transplantation experiments,donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context,stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system,EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion,and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x),in contrast,was not significantly induced via WT-EpoR,EpoR-HM,or EpoR-H alleles. In Kit+ CD71+ erythroblasts,EpoR-PY343 signals furthermore enhanced SCF growth effects,and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit- CD71+ erythroblasts,oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis,therefore,requires stage-specific EpoR-PY343-Stat5 signals,some of which selectively bolster SCF and oncostatin-M action. View Publication

BACKGROUND Hematopoietic stem cells (HSC) have traditionally been frozen using the cryoprotectant DMSO in dextran-40,saline or albumin. However,the process of freezing and thawing results in loss of HSC numbers and/or function. METHODS This study investigated the use of CryoStor for the freezing of HSC from cord blood (CB). CB donations (n = 30) were collected under an Institutional Ethics Committee-approved protocol,volume reduced and frozen using three different methods of cryoprotection. Aliquots were frozen with either 10% DMSO in dextran-40,10% DMSO in CryoStor or 5% DMSO in CryoStor. Prior to freezing samples were separated for nucleated cell (NC) and CD34+ counts and assessment of CD34+ viability. Aliquots were frozen and kept in vapor phase nitrogen for a minimum of 72 h. Vials were rapidly thawed at 37 degrees C and tested for NC and CD34+ counts and CD34+ viability and colony-forming unit (CFU) assay. RESULTS Cells frozen with CryoStor in 10% DMSO had significantly improved NC (P < 0.001),CD34+ recovery,viable CD34+ (P < 0.001) and CFU numbers (P < 0.001) compared with dextran in 10% DMSO. CryoStor in 5% DMSO resulted in significantly improved NC (P < 0.001) and CFU (P < 0.001). DISCUSSION These results suggest that improved HSC recovery,viability and functionality can be obtained using CryoStor with 10% DMSO and that similar if not better numbers can be obtained with 5% DMSO compared with dextran-40 with 10% DMSO. View Publication

Targeted cancer therapies exploit the continued dependence of cancer cells on oncogenic mutations. Such agents can have remarkable activity against some cancers,although antitumor responses are often heterogeneous,and resistance remains a clinical problem. To gain insight into factors that influence the action of a prototypical targeted drug,we studied the action of imatinib (STI-571,Gleevec) against murine cells and leukemias expressing BCR-ABL,an imatinib target and the initiating oncogene for human chronic myelogenous leukemia (CML). We show that the tumor suppressor p53 is selectively activated by imatinib in BCR-ABL-expressing cells as a result of BCR-ABL kinase inhibition. Inactivation of p53,which can accompany disease progression in human CML,impedes the response to imatinib in vitro and in vivo without preventing BCR-ABL kinase inhibition. Concordantly,p53 mutations are associated with progression to imatinib resistance in some human CMLs. Our results identify p53 as a determinant of the response to oncogene inhibition and suggest one way in which resistance to targeted therapy can emerge during the course of tumor evolution. View Publication

A JAK2(V617F) mutation is frequently found in several BCR/ABL-negative myeloproliferative disorders. To address the contribution of this mutant to the pathogenesis of these different myeloproliferative disorders,we used an adoptive transfer of marrow cells transduced with a retrovirus expressing JAK2(V617F) in recipient irradiated mice. Hosts were analyzed during the 6 months after transplantation. For a period of 3 months,mice developed polycythemia,macrocytosis and usually peripheral blood granulocytosis. Transient thrombocytosis was only observed in a low-expresser group. All mice displayed trilineage hyperplasia in marrow and spleen along with an amplification of myeloid and erythroid progenitor cells and a formation of endogenous erythroid colonies. After 3 to 4 months,polycythemia regressed,abnormally shaped red blood cells and platelets were seen in circulation,and a deposition of reticulin fibers was observed in marrow and spleen. Development of fibrosis was associated with anemia,thrombocytopenia,high neutrophilia,and massive splenomegaly. These features mimic human polycythemia vera and its evolution toward myelofibrosis. This work demonstrates that JAK2(V617F) is sufficient for polycythemia and fibrosis development and offers an in vivo model to assess novel therapeutic approaches for JAK2(V617F)-positive pathologies. Questions remain regarding the exact contribution of JAK2(V617F) in other myeloproliferative disorders. View Publication