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Here a new,intrinsically pluripotent,CD45-negative population from human cord blood,termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 10(15) cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts,chondroblasts,adipocytes,and hematopoietic and neural cells including astrocytes and neurons that express neurofilament,sodium channel protein,and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model,the preimmune fetal sheep,resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals. View Publication

During ontogenesis and the entire adult life hematopoietic stem and progenitor cells have the capability to migrate. In comparison to the process of peripheral leukocyte migration in inflammatory responses,the molecular and cellular mechanisms governing the migration of these cells remain poorly understood. A common feature of migrating cells is that they need to become polarized before they migrate. Here we have investigated the issue of cell polarity of hematopoietic stem/progenitor cells in detail. We found that human CD34(+) hematopoietic cells (1) acquire a polarized cell shape upon cultivation,with the formation of a leading edge at the front pole and a uropod at the rear pole; (2) exhibit an amoeboid movement,which is similar to the one described for migrating peripheral leukocytes; and (3) redistribute several lipid raft markers including cholesterol-binding protein prominin-1 (CD133) in specialized plasma membrane domains. Furthermore,polarization of CD34(+) cells is stimulated by early acting cytokines and requires the activity of phosphoinositol-3-kinase as previously reported for peripheral leukocyte polarization. Together,our data reveal a strong correlation between polarization and migration of peripheral leukocytes and hematopoietic stem/progenitor cells and suggest that they are governed by similar mechanisms. View Publication

Human hematopoietic stem cells (HSCs) are commonly purified by the expression of cell surface markers such as CD34. Because cell phenotype can be altered by cell cycle progression or ex vivo culture,purification on the basis of conserved stem cell function may represent a more reliable way to isolate various stem cell populations. We have purified primitive HSCs from human umbilical cord blood (UCB) by lineage depletion (Lin(-)) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. ALDH(hi)Lin(-) cells contained 22.6% +/- 3.0% of the Lin(-) population and highly coexpressed primitive HSC phenotypes (CD34(+) CD38(-) and CD34(+)CD133(+)). In vitro hematopoietic progenitor function was enriched in the ALDH(hi)Lin(-) population,compared with ALDH(lo)Lin(-) cells. Multilineage human hematopoietic repopulation was observed exclusively after transplantation of ALDH(hi)Lin(-) cells. Direct comparison of repopulation with use of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID beta2 microglobulin (beta2M) null models demonstrated that 10-fold greater numbers of ALDH(hi)-Lin(-) cells were needed to engraft the NOD/SCID mouse as compared with the more permissive NOD/SCID beta2M null mouse,suggesting that the ALDH(hi)Lin(-) population contained committed progenitors as well as primitive repopulating cells. Cell fractionation according to lineage depletion and ALDH activity provides a viable and prospective purification of HSCs on the basis of cell function rather than cell surface phenotype. View Publication

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors,useful for both basic research and clinical applications. Besides their characterization in colony assays,protocols exist for the cultivation of lymphoid,myeloid,and erythroid cells. With the possible exception of mast cells,however,long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here,we describe for the first time an efficient yet easy strategy to generate mass cultures of pure,immature erythroid progenitors from mouse ES cells (ES-EPs),using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived,adult,definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions,ES-EPs differentiated into mature,enucleated erythrocytes. Importantly,ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition,the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells. View Publication

Hematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However,the mechanisms involved in controlling the number and abilities of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in coculture with CD34+ cells and human stromal cells. Ihh mRNA was expressed in primary and telomerized human (hTERT) stromal cells,and its receptor molecules were detected in CD34+ cells. Ihh gene transfer into hTERT stromal cells enhanced their hematopoietic supporting potential,which was elevated compared with control stromal cells,as indicated by the colony-forming units in culture (CFU-Cs) (26-fold +/- 2-fold versus 59-fold +/- 3-fold of the initial cell number; mixed colony-forming units [CFU-Mix's],63-fold +/- 37-fold versus 349-fold +/- 116-fold). Engraftments of nonobese diabetic/severe combined immunodeficiency-beta2m-/- (NOD/SCID-beta2-/-) repopulating cells (RCs) expanded on Ihh stromal cells were significantly higher compared with control coculture results,and engraftment was neutralized by addition of an antihedgehog antibody. Limiting dilution analysis indicated that NOD/SCID-beta2m-/- RCs proliferated efficiently on Ihh stromal cells,compared with control stromal cells. These results indicate that Ihh gene transfer could enhance the primitive hematopoietic support ability of human stromal cells. View Publication

Primitive hematopoietic cells from several species are known to efflux both Hoechst 33342 and Rhodamine-123. We now show that murine hematopoietic stem cells (HSCs) defined by long-term multilineage repopulation assays efflux both dyes variably according to their developmental or activation status. In day 14.5 murine fetal liver,very few HSCs efflux Hoechst 33342 efficiently,and they are thus not detected as side population" (SP) cells. HSCs in mouse fetal liver also fail to efflux Rhodamine-123. Both of these features are retained by most of the HSCs present until 4 weeks after birth but are reversed by 8 weeks of age or after a new HSC population is regenerated in adult mice that receive transplants with murine fetal liver cells. Activation of adult HSCs in vivo following 5-fluorouracil treatment� View Publication

The development of immunodeficient mouse xenograft models has greatly facilitated the investigation of some human hematopoietic malignancies,but application of this approach to the myelodysplastic syndromes (MDSs) has proven difficult. We now show that cells from most MDS patients (including all subtypes) repopulate nonobese diabetic-severe combined immunodeficient (scid)/scid-beta2 microglobulin null (NOD/SCID-beta2m(-/-)) mice at least transiently and produce abnormal differentiation patterns in this model. Normal marrow transplants initially produce predominantly erythroid cells and later predominantly B-lymphoid cells in these mice,whereas most MDS samples produced predominantly granulopoietic cells. In 4 of 4 MDS cases,the regenerated cells showed the same clonal markers (trisomy 8,n = 3; and 5q-,n = 1) as the original sample and,in one instance,regenerated trisomy 8(+) B-lymphoid as well as myeloid cells were identified. Interestingly,the enhanced growth of normal marrow obtained in NOD/SCID-beta2m(-/-) mice engineered to produce human interleukin-3,granulocyte-macrophage colony-stimulating factor,and Steel factor was seen only with 1 of 7 MDS samples. These findings support the concept that human MDS originates in a transplantable multilineage hematopoietic stem cell whose genetic alteration may affect patterns of differentiation and responsiveness to hematopoietic growth factors. They also demonstrate the potential of this new murine xenotransplant model for future investigations of MDS. View Publication

Interleukin-6 (IL-6) is a critical factor in the regulation of stromal function and hematopoiesis. In vivo bromodeoxyuridine incorporation analysis indicates that the percentage of Lin(-)Sca-1(+) hematopoietic progenitors undergoing DNA synthesis is diminished in IL-6-deficient (IL-6(-/-)) bone marrow (BM) compared with wild-type BM. Reduced proliferation of IL-6(-/-) BM progenitors is also observed in IL-6(-/-) long-term BM cultures,which show defective hematopoietic support as measured by production of total cells,granulocyte macrophage-colony-forming units (CFU-GMs),and erythroid burst-forming units (BFU-Es). Seeding experiments of wild-type and IL-6(-/-) BM cells on irradiated wild-type or IL-6-deficient stroma indicate that the hematopoietic defect can be attributed to the stromal and not to the hematopoietic component. In IL-6(-/-) BM,stromal mesenchymal precursors,fibroblast CFUs (CFU-Fs),and stroma-initiating cells (SICs) are reduced to almost 50% of the wild-type BM value. Moreover,IL-6(-/-) stromata show increased CD34 and CD49e expression and reduced expression of the membrane antigens vascular cell adhesion molecule-1 (VCAM-1),Sca-1,CD49f,and Thy1. These data strongly suggest that IL-6 is an in vivo growth factor for mesenchymal precursors,which are in part implicated in the reduced longevity of the long-term repopulating stem cell compartment of IL-6(-/-) mice. View Publication

OBJECTIVE: Ionizing radiation (IR) and busulfan (BU) are commonly used as preconditioning regimens for bone marrow transplantation (BMT). We examined whether induction of apoptosis in murine bone marrow (BM) hematopoietic cells contributes to IR- and BU-induced suppression of their hematopoietic function. METHODS: The hematopoietic functions of hematopoietic stem cells (HSCs) and progenitors were analyzed by the cobblestone area-forming cell (CAFC) assay. Apoptosis was determined by measuring 3,3'-dihexyloxacarbocyanine iodide (DiCO6) uptake,annexin V staining,and/or sub-G(0/1) cells. Four cell types were studied: murine BM mononuclear cells (BM-MNCs),linage-negative hematopoietic cells (Lin-) cells),Lin- Scal+ c-kit+ cells,and Lin- Scal- c-kit+ cells by flow cytometry. RESULTS: Exposure of BM-MNCs to IR (4 Gy) or incubation of the cells with BU (30 microM) resulted in a significant reduction in CAFC frequency (ptextless0.001). The survival fractions of various day-types of CAFC for the irradiated cells were less than 10%,while that for BU-treated cells was 71.3% on day 7 and progressively declined to 5.3% on day 35. Interestingly,IR significantly induced apoptosis in BM-MNCs,Lin- cells,HSCs,and progenitors,whereas BU failed to increase apoptosis in these cells. In addition,preincubation of BM-MNCs with z-Val-Ala-Asp (OCH3)-fluoromethylketone,methyl ester (z-VAD) attenuated IR-induced reduction in CAFC but not that induced by BU. CONCLUSION: IR and BU differentially suppress the hematopoietic function of HSCs and progenitors by fundamentally different mechanisms. IR inhibits the function primarily by the induction of HSC and progenitor apoptosis. In contrast,BU suppresses HSC and progenitor function via an apoptosis-independent mechanism. View Publication

We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental. View Publication

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood,thymus,and spleen of IFN-beta-/- mice,activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production,relative to IFN-beta+/+ mice. Notably,constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages,respectively,of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice,associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1,IgM,and CD23 expression. Circulating IgM-,Mac-1-,and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice,shown by the reduction of colony-forming units,granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether,our data suggest that,in addition to the direct growth-inhibitory effects on tumor cells,IFN-beta is required during different stages of maturation in the development of the immune system. View Publication

Imatinib is a novel tyrosine kinase inhibitor used for the treatment of Philadelphia chromosome-positive leukemias and other malignancies. Side effects are mostly moderate; however,a dose-dependent hematologic toxicity affecting all hematopoietic lineages is observed clinically. The aim of this study was to investigate the effect of imatinib on normal hematopoietic stem and progenitor cells in vitro. A dose-dependent decrease in proliferation potential was found when CD34+ cells were expanded in serum-free medium supplemented with 6 growth factors and imatinib. Functionally,a decrease in colony-forming capacity was observed under increasing doses of imatinib. However,no such effect on more primitive cobblestone area-forming cells was detectable. Both withdrawal of stem cell factor from our expansion cultures or functional inhibition of c-kit led to a similar degree of inhibition of expansion,whereas the effect of imatinib was substantially greater at all dose levels tested. These data suggest a significant inhibitory effect of imatinib on normal CD34+ progenitor (but not stem) cells that is largely independent of c-kit signaling. View Publication