参考文献

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates,specifically,hematopoietic,mesodermal,and neurectodermal. In this study,we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore,we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells,expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo,and potentially for understanding and treating neurodegenerative and psychiatric diseases. View Publication

OBJECTIVE: +HOX genes are expressed in the hematopoietic system and increasing data point to their involvement in the control of proliferation and/or differentiation. Genes belonging to the C cluster are preferentially expressed in developing and differentiated lymphoid lineages. However,recent studies demonstrated,by RT-PCR,that the HOXC4 gene is also actively transcribed in the most undifferentiated hematopoietic cells (CD34(+)38(low)) and in more mature myeloid and erythroid progenitors. We evaluated the expression of HOXC4 protein on human CD34(+) cells and the in vitro effect of its overexpression on proliferation and differentiation. MATERIALS AND METHODS: We assessed the expression of HOXC4 on human CD34(+) cells using a polyclonal antibody raised against the C-terminal portion of the protein expressed using the baculovirus system. Overexpression of HOXC4 in human CD34(+) cells was obtained by retroviral gene transfer; its effect on clonogenic (CFU-GM,BFU-E,and CFU-GEMM) and early progenitors (LTC-IC) was evaluated. RESULTS: The HOXC4 protein is indeed expressed in human CD34(+) cells,and its overexpression in human CD34(+) cells increases the proliferation potential of clonogenic and early progenitors. CFU-GM showed a median threefold expansion (range: 1.1-19.4; p textless 0.002) compared with control transduced with the vector alone. The increment of BFU-E was higher (median ninefold,range 2.5-35; p textless 0. 0009) and erythroid colonies presented a larger size with normal morphology. An even more marked effect was observed on LTC-IC (median 13,onefold; range 4.1-102.1,p textless 0.0001). CONCLUSION: We demonstrate that HOXC4 is expressed in CD34(+) cells and that its overexpression induces an in vitro expansion of committed as well as very early hematopoietic progenitors. The most striking effect was obtained on LTC-IC with an expansion of 13.1-fold. The enforced expression of HOXC4 induced a significant increase (p textless 0.009) in the number of erythroid colonies compared with CFU-GM,although without perturbing,at least in vitro,the maturation program of the cells. On the other hand,the effect of the gene overexpression did not induce any skewing in the colony types derived from the myeloid lineage. View Publication

Hematologic malignancies such as acute and chronic myeloid leukemia are characterized by the malignant transformation of immature CD34(+) progenitor cells. Transformation is associated with elevated expression of the Wilm's tumor gene encoded transcription factor (WT1). Here we demonstrate that WT1 can serve as a target for cytotoxic T lymphocytes (CTL) with exquisite specificity for leukemic progenitor cells. HLA-A0201- restricted CTL specific for WT1 kill leukemia cell lines and inhibit colony formation by transformed CD34(+) progenitor cells isolated from patients with chronic myeloid leukemia (CML),whereas colony formation by normal CD34(+) progenitor cells is unaffected. Thus,the tissue-specific transcription factor WT1 is an ideal target for CTL-mediated purging of leukemic progenitor cells in vitro and for antigen-specific therapy of leukemia and other WT1-expressing malignancies in vivo. View Publication

We studied the suitability of collagen-based semisolid medium for assay of endogenous erythroid colony formation performed in myeloproliferative disorders. Bone marrow (BM) mononuclear cells (MNC) from 103 patients suspected of having polycythemia vera (PV,76 patients) or essential thrombocythemia (ET,27 patients) were grown in collagen-based,serum-free,cytokine-free semisolid medium. Colony analysis at day 8 or 10 showed that this collagen assay is specific,as endogenous growth of erythroid colonies was never observed in cultures of 16 healthy donors and 6 chronic myelogenous leukemia (CML) patients. Endogenous erythroid colony formation was observed in 53.3% of patients suspected of PV,with only 15.4% of positive cultures for patients with 1 minor PV criterion and 72% (p = 0.009) of positive cultures for patients with textgreater or =2 minor or 1 major PV criterion. Similarly,endogenous growth of erythroid colonies was found in 44.4% of patients suspected of ET,with 31.6% of positive cultures for patients with 1 ET criterion versus 75% for patients with textgreater or =2 ET criteria. In addition,we found that in collagen gels,tests of erythropoietin (EPO) hypersensitivity in the presence of 0.01 or 0.05 U/ml of EPO and tests of endogenous colony-forming units-megakaryocyte (CFU-MK) formation cannot be used to detect PV or ET,as these tests were positive for,respectively,21.4% and 50% of healthy donors and 83% and 50% of CML patients. A retrospective analysis suggests that collagen assays are more sensitive than methylcellulose assays to assess endogenous growth of erythroid colonies. In summary,serum-free collagen-based colony assays are simple and reliable assays of endogenous growth of erythroid colonies in myeloproliferative diseases. They also appear to be more sensitive than methylcellulose-based assays. View Publication

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells,up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking,however,decreased to 0.1% or lower within 2 weeks. In contrast,EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks,the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis,and EGFP expression was observed in CD4(+),CD8(+),CD20(+),and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452) View Publication

The molecular and cellular requirements for the development of different populations of human dendritic cells (DC) were studied. Conditions were defined that support DC production from lymphoid progenitors but that fail to induce DC formation from peripheral monocytes. The production of these lymphoid-related DC was severely blocked when hematopoietic progenitors overexpressed Ik7,a mutant dominant-negative Ikaros protein. In contrast,Ik7 did not block the formation of DC in conditions supporting the development of monocyte-derived DC. Furthermore,Ik7 did not block the formation of monocyte/macrophages and enhanced granulopoiesis. One of the molecular mechanisms mediated by Ik7 appears to be down-regulation of the flt3-receptor mRNA. Thus,distinct signals control the formation of DC demonstrating that some aspects of DC diversity are determined in part by distinct molecular cues at the hematopoietic level. (Blood. 2000;95:128-137) View Publication

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted,self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani,K. Hobson,and M. S. Rao,1997,Dev. Biol. 186,202-223; M. S. Rao and M. Mayer-Proschel,1997,Dev. Biol. 188,48-63; M. Mayer-Proschel,A. J. Kalyani,T. Mujtaba,and M. S. Rao,1997,Neuron 19,773-785). We now show that cells identical to rat NEPs,NRPs,and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs,respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive,undergo self-renewal in defined medium,and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus,lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells. View Publication

To study molecular events involved in B lymphocyte development and V(D)J rearrangement,we have established an efficient system for the differentiation of embryonic stem (ES) cells into mature Ig-secreting B lymphocytes. Here,we show that B lineage cells generated in vitro from ES cells are functionally analogous to normal fetal liver-derived or bone marrow-derived B lineage cells at three important developmental stages: first,they respond to Flt-3 ligand during an early lymphopoietic progenitor stage; second,they become targets for Abelson murine leukemia virus (A-MuLV) infection at a pre-B cell stage; third,they secrete Ig upon stimulation with lipopolysaccharide at a mature mitogen-responsive stage. Moreover,the ES cell-derived A-MuLV-transformed pre-B (EAB) cells are phenotypically and functionally indistinguishable from standard A-MuLV-transformed pre-B cells derived from infection of mouse fetal liver or bone marrow. Notably,EAB cells possess functional V(D)J recombinase activity. In particular,the generation of A-MuLV transformants from ES cells will provide an advantageous system to investigate genetic modifications that will help to elucidate molecular mechanisms in V(D)J recombination and in A-MuLV-mediated transformation. View Publication

Because hematopoietic stem cells are rich in aldehyde dehydrogenase (ALDH) activity,we developed a fluorescent substrate for ALDH,termed BODIPY aminoacetaldehyde (BAAA),and tested its potential for isolating primitive human hematopoietic cells. A population of cells with low orthogonal light scattering and bright fluorescence intensity (SSC(lo)ALDH(br) cells) could be readily fractionated from human umbilical cord blood cells costained with BAAA and the multidrug-resistance inhibitor verapamil. The SSC(lo)ALDH(br) population was depleted of lineage-committed cells,40-90% pure for CD34(+)CD38(lo/-) cells,and enriched 50- to 100-fold for primitive hematopoietic progenitors detected in short- and long-term culture analyses. Together,these observations indicate that fractionating human hematopoietic stem cells on the basis of ALDH activity using BAAA is an effective method for isolating primitive human hematopoietic progenitors. This technique may be useful for isolating stem cells from other tissues as well. View Publication

Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation,such as the TCR/CD3 complex and the costimulatory receptor CD28,are capable of mediating T-cell activation resulting in tumor cell killing. In this study,we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2,when used alone rather than in combination,markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore,these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and,in particular,of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients. View Publication