技术资料

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

Autosomal recessive osteopetrosis (OP) is a rare,lethal disorder in which osteoclasts are absent or nonfunctional,resulting in a bone marrow cavity insufficient to support hematopoiesis. Because osteoclasts are derived from hematopoietic precursors,allogeneic hematopoietic cell transplantation can cure the bony manifestations of the disorder. However,high rates of graft failure have been observed in this population. It is not possible to harvest bone marrow from these patients for reinfusion should graft failure be observed. We report that 8 of 10 patients with OP had high numbers of circulating CD34(+) cells (3% +/- 0.9%). This increased proportion of peripheral CD34(+) cells made it possible to harvest 2 x 10(6) CD34(+) cells per kilogram with a total volume of blood ranging from 8.3 to 83.7 mL (1.3-11.6 mL/kg). In addition,colony-forming assays documented significantly more colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid in the blood of osteopetrotic patients compared with controls; the numbers of colony-forming units approximated those found in control marrow. We conclude that OP patients with high levels of circulating CD34(+) are candidates for peripheral blood autologous harvest by limited exchange transfusion. These cells are then available for reinfusion should graft failure be observed in patients for whom retransplantation is impractical. View Publication

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA,TRKB,TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase-polymerase chain reaction (RT-PCR),we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB(+) cases (16/30). Activation of TRKA or TRKB by NGF and BDNF,respectively,efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover,activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target. View Publication

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies,but is absent on normal tissues,including hematopoietic progenitor cells,and may therefore be an appropriate candidate for T cell-mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity,PRAME-specific cytotoxic T lymphocytes (CTLs),we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal,PRAME-specific CTL lines and elicited high-avidity CTLs,with a high proportion of cells recognizing a previously uninvestigated HLA-A*02-restricted epitope,P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME(+) hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts,but also against leukemic progenitor cells as assessed by colony-forming-inhibition assays,which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors,indicating that this approach may be of value for immunotherapy of PRAME(+) hematologic malignancies. View Publication

Disentangling the complex interactions that govern stem cell fate choices of self-renewal,differentiation,or death presents a formidable challenge. Image-based phenotype-driven screening meets this challenge by providing means for rapid testing of many small molecules simultaneously. Pluripotent embryonal carcinoma (EC) cells offer a convenient substitute for embryonic stem (ES) cells in such screens because they are simpler to maintain and control. The authors developed an image-based screening assay to identify compounds that affect survival or differentiation of the human EC stem cell line NTERA2 by measuring the effect on cell number and the proportion of cells expressing a pluripotency-associated marker SSEA3. A pilot screen of 80 kinase inhibitors identified several compounds that improved cell survival or induced differentiation. The survival compounds Y-27632,HA-1077,and H-8 all strongly inhibit the kinases ROCK and PRK2,highlighting the important role of these kinases in EC cell survival. Two molecules,GF109203x and rottlerin,induced EC differentiation. The effects of rottlerin were also investigated in human ES cells. Rottlerin inhibited the self-renewal ability of ES cells,caused the cell cycle arrest,and repressed the expression of pluripotency-associated genes. View Publication

The fabrication of high-density polymer microarray is described,allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance. View Publication

Fluoride is a ubiquitous natural substance that is often used in dental products to prevent dental caries. The biphasic actions of fluoride imply that excessive systemic exposure to fluoride can cause harmful effects on embryonic development in both animal models and humans. However,insufficient information is available on the effects of fluoride on human embryonic stem cells (hESCs),which is a novel in vitro humanized model for analyzing the embryotoxicities of chemical compounds. Therefore,we investigated the effects of sodium fluoride (NaF) on the proliferation,differentiation and viability of H9 hESCs. For the first time,we showed that 1 mM NaF did not significantly affect the proliferation of hESCs but did disturb the gene expression patterns of hESCs during embryoid body (EB) differentiation. Higher doses of NaF (2 mM and above) markedly decreased the viability and proliferation of hESCs. The mode and underlying mechanism of high-dose NaF-induced cell death were further investigated by assessing the sub-cellular morphology,mitochondrial membrane potential (MMP),caspase activities,cellular reactive oxygen species (ROS) levels and activation of mitogen-activated protein kinases (MAPKs). High-dose NaF caused the death of hESCs via apoptosis in a caspase-mediated but ROS-independent pathway,coupled with an increase in the phospho-c-Jun N-terminal kinase (p-JNK) levels. Pretreatment with a pJNK-specific inhibitor (SP600125) could effectively protect hESCs from NaF-induced cell death in a concentration- and time-dependent manner. These findings suggest that NaF might interfere with early human embryogenesis by disturbing the specification of the three germ layers as well as osteogenic lineage commitment and that high-dose NaF could cause apoptosis through a JNK-dependent pathway in hESCs. View Publication

Chemical compounds have emerged as powerful tools for modulating ESC functions and deriving induced pluripotent stem cells (iPSCs),but documentation of compound-induced efficient directed differentiation in human ESCs (hESCs) and human iPSC (hiPSCs) is limited. By screening a collection of chemical compounds,we identified compound C (also denoted as dorsomorphin),a protein kinase inhibitor,as a potent regulator of hESC and hiPSC fate decisions. Compound C suppresses mesoderm,endoderm,and trophoectoderm differentiation and induces rapid and high-efficiency neural conversion in both hESCs and hiPSCs,88.7% and 70.4%,respectively. Interestingly,compound C is ineffective in inducing neural conversion in mouse ESCs (mESCs). Large-scale kinase assay revealed that compound C targets at least seven transforming growth factor beta (TGF-β) superfamily receptors,including both type I and type II receptors,and thereby blocks both the Activin and bone morphogenesis protein (BMP) signaling pathways in hESCs. Dual inhibition of Activin and BMP signaling accounts for the effects of compound C on hESC differentiation and neural conversion. We also identified muscle segment homeobox gene 2 (MSX2) as a downstream target gene of compound C and a key signaling intermediate of the BMP pathway in hESCs. Our findings provide a single-step cost-effective method for efficient derivation of neural progenitor cells in adherent culture from human pluripotent stem cells. Therefore,it will be uniquely suitable for the production of neural progenitor cells in large scale and should facilitate the use of stem cells in drug screening and regenerative medicine and study of early human neural development. View Publication

Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development,modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (˜70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (textgreater250%) MN production in chemically defined adherent cultures. Furthermore,we show that Islet-1 is essential for formation of mature and functional human MNs,but,unlike its mouse counterpart,does not regulate cell survival or suppress the V2a interneuron fate. Together,our discoveries improve the strategy for MN derivation,advance our understanding of human neural specification and MN development,and provide invaluable tools for human developmental studies,drug discovery and regenerative medicine. View Publication

The reprogramming of human somatic cells to induced pluripotent stem (hiPS) cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells,including fibroblasts,keratinocytes and peripheral blood cells,with variable reprogramming efficiencies and kinetics. Here,we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS) cells,with similar efficiencies to keratinocytes,which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES) cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure. View Publication

Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and beta-thalassemia. Here,we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling betaA-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene,and erythroid cells derived in vitro from these in vivo-regenerated cells produced high levels of betaA-T87Q-globin protein. Linker-mediated PCR analysis identified multiple transgene-positive clones in all mice analyzed with 2.1 +/- 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes,including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust,erythroid-specific production of therapeutically relevant levels of beta-globin protein. However,the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications. View Publication

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However,HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs,we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein,and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells,HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo,which resulted in a marked enhancement of the primitive CD34+ subpopulation (P =.01). However,high HOXB4 expression substantially impaired the myeloerythroid differentiation program,and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P textless.03) and in vivo (P =.01). Furthermore,HOXB4 overexpression also significantly reduced B-cell output (P textless.01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials. View Publication