技术资料

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

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However,little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb),97A6,that specifically detects human Ba,MC (lung,skin),and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils,eosinophils,monocytes,lymphocytes,platelets) did not react with MoAb 97A6,and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (textgreater98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40),Ba-eosinophil colonies (7 of 40),Ba-macrophage colonies (3 of 40),and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast,no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells,starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (textgreater30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors,MC progenitors,Ba progenitors,as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors. View Publication

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Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However,the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However,5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood,as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted,mediated by CD8(+) lymphocytes,and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959) View Publication

The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly,we found a significant reduction in proliferation and an accumulation in the G(2)/M phase of Cre-expressing cells. To minimize the toxic effect of Cre,we designed a lentiviral vector that integrates into the host genome,expresses Cre in the target cell,and is subsequently deleted from the genome in a Cre-dependent manner. Thus,the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression,transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo. View Publication

In the present study,we report a new method for enrichment and analysis of fetal CD34+ stem cells after culture in order to determine whether it is feasible for noninvasive prenatal diagnosis. We also determined whether fetal CD34+ stem cells persist in maternal blood after delivery and assessed whether they have an impact on noninvasive prenatal diagnosis of genetic abnormalities. Peripheral blood samples were obtained from 35 pregnant women,13 non-pregnant women who had given birth to male offsprings,12 women who had never been pregnant,and eight pregnant women with male fetuses. CD34+ stem cells were enriched and either cultured for prenatal diagnosis or analyzed with fluorescence in situ hybridization (FISH)/polymerase chain reaction (PCR) to determine peristance in maternal blood. Fetal/maternal cells can be isolated and grown in vitro" to provide enough cells for a more accurate fetal sex or aneuploid prediction than is provided by unenriched and uncultured CD34+ stem cells. The presence of fetal cells in maternal blood samples from mothers who had given birth to male offspring was found in 3 of 13 blood samples. PCR was positive for Y chromosome in one woman who had never been pregnant. Analysis of cultured CD34+ stem cells from mothers with Y PCR positivity did not detect any male cells in any samples. Even if PCR positivity is due to persistence of fetal stem cells from previous pregnancies� View Publication

Patients with heart failure are predisposed to infections and anemia,possibly due to reduced hematopoiesis. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in heart failure,and it inhibits normal hematopoiesis,partly due to apoptosis through the effector molecule Fas. We examined bone marrow progenitor cells of mice with heart failure induced by acute myocardial infarction. The fraction of progenitor cells in mice with heart failure was only approximately 40% of control. Measured with in vitro clonal assays,the proliferative capacity of the progenitor cells in mice with heart failure was reduced to approximately 50% of control. Flow cytometry with specific markers revealed a threefold increase in apoptosis among progenitor cells from mice with heart failure. In these mice,TNF-alpha/Fas expression was increased in bone marrow natural killer (NK) and T cells,and these lymphocytes showed increased cytolytic activity in vitro against progenitor cells. We conclude that the TNF-alpha/Fas pathway in lymphocytes is activated in the bone marrow during heart failure,which may play a pathogenic role in the observed decrease in hematopoiesis. View Publication

Once set,the inactive status of the X chromosome in female somatic cells is preserved throughout subsequent cell divisions. The inactive status of the X chromosome is characterized by many features,including late replication. In contrast to induced pluripotent stem cells (iPSCs) in mice,the X chromosome in human female iPSCs usually remains inactive after reprogramming of somatic cells to the pluripotent state,although recent studies point to the possibility of reactivation of the X chromosome. Here,we demonstrated that,during reprogramming,the inactive X chromosome switches from late to synchronous replication,with restoration of the transcription of previously silenced genes. This process is accompanied by accumulation of a new epigenetic mark or intermediate of the DNA demethylation pathway,5-hydroxymethylcytosine (5hmC),on the activated X chromosome. Our results indicate that the active status of the X chromosome is better confirmed by early replication and the reappearance of 5hmC,rather than by appearance of histone marks of active chromatin,removal of histone marks of inactive chromatin,or an absence of XIST coating. View Publication

Pluripotent stem cells have the potential to become most of the cell types that make up an organism. However,the signals that trigger these cells to turn into neurons rather than lung cells or muscle cells,for example,are not fully understood. Proteins called growth factors are known to have a role in this process,as are transcription factors,but it is not clear if other factors are also involved. In an attempt to identify additional mechanisms that could contribute to the formation of neurons,Sun et al. screened more than 2,000 small molecules for their ability to transform mouse pluripotent stem cells into neurons in cell culture. Surprisingly,they found that a compound called selamectin,which is used to treat parasitic flatworm infections,also triggered stem cells to turn into neurons. Selamectin works by blocking a particular type of ion channel in flatworms,but this ion channel is not found in vertebrates,which means that selamectin must be promoting the formation of neurons in mice via a different mechanism. Given that a drug related to selamectin is known to act on a subtype of receptors for the neurotransmitter GABA,Sun et al. wondered whether these receptors—known as GABAA receptors—might also underlie the effects of selamectin. Consistent with this idea,drugs that increased GABAA activity stimulated the formation of neurons,whereas drugs that reduced GABAA function blocked the effects of selamectin. In addition,Sun et al. showed that selamectin triggers human embryonic stem cells to become neurons,and that it also promotes the formation of new neurons in developing zebrafish in vivo. As well as revealing an additional mechanism for the formation of neurons from stem cells,the screening technique introduced by Sun et al. could help to identify further pro-neuronal molecules,which could aid the treatment of neurodevelopmental and neurodegenerative disorders. DOI: [http://dx.doi.org/10.7554/eLife.00508.002][1] [1]: /lookup/doi/10.7554/eLife.00508.002 View Publication

Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population,and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular,disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors,aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits,we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as globulomers"� View Publication

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular,primitive leukemia cells,often termed leukemia stem cells,are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins,presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation,CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise,we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly,these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism,which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1),as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism,an intrinsic property of primary human AML cells. View Publication

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However,purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here,we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins,gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells,MLC-2v selected CMs had larger action potential amplitudes and durations. In addition,by immunofluorescence,we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However,only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach,it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. ?? 2013 Elsevier B.V. View Publication