技术资料

SALL4,a human homolog to Drosophila spalt,is a novel zinc finger transcriptional factor essential for development. We cloned SALL4 and its isoforms (SALL4A and SALL4B). Through immunohistochemistry and real-time reverse-transcription-polymerase chain reaction (RT-PCR),we demonstrated that SALL4 was constitutively expressed in human primary acute myeloid leukemia (AML,n = 81),and directly tested the leukemogenic potential of constitutive expression of SALL4 in a murine model. SALL4B transgenic mice developed myelodysplastic syndrome (MDS)-like features and subsequently AML that was transplantable. Increased apoptosis associated with dysmyelopoiesis was evident in transgenic mouse marrow and colony-formation (CFU) assays. Both isoforms could bind to beta-catenin and synergistically enhanced the Wnt/beta-catenin signaling pathway. Our data suggest that the constitutive expression of SALL4 causes MDS/AML,most likely through the Wnt/beta-catenin pathway. Our murine model provides a useful platform to study human MDS/AML transformation,as well as the Wnt/beta-catenin pathway's role in the pathogenesis of leukemia stem cells. View Publication

Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here,we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors,enabling a rapid surge" capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates� View Publication

OBJECTIVE: In vitro models of hematopoiesis used in investigative hematopathology and in safety studies on candidate drugs,involve clonogenic assays on colony-forming unit granulocyte macrophage (CFU-GM). These assays require live and unstained colonies to be counted. Most laboratories still rely on visual scoring,which is time-consuming and error-prone. As a consequence,automated scoring is highly desired. An algorithm that recognizes and scores CFU-GM colonies by data fusion has been developed. Some preliminary results are presented in this article. METHODS: CFU-GM assays were carried out on hematopoietic progenitors (human umbilical cord blood cells) grown in methylcellulose. Colony images were acquired by a digital camera and stored. RESULTS: The classifier was designed to process images of layers sampled from a three-dimensional (3D) domain and forming a stack. Structure and texture information was extracted from each image. Classifier training was based on a 3D colony model applied to the image stack. The number of scored colonies (assigned class) was required to match the count supplied by the human expert (class of belonging). The trained classifier was validated on one more stack and then applied to a stack with overlapping colonies. Scoring in distortion- and caustic-affected border areas was also successfully demonstrated. Because of hardware limitations,compact colonies in some cases were missed. CONCLUSIONS: The industry's scoring methods all rely on structure alone and process 2D data. Instead,the classifier here fuses data from a whole stack and is capable,in principle,of high-throughput screening. View Publication

Chemokine stromal derived factor 1 (SDF-1) is involved in trafficking of hematopoietic stem cells (HSCs) from the bone marrow (BM) to peripheral blood (PB) and has been found to enhance postischemia angiogenesis. This study was aimed at investigating whether SDF-1 plays a role in differentiation of BM-derived c-kit(+) stem cells into endothelial progenitor cells (EPCs) and in ischemia-induced trafficking of stem cells from PB to ischemic tissues. We found that SDF-1 enhanced EPC number by promoting alpha(2),alpha(4),and alpha(5) integrin-mediated adhesion to fibronectin and collagen I. EPC differentiation was reduced in mitogen-stimulated c-kit(+) cells,while cytokine withdrawal or the overexpression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4) restored such differentiation,suggesting a link between control of cell cycle and EPC differentiation. We also analyzed the time course of SDF-1 expression in a mouse model of hind-limb ischemia. Shortly after femoral artery dissection,plasma SDF-1 levels were up-regulated,while SDF-1 expression in the bone marrow was down-regulated in a timely fashion with the increase in the percentage of PB progenitor cells. An increase in ischemic tissue expression of SDF-1 at RNA and protein level was also observed. Finally,using an in vivo assay such as injection of matrigel plugs,we found that SDF-1 improves formation of tubulelike structures by coinjected c-kit(+) cells. Our findings unravel a function for SDF-1 in increase of EPC number and formation of vascular structures by bone marrow progenitor cells. View Publication

Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities,additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac,known as SDX-308,and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101,a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation,and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308,as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-kappaB activation and osteoclast formation in an osteoclast cellular model,RAW 264.7. SDX-308 effectively suppressed TNF-alpha-induced IKK-gamma and IkappaB-alpha phosphorylation and degradation and subsequent NF-kappaB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity,potentially by controlling NF-kappaB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study. 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

The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDH(hi)Lin- cells). Here,we further dissected the ALDH(hi)-Lin- population by selection for CD133,a surface molecule expressed on progenitors from hematopoietic,endothelial,and neural lineages. ALDH(hi)CD133+Lin- cells were primarily CD34+,but also included CD34-CD38-CD133+ cells,a phenotype previously associated with repopulating function. Both ALDH(hi)CD133-Lin- and ALDH(hi)CD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro,whereas only the ALDH(hi)CD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID beta2M-null mice that received transplants of ALDH(hi)CD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells,suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDH(hi)CD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial,secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies. View Publication

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs),and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the $\beta$-globin gene (HBB). Sickle hemoglobin damages erythrocytes,causing vasoocclusion,severe pain,progressive organ damage,and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA,together with a single-stranded DNA oligonucleotide donor (ssODN),to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice,ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing,enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells,and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases. 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

The translocation t(11;18)(q21;q21) that generates an API2-MALT1 fusion protein is the most common structural abnormality among the genetic defects reported in mucosa-associated lymphoid tissue (MALT)-type lymphomas,and its presence correlates with the apparent lack of further genetic instability or chromosomal imbalances. Hence,constitutive nuclear factor-kappaB (NF-kappaB) activation induced by the API2-MALT1 fusion protein is considered essential for B-cell transformation. To examine its role in B-cell development and lymphomagenesis,Emu-API2-MALT1 transgenic mice were produced. Our data show that expression of the API2-MALT1 fusion protein alone is not sufficient for the development of lymphoma masses within 50 weeks. Nevertheless,API2-MALT1 expression affected B-cell maturation in the bone marrow and triggered the specific expansion of splenic marginal zone B cells. Polyubiquitination of IkappaB kinase gamma (IKKgamma),indicative for enhanced NF-kappaB activation,was increased in splenic lymphocytes and promoted the survival of B cells ex vivo. In addition,we show that the API2-MALT1 fusion resided in the cholesterol- and sphingolipid-enriched membrane microdomains,termed lipid rafts. We provide evidence that association of the MALT1 COOH terminal with the lipid rafts,which is mediated by the API2 portion,is sufficient to trigger NF-kappaB activation via enhanced polyubiquitination of IKKgamma. Taken together,these data support the hypothesis that the API2-MALT1 fusion protein can contribute to MALT lymphoma formation via increased NF-kappaB activation. View Publication

Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences,highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date,alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells,the transfer of intact viral genomes is unacceptable for clinical applications,due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins,which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover,intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements,we were able to generate a self-inactivating (SIN) alpharetroviral vector,whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection. View Publication

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