技术资料

AIM Human embryonic stem cells (hESCs) represent a novel cell source to treat diseases such as heart failure and for use in drug screening. In this study,we aim to promote efficient generation of cardiomyocytes from hESCs by combining the current optimal techniques of controlled growth of undifferentiated cells and specific induction for cardiac differentiation. We also aim to examine whether these methods are scalable and whether the differentiated cells can be cryopreserved. METHODS & RESULTS hESCs were maintained without conditioned medium or feeders and were sequentially treated with activin A and bone morphogenetic protein-4 in a serum-free medium. This led to differentiation into cell populations containing high percentages of cardiomyocytes. The differentiated cells expressed appropriate cardiomyocyte markers and maintained contractility in culture,and the majority of the cells displayed working chamber (atrial and ventricular) type electrophysiological properties. In addition,the cell growth and differentiation process was adaptable to large culture formats. Moreover,the cardiomyocytes survived following cryopreservation,and viable cardiac grafts were detected after transplantation of cryopreserved cells into rat hearts following myocardial infarctions. CONCLUSION These results demonstrate that cardiomyocytes of high quality can be efficiently generated and cryopreserved using hESCs maintained in serum-free medium,a step forward towards the application of these cells to human clinical use or drug discovery. View Publication

With the aim of finding small molecules that stimulate erythropoiesis earlier than erythropoietin and that enhance erythroid colony-forming unit (CFU-E) production,we studied the mechanism by which glucocorticoids increase CFU-E formation. Using erythroid burst-forming unit (BFU-E) and CFU-E progenitors purified by a new technique,we demonstrate that glucocorticoids stimulate the earliest (BFU-E) progenitors to undergo limited self-renewal,which increases formation of CFU-E cells textgreater 20-fold. Interestingly,glucocorticoids induce expression of genes in BFU-E cells that contain promoter regions highly enriched for hypoxia-induced factor 1α (HIF1α) binding sites. This suggests activation of HIF1α may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed,HIF1α activation by a prolyl hydroxylase inhibitor (PHI) synergizes with glucocorticoids and enhances production of CFU-Es 170-fold. Because PHIs are able to increase erythroblast production at very low concentrations of glucocorticoids,PHI-induced stimulation of BFU-E progenitors thus represents a conceptually new therapeutic window for treating erythropoietin-resistant anemia. View Publication

Greater than 75% of all hematologic malignancies derive from germinal center (GC) or post-GC B cells,suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID),GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair,homologous recombination,base excision repair,and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells,reflecting activation of a process we have termed somatic hyperrepair (SHR). Using an in vitro system,we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators,SHR induction strictly required signals provided by contact with activated CD4+ T cells,and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression,as GC B cells from AID-/-mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process,our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells. View Publication

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors,but the functional capacity of DC must be assessed in detail,especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant,providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol,resulting in full maturation of monocyte-derived DC(s). Both RIG-I-dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1-specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors,or TLRs,do not show these functional features. Therefore,SeV-infected DC have the potential for DC-directed immunotherapy. View Publication

Intestinal ischemia-reperfusion (IR)-induced damage requires complement receptor 2 (CR2) for generation of the appropriate natural Ab repertoire. Pathogenic Abs recognize neoantigens on the ischemic tissue,activate complement,and induce intestinal damage. Because C3 cleavage products act as ligands for CR2,we hypothesized that CR2(hi) marginal zone B cells (MZBs) require C3 for generation of the pathogenic Abs. To explore the ability of splenic CR2(+) B cells to generate the damaging Ab repertoire,we adoptively transferred either MZBs or follicular B cells (FOBs) from C57BL/6 or Cr2(-/-) mice into Rag-1(-/-) mice. Adoptive transfer of wild type CR2(hi) MZBs but not CR2(lo) FOBs induced significant damage,C3 deposition,and inflammation in response to IR. In contrast,similarly treated Rag-1(-/-) mice reconstituted with either Cr2(-/-) MZB/B1 B cells (B1Bs) or FOBs lacked significant intestinal damage and displayed limited complement activation. To determine whether C3 cleavage products are critical in CR2-dependent Ab production,we evaluated the ability of the natural Ab repertoire of C3(-/-) mice to induce damage in response to IR. Infusion of C3(-/-) serum into Cr2(-/-) mice restored IR-induced tissue damage. Furthermore,Rag-1(-/-) mice sustained significant damage after infusion of Abs from C3(-/-) but not Cr2(-/-) mice. Finally,adoptive transfer of MZBs from C3(-/-) mice into Rag-1(-/-) mice resulted in significant tissue damage and inflammation. These data indicate that CR2 expression on MZBs is sufficient to induce the appropriate Abs required for IR-induced tissue damage and that C3 is not critical for generation of the pathogenic Abs. View Publication

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this,time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis,including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique,in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work,and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality,fast chromosomal analysis of human ES and iPS cells. View Publication

RARA (retinoic acid receptor alpha) haploinsufficiency is an invariable consequence of t(15;17)(q22;q21) translocations in acute promyelocytic leukemia (APL). Retinoids and RARA activity have been implicated in hematopoietic self-renewal and neutrophil maturation. We and others therefore predicted that RARA haploinsufficiency would contribute to APL pathogenesis. To test this hypothesis,we crossed Rara(+/-) mice with mice expressing PML (promyelocytic leukemia)-RARA from the cathepsin G locus (mCG-PR). We found that Rara haploinsufficiency cooperated with PML-RARA,but only modestly influenced the preleukemic and leukemic phenotype. Bone marrow from mCG-PR(+/-) × Rara(+/-) mice had decreased numbers of mature myeloid cells,increased ex vivo myeloid cell proliferation,and increased competitive advantage after transplantation. Rara haploinsufficiency did not alter mCG-PR-dependent leukemic latency or penetrance,but did influence the distribution of leukemic cells; leukemia in mCG-PR(+/-) × Rara(+/-) mice presented more commonly with low to normal white blood cell counts and with myeloid infiltration of lymph nodes. APL cells from these mice were responsive to all-trans retinoic acid and had virtually no differences in expression profiling compared with tumors arising in mCG-PR(+/-) × Rara(+/+) mice. These data show that Rara haploinsufficiency (like Pml haploinsufficiency and RARA-PML) can cooperate with PML-RARA to influence the pathogenesis of APL in mice,but that PML-RARA is the t(15;17) disease-initiating mutation. View Publication

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation,overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities,which indicates the presence of other mechanisms for Evi-1 activation. In this study,we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL-mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population,in which Evi-1 particularly contributes to the propagation of MLL-ENL-immortalized cells. Furthermore,gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell-like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell-derived MLL leukemic cells. View Publication

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular bulk degradation pathway that plays critical roles in eliminating intracellular pathogens,presenting endogenous Ags,and regulating T lymphocyte survival and proliferation. In this study,we have investigated the role of autophagy in regulating the endoplasmic reticulum (ER) compartment in T lymphocytes. We found that ER content is expanded in mature autophagy-related protein (Atg) 7-deficient T lymphocytes. Atg7-deficient T cells stimulated through the TCR display impaired influx,but not efflux,of calcium,and ER calcium stores are increased in Atg7-deficient T cells. Treatment with the ER sarco/ER Ca(2+)-ATPase pump inhibitor thapsigargin rescues the calcium influx defect in Atg7-deficient T lymphocytes,suggesting that this impairment is caused by an intrinsic defect in ER. Furthermore,we found that the stimulation-induced redistribution of stromal interaction molecule-1,a critical event for the store-operated Ca(2+) release-activated Ca(2+) channel opening,is impaired in Atg7-deficient T cells. Together,these findings indicate that the expanded ER compartment in Atg7-deficient T cells contains increased calcium stores,and the inability of these stores to be depleted causes defective calcium influx in these cells. Our results demonstrate that autophagy plays an important role in maintaining ER and calcium homeostasis in T lymphocytes. View Publication

In elderly subjects and in patients with chronic inflammatory diseases,there is an increased subset of monocytes with a CD14(+)CD16(+) phenotype,whose origin and functional relevance has not been well characterized. In this study,we determined whether prolonged survival of human CD14(++)CD16(-) monocytes promotes the emergence of senescent cells,and we analyzed their molecular phenotypic and functional characteristics. We used an in vitro model to prolong the life span of healthy monocytes. We determined cell senescence,intracellular cytokine expression,ability to interact with endothelial cells,and APC activity. CD14(+)CD16(+) monocytes were senescent cells with shortened telomeres (215 ± 37 relative telomere length) versus CD14(++)CD16(-) cells (339 ± 44 relative telomere length; p textless 0.05) and increased expression of β-galactosidase (86.4 ± 16.4% versus 10.3 ± 7.5%,respectively; p = 0.002). CD14(+)CD16(+) monocytes exhibited features of activated cells that included expression of CD209,release of cytokines in response to low-intensity stimulus,and increased capacity to sustain lymphocyte proliferation. Finally,compared with CD14(++)CD16(-) cells,CD14(+)CD16(+) monocytes showed elevated expression of chemokine receptors and increased adhesion to endothelial cells (19.6 ± 8.1% versus 5.3 ± 4.1%; p = 0.033). In summary,our data indicated that the senescent CD14(+)CD16(+) monocytes are activated cells,with increased inflammatory activity and ability to interact with endothelial cells. Therefore,accumulation of senescent monocytes may explain,in part,the development of chronic inflammation and atherosclerosis in elderly subjects and in patients with chronic inflammatory diseases. View Publication

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species,the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest,followed by an immunodepletion step using microbeads coated with CD11b,CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion,a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1),CD90,CD105 and CD73 cell surface markers,but negative for the hematopoietic surface markers CD14,CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic,osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time. View Publication

BACKGROUND AND PURPOSE: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here,we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs.backslashnbackslashnEXPERIMENTAL APPROACH: Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points,day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C.backslashnbackslashnKEY RESULTS: Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2,TUBB III,PAX6,TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2,PITX2,GATA5,MYL4,TNNT2,COL1A1 and COL1A2. In addition,no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated.backslashnbackslashnCONCLUSIONS AND IMPLICATIONS: Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments. View Publication