技术资料

Lysophosphatidic acid (LPA) is enriched in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests cancer-associated myofibroblasts play a pivotal role in tumorigenesis through secreting stromal cell-derived factor-1 (SDF-1). In the present study,we demonstrate that LPA induces expression of alpha-smooth muscle actin (alpha-SMA),a marker for myofibroblasts,in human adipose tissue-derived mesenchymal stem cells (hADSCs). The LPA-induced expression of alpha-SMA was completely abrogated by pretreatment of the cells with Ki16425,an antagonist of LPA receptors,or by silencing LPA(1) or LPA(2) isoform expression with small interference RNA (siRNA). LPA elicited phosphorylation of Smad2/3,and siRNA-mediated depletion of endogenous Smad2/3 or adenoviral expression of Smad7,an inhibitory Smad,abrogated the LPA induced expression of alpha-SMA and phosphorylation of Smad2/3. LPA-induced secretion of transforming growth factor (TGF)-beta1 in hADSCs,and pretreatment of the cells with SB431542,a TGF-beta type I receptor kinase inhibitor,or anti-TGF-beta1 neutralizing antibody inhibited the LPA-induced expression of alpha-SMA and phosphorylation of Smad2. Furthermore,ascites from ovarian cancer patients or conditioned medium from ovarian cancer cells induced expression of alpha-SMA and phosphorylation of Smad2,and pretreatment of the cells with Ki16425 or SB431542 abrogated the expression of alpha-SMA and phosphorylation of Smad2. In addition,LPA increased the expression of SDF-1 in hADSCs,and pretreatment of the cells with Ki16425 or SB431562 attenuated the LPA-stimulated expression of SDF-1. These results suggest that cancer-derived LPA stimulates differentiation of hADSCs to myofibroblast-like cells and increases SDF-1 expression through activating autocrine TGF-beta1-Smad signaling pathway. View Publication

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However,development of cell-based regenerative therapies has been hindered by the lack of preclinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII mice,we characterized the distribution of lineage-depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase (ALDH) activity with CD133 coexpression. ALDH(hi) or ALDH(hi)CD133+ cells produced robust hematopoietic reconstitution and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that coexpressed human leukocyte antigen (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues,including islet and ductal regions of mouse pancreata. In contrast,variable phenotypes were detected in the chimeric liver,with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels and CD45-/HLA- cells with diluted GUSB expression predominant in the liver parenchyma. However,true nonhematopoietic human (HLA+/CD45-) cells were rarely detected in other peripheral tissues,suggesting that these GUSB+/HLA-/CD45- cells in the liver were a result of downregulated human surface marker expression in vivo,not widespread seeding of nonhematopoietic cells. However,relying solely on continued expression of cell surface markers,as used in traditional xenotransplantation models,may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes,indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. View Publication

Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells,capable of germline transmission,from mouse somatic cells by transduction of four defined transcription factors. Here,we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4,Sox2,Klf4,and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology,proliferation,surface antigens,gene expression,epigenetic status of pluripotent cell-specific genes,and telomerase activity. Furthermore,these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts. View Publication

Cell therapy is a novel promising option for treatment of ischemic diseases. Administered endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. However,the number of cells that home to ischemic tissues is restricted. The GTPase Rap1 plays an important role in the regulation of adhesion and chemotaxis. We investigated whether pharmacologic activation of Epac1,a nucleotide exchange protein for Rap1,which is directly activated by cAMP,can improve the adhesive and migratory capacity of distinct progenitor cell populations. Stimulation of Epac by a cAMP-analog increased Rap1 activity and stimulated the adhesion of human EPCs,CD34(+) hematopoietic progenitor cells,and mesenchymal stem cells (MSCs). Specifically,short-term stimulation with a specific Epac activator increased the beta2-integrin-dependent adhesion of EPCs to endothelial cell monolayers,and of EPC and CD34(+) cells to ICAM-1. Furthermore,the Epac activator enhanced the beta1-integrin-dependent adhesion of EPCs and MSCs to the matrix protein fibronectin. In addition,Epac1 activation induced the beta1- and beta2-integrin-dependent migration of EPCs on fibronectin and fibrinogen. Interestingly,activation of Epac rapidly increased lateral mobility of beta1- and beta2-integrins,thereby inducing integrin polarization,and stimulated beta1-integrin affinity,whereas the beta2-integrin affinity was not increased. Furthermore,prestimulation of EPCs with the Epac activator increased homing to ischemic muscles and neovascularization-promoting capacity of intravenously injected EPCs in the model of hind limb ischemia. These data demonstrate that activation of Epac1 increases integrin activity and integrin-dependent homing functions of progenitor cells and enhances their in vivo therapeutic potential. These results may provide a platform for the development of novel therapeutic approaches to improve progenitor cell homing. View Publication

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4,SOX2,NANOG,and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes,express telomerase activity,express cell surface markers and genes that characterize human ES cells,and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development,as well as for applications in transplantation medicine,once technical limitations (for example,mutation through viral integration) are eliminated. View Publication

Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling-dorsomorphin,which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2,ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation,target gene transcription and osteogenic differentiation. Using dorsomorphin,we examined the role of BMP signaling in iron homeostasis. In vitro,dorsomorphin inhibited BMP-,hemojuvelin- and interleukin 6-stimulated expression of the systemic iron regulator hepcidin,which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo,systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver,whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation,normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis. View Publication

Human bone marrow multipotent mesenchymal stromal cells are progenitor cells that can be expanded in vitro and differentiate into various cells of mesodermal origin. They contribute to the bone marrow reticular niche,where mature B cells and long-lived plasma cells are maintained. Multipotent mesenchymal stromal cells were recently shown to modulate T- and B-cell proliferation and differentiation,dendritic cell maturation,and natural killer activity. These immunoregulatory properties encouraged a possible use of these cells to modulate autoimmune responses in humans. We studied the influence of bone marrow mesenchymal stem cells on highly purified B-cell subsets isolated from healthy donors and total B cells from pediatric systemic lupus erythematosus patients. Bone marrow mesenchymal stem cells promoted proliferation and differentiation into immunoglobulin-secreting cells of transitional and naive B cells stimulated with an agonist of Toll-like receptor 9,in the absence of B cell receptor triggering. They strongly enhanced proliferation and differentiation into plasma cells of memory B-cell populations. A similar effect was observed in response to polyclonal stimulation of B cells isolated from pediatric patients with systemic lupus erythematosus. This study casts important questions on bone marrow mesenchymal stem cells as a therapeutic tool in autoimmune diseases in which B-cell activation is crucially implicated in the pathogenesis of the disease. View Publication

The functional significance of the electrophysiological properties of neural precursor cells (NPCs) was investigated using dissociated neurosphere-derived NPCs from the forebrain subventricular zone (SVZ) of adult mice. NPCs exhibited hyperpolarized resting membrane potentials,which were depolarized by the K(+) channel inhibitor,Ba(2+). Pharmacological analysis revealed two distinct K(+) channel families: Ba(2+)-sensitive K(ir) channels and tetraethylammonium (TEA)-sensitive K(v) (primarily K(DR)) channels. Ba(2+) promoted mitogen-stimulated NPC proliferation,which was mimicked by high extracellular K(+),whereas TEA inhibited proliferation. Based on gene and protein levels in vitro,we identified K(ir)4.1,K(ir)5.1 and K(v)3.1 channels as the functional K(+) channel candidates. Expression of these K(+) channels was immunohistochemically found in NPCs of the adult mouse SVZ,but was negligible in neuroblasts. It therefore appears that expression of K(ir) and K(v) (K(DR)) channels in NPCs and related changes in the resting membrane potential could contribute to NPC proliferation and neuronal lineage commitment in the neurogenic microenvironment. View Publication

Thiazolidinediones (TZD) are widely prescribed for the treatment of Type 2 diabetes. Increased loss of bone mass and a higher incidence of fractures have been associated with the use of this class of drugs in post-menopausal women. In vitro studies performed in rodent cell models indicated that rosiglitazone (RGZ),one of the TZD,inhibited osteoblastogenesis and induced adipogenesis in bone marrow progenitor cells. The objective of the present study was to determine for the first time the RGZ-dependent shift from osteoblastogenesis toward adipogenesis using a human cell model. To this purpose,bone marrow-derived mesenchymal stem cells were characterized and induced to differentiate along osteogenic and adipogenic lineages. We found that the exposure to RGZ potentiated adipogenic differentiation and shifted the differentiation toward an osteogenic phenotype into an adipogenic phenotype,as assessed by the appearance of lipid droplets. Accordingly,RGZ markedly increased the expression of the typical marker of adipogenesis fatty-acid binding protein 4,whereas it reduced the expression of Runx2,a marker of osteoblastogenesis. This is the first demonstration that RGZ counteracts osteoblastogenesis and induces a preferential differentiation into adipocytes in human mesenchymal stem cells. View Publication

Aberrant expression of Jagged1 and Notch1 are associated with poor outcome in breast cancer. However,the reason that Jagged1 and/or Notch overexpression portends a poor prognosis is unknown. We identify Slug,a transcriptional repressor,as a novel Notch target and show that elevated levels of Slug correlate with increased expression of Jagged1 in various human cancers. Slug was essential for Notch-mediated repression of E-cadherin,which resulted in beta-catenin activation and resistance to anoikis. Inhibition of ligand-induced Notch signaling in xenografted Slug-positive/E-cadherin-negative breast tumors promoted apoptosis and inhibited tumor growth and metastasis. This response was associated with down-regulated Slug expression,reexpression of E-cadherin,and suppression of active beta-catenin. Our findings suggest that ligand-induced Notch activation,through the induction of Slug,promotes tumor growth and metastasis characterized by epithelial-to-mesenchymal transition and inhibition of anoikis. View Publication

Reversine (RV) is the synthetic purine identified from a protein kinase-based screen of purine mimetics and it has been shown to induce muscle myoblast differentiation into progenitor cells that can be further converted into other cell lineages. Since protein kinases play a pivotal role in cell cycle control,we hypothesize that RV might affect the proliferation of cancer cells. Herein we report that RV inhibited growth of cultured human tumor cells,respectively,PC-3,HeLa,CWR22Rv1,and DU-145 cells,and induced accumulation of polyploidal cells with textgreater or =4N DNA content. However,RV was without effect on growth of normal prostate epithelial cells. RV-treated PC-3 cells showed enlarged nuclei and an estimated 100-fold increase in cell size. Moreover,PC-3 cells treated with RV for 2-4 days were accompanied by a marked increase in the expression of p21(WAF1),a modest elevation in the levels of cyclin D3 and CDK6 and concomitantly,also a substantial reduction in cyclin B and CDK1. These results suggest that RV may induce polyploidy and increase in cell size by up-regulating p21(WAF1) and cyclin D3/CDK6,while simultaneously suppressing the expression of cyclin B and CDK1. View Publication

Under standard culture conditions,tumor cells are exposed to 20% O(2),whereas the mean tumor oxygen levels within the tumor are much lower. We demonstrate,using low-passaged human tumor cell cultures established from glioma,that a reduction in the oxygen level in these cell cultures dramatically increases the percentage of CD133 expressing cells. View Publication