技术资料

INTRODUCTION: Various synthetic bone-graft substitutes are used commercially as osteoconductive scaffolds in the treatment of bone defects and fractures. The role of bone-graft substitutes is changing from osteoconductive conduits for growth to an delivery system for biologic fracture treatments. Achieving optimal bone regeneration requires biologics (e.g. MSC) and using the correct scaffold incorporated into a local environment for bone regeneration. The need for an unlimited supply with high quality bone-graft substitutes continue to find alternatives for bone replacement surgery. MATERIALS AND METHODS: This in vitro study investigates cell seeding efficiency,metabolism,gene expression and growth behaviour of MSC sown on six commercially clinical available bone-graft substitutes in order to define their biological properties: synthetic silicate-substituted porous hydroxyapatite (Actifuse ABX),synthetic alpha-TCP (Biobase),synthetic beta-TCP (Vitoss),synthetic beta-TCP (Chronos),processed human cancellous allograft (Tutoplast) and processed bovines hydroxyapatite ceramic (Cerabone). 250,000 MSC derived from human bone marrow (n=4) were seeded onto the scaffolds,respectively. On days 2,6 and 10 the adherence of MSC (fluorescence microscopy) and cellular activity (MTT assay) were analysed. Osteogenic gene expression (cbfa-1) was analysed by RT-PCR and scanning electron microscopy was performed. RESULTS: The highest number of adhering cells was found on Tutoplast (e.g. day 6: 110.0+/-24.0 cells/microscopic field; ptextless0.05) followed by Chronos (47.5+/-19.5,ptextless0.05),Actifuse ABX (19.1+/-4.4),Biobase (15.7+/-9.9),Vitoss (8.8+/-8.7) and Cerabone (8.1+/-2.2). MSC seeded onto Tutoplast showed highest metabolic activity and gene expression of cbfa-1. These data are confirmed by scanning electron microscopy. The cell shapes varied from round-shaped cells to wide spread cells and cell clusters,depending on the bone-graft substitutes. Processed human cancellous allograft is a well-structured and biocompatible scaffold for ingrowing MSC in vitro. Of all other synthetical scaffolds,beta-tricalcium phosphate (Chronos) have shown the best growth behaviour for MSC. DISCUSSION: Our results indicate that various bone-graft substitutes influence cell seeding efficiency,metabolic activity and growth behaviour of MSC in different manners. We detected a high variety of cellular integration of MSC in vitro,which may be important for bony integration in the clinical setting. View Publication

Transforming growth factor (TGF)-beta superfamily proteins play a key role in the regulation of human embryonic stem cells (hESCs). Those of the TGFbeta/activin/nodal branch seem to support self-renewal and pluripotency,whereas those of the bone morphogenic protein (BMP) branch induce differentiation. In contrast to this generalization,we found that hESC remained undifferentiated after knockdown of SMAD4 with inducible short hairpin RNA interference,although the knockdown inhibited TGFbeta signaling and rendered the cells nonresponsive to BMP-induced differentiation. Moreover,the rapid differentiation of hESC after pharmacological inhibition of TGFbeta/activin/nodal receptor signaling was restricted after SMAD4 knockdown. These results suggest that TGFbeta/activin/nodal signaling supports the undifferentiated phenotype of hESC by suppressing BMP activity. During long-term culture,SMAD4 knockdown cell populations became less stable and more permissive to neural induction,a situation that was rescued by re-establishment of SMAD4 expression. These results suggest that SMAD4 is not required for maintenance of the undifferentiated state of hESC,but rather to stabilize that state. View Publication

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34(+) cells. This pure population of immature erythroblasts can be expanded to obtain 4x10(8) erythroblasts from 1x10(8) PBMC after 13-14 days in culture. Upon synchronized differentiation,high levels of enucleation (80-90%) and low levels of cell death (textless10%) are achieved. We compared the yield of erythroblasts obtained from PBMC,CD34(+) cells or PBMC depleted of CD34(+) cells and show that CD34(-) cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available. View Publication

Metabolic studies of human embryonic stem cells (hESCs) can provide important information for stem cell bioprocessing. To this end,we have examined growth and metabolism of hESCs in both traditional 2-dimensional (2D) colony cultures and 3-dimensional microcarrier cultures using a conditioned medium and 3 serum-free media. The 2D colony cultures plateaued at cell densities of 1.1-1.5 × 10�?� cells/mL at day 6 due to surface limitation. Microcarrier cultures achieved 1.5-2 × 10�?� cells/mL on days 8-10 before reaching a plateau; this growth arrest was not due to surface limitation,but probably due to metabolic limitations. Metabolic analysis of the cultures showed that amino acids (including glutamine) and glucose are in excess and are not limiting cell growth; on the other hand,the high levels of waste products (25 mM lactate and 0.8 mM ammonium) and low pH (6.6) obtained at the last stages of cell propagation could be the causes for growth arrest. hESCs cultured in media supplemented with lactate (up to 28 mM) showed reduced cell growth,whereas ammonium (up to 5 mM) had no effect. Lactate and,to a lesser extent,ammonia affected pluripotency as reflected by the decreasing population of cells expressing pluripotent marker TRA-1-60. Feeding hESC cultures with low concentrations of glucose resulted in lower lactate levels (∼10%) and a higher pH level of 6.7,which leads to a 40% increase in cell density. We conclude that the high lactate levels and the low pH during the last stages of high-density hESC culture may limit cell growth and affect pluripotency. To overcome this limitation,a controlled feed of low levels of glucose and online control of pH can be used. View Publication

PURPOSE: The existence of cancer stem cells (CSCs) in breast cancer has profound implications for cancer prevention. In this study,we evaluated sulforaphane,a natural compound derived from broccoli/broccoli sprouts,for its efficacy to inhibit breast CSCs and its potential mechanism. EXPERIMENTAL DESIGN: Aldefluor assay and mammosphere formation assay were used to evaluate the effect of sulforaphane on breast CSCs in vitro. A nonobese diabetic/severe combined immunodeficient xenograft model was used to determine whether sulforaphane could target breast CSCs in vivo,as assessed by Aldefluor assay,and tumor growth upon cell reimplantation in secondary mice. The potential mechanism was investigated using Western blotting analysis and beta-catenin reporter assay. RESULTS: Sulforaphane (1-5 micromol/L) decreased aldehyde dehydrogenase-positive cell population by 65% to 80% in human breast cancer cells (P textless 0.01) and reduced the size and number of primary mammospheres by 8- to 125-fold and 45% to 75% (P textless 0.01),respectively. Daily injection with 50 mg/kg sulforaphane for 2 weeks reduced aldehyde dehydrogenase-positive cells by textgreater50% in nonobese diabetic/severe combined immunodeficient xenograft tumors (P = 0.003). Sulforaphane eliminated breast CSCs in vivo,thereby abrogating tumor growth after the reimplantation of primary tumor cells into the secondary mice (P textless 0.01). Western blotting analysis and beta-catenin reporter assay showed that sulforaphane downregulated the Wnt/beta-catenin self-renewal pathway. CONCLUSIONS: Sulforaphane inhibits breast CSCs and downregulates the Wnt/beta-catenin self-renewal pathway. These findings support the use of sulforaphane for the chemoprevention of breast cancer stem cells and warrant further clinical evaluation. View Publication

It has been shown that genetic inhibition of p53 leads to enhanced proliferation of hematopoietic stem cells (HSCs). This could,in theory,contribute to the increased frequency of tumor development observed in p53-deficient mice and humans. In our previous work,we identified chemical p53 inhibitors (PFTs) that suppress the transactivation function of p53 and protect cultured cells and mice from death induced by gamma irradiation (IR). Here we found that when applied to bone marrow cells in vitro or injected into mice,PFTb impeded IR-induced reduction of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) population sizes. In addition,we showed that PFTb stimulated HSC and HPC proliferation in the absence of IR in vitro and in vivo and mobilized HSCs to the peripheral blood. Importantly,however,PFTb treatment did not affect the timing or frequency of tumor development in irradiated p53 heterozygous mice used as a model for determination of carcinogenicity. Thus,although PFTb administration led to increased numbers of HSCs and HPCs,it was not carcinogenic in mice. These findings suggest that chemical p53 inhibitors may be clinically useful as safe and effective stimulators of hematopoiesis. View Publication

First developed for hematologic disorders,the concept of cancer stem cells (CSCs) was expanded to solid tumors,including colorectal cancer (CRC). The traditional model of colon carcinogenesis includes several steps that occur via mutational activation of oncogenes and inactivation of tumor suppressor genes. Intestinal epithelial cells exist for a shorter amount of time than that required to accumulate tumor-inducing genetic changes,so researchers have investigated the concept that CRC arises from the long-lived stem cells,rather than from the differentiated epithelial cells. Colon CSCs were originally identified through the expression of the CD133 glycoprotein using an antibody directed to its epitope AC133. It is not clear if CD133 is a marker of colon CSCs-other cell surface markers,such as epithelial-specific antigen,CD44,CD166,Musashi-1,CD29,CD24,leucine-rich repeat-containing G-protein-coupled receptor 5,and aldehyde dehydrogenase 1,have been proposed. In addition to initiating and sustaining tumor growth,CSCs are believed to mediate cancer relapse after chemotherapy. How can we identify and analyze colon CSCs and what agents are being designed to kill this chemotherapy-refractory population? View Publication

Current induction schemes directing hematopoietic differentiation of human embryonic stem cells (hESCs) are not well defined to mimic the sequential stages of hematopoietic development in vivo. Here,we report a 3-stage method to direct differentiation of hESCs toward hematopoietic progenitors in chemically defined mediums. In the first 2 stages,we efficiently generated T-positive primitive streak/mesendoderm cells and kinase domain receptor-positive (KDR(+)) platelet-derived growth factor receptor α-negative (PDGFRα(-)) hemato-vascular precursors sequentially. In the third stage,we found that cells in a spontaneous differentiation condition mainly formed erythroid colonies. Addition of all-trans retinoic acid (RA) greatly enhanced generation of hematopoietic progenitors in this stage while suppressing erythroid development. The RA-treated cells highly expressed definitive hematopoietic genes,formed large numbers of multilineage and myeloid colonies,and gave rise to greater than 45% CD45(+) hematopoietic cells. When hematopoietic progenitors were selected with CD34 and C-Kit,greater than 95% CD45(+) hematopoietic cells could be generated. In addition,we found that endogenous RA signaling at the second stage was required for vascular endothelial growth factor/basic fibroblast growth factor-induced hemato-vascular specification,whereas exogenously applied RA efficiently induced KDR(-)PDGFRα(+) paraxial mesoderm cells. Our study suggests that RA signaling plays diverse roles in human mesoderm and hematopoietic development. View Publication

CD200 is a transmembrane protein broadly expressed on a variety of cell types,which delivers immunoregulatory signals through binding to receptors (CD200Rs) expressed on monocytes/myeloid cells and T lymphocytes. Signals delivered through the CD200:CD200R axis have been shown to play an important role in the regulation of anti-tumor immunity,and overexpression of CD200 has been reported in a number of malignancies,including CLL,as well as on cancer stem cells. We investigated the effect of CD200 blockade in vitro on a generation of CTL responses against a poorly immunogenic CD200+ lymphoma cell line and fresh cells obtained from CLL patients using anti-CD200 mAb and CD200-specific siRNAs. Suppression of functional expression of CD200 augmented killing of the CD200+ cells,as well as production of the inflammatory cytokines IFN-gamma and TNF-alpha by effector PBMCs. Killing was mediated by CD8+ cytotoxic T cells,and CD4+ T cells play an important role in CD200-mediated suppression of CTL responses. Our data suggest that CD200 blockade may represent a novel approach to clinical treatment of CLL. View Publication

Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties,and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4,Klf4,and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin,a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression,transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs,these epigenetically converted cells have growth properties,an X-chromosome activation state (XaXa),a gene expression profile,and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally,the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated naïve" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific� View Publication

HOXA9-mediated up-regulation of miR-155 was noted during an array-based analysis of microRNA expression in Hoxa9(-/-)bone marrow (BM) cells. HOXA9 induction of miR-155 was confirmed in these samples,as well as in wild-type versus Hoxa9-deficient marrow,using northern analysis and qRT-PCR. Infection of wild-type BM with HOXA9 expressing or GFP(+) control virus further confirmed HOXA9-mediated regulation of miR-155. miR-155 expression paralleled Hoxa9 mRNA expression in fractionated BM progenitors,being highest in the stem cell enriched pools. HOXA9 capacity to induce myeloid colony formation was blunted in miR-155-deficient BM cells,indicating that miR-155 is a downstream mediator of HOXA9 function in blood cells. Pu.1,an important regulator of myelopoiesis,was identified as a putative down stream target for miR-155. Although miR-155 was shown to down-regulate the Pu.1 protein,HOXA9 did not appear to modulate Pu.1 expression in murine BM cells. View Publication

Natural killer cells are lymphocytes specialized to participate in host defense through their innate ability to mediate cytotoxicity by secreting the contents of preformed secretory lysosomes (lytic granules) directly onto a target cell. This form of directed secretion requires the formation of an immunological synapse and occurs stepwise with actin reorganization preceding microtubule-organizing center (MTOC) polarization to the synapse. Because MTOC polarization to the synapse is required for polarization of lytic granules,we attempted to define their interrelationship. We found that compared with the time required for MTOC polarization,lytic granules converged to the MTOC rapidly. The MTOC-directed movement of lytic granules was independent of actin and microtubule reorganization,dependent on dynein motor function,occurred before MTOC polarization,and did not require a commitment to cytotoxicity. This defines a novel paradigm for rapid MTOC-directed transport as a prerequisite for directed secretion,one that may prepare,but not commit cells for precision secretory function. View Publication