技术资料

Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible,defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines,MEL1,MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up. View Publication

BACKGROUND AND AIMS: Atherosclerosis is known to be an inflammatory disease and there is increasing evidence that chylomicron remnants (CMR),the lipoproteins which carry dietary fats in the blood,cause macrophage foam cell formation and inflammation. In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is increased,and clearance of CMR from blood may be delayed,however,whether CMR contribute directly to monocyte activation and subsequent egress into the arterial wall has not been established. Here,the contribution of CMR to activation of monocyte pro-inflammatory pathways was assessed using an in vitro model. METHODS AND RESULTS: Primary human monocytes and CMR-like particles (CRLP) were used to measure several endpoints of monocyte activation. Treatment with CRLP caused rapid and prolonged generation of reactive oxygen species by monocytes. The pro-inflammatory chemokines MCP-1 and IL-8 were secreted in nanogram quantities by the cells in the absence of CRLP. IL-8 secretion was transiently increased after CRLP treatment,and CRLP maintained secretion in the presence of pharmacological inhibitors of IL-8 production. In contrast,exposure to CRLP significantly reduced MCP-1 secretion. Chemotaxis towards MCP-1 was increased in monocytes pre-exposed to CRLP and was reversed by addition of exogenous MCP-1. CONCLUSION: Our findings indicate that CRLP activate human monocytes and augment their migration in vitro by reducing cellular MCP-1 expression. Our data support the current hypothesis that CMR contribute to the inflammatory milieu of the arterial wall in early atherosclerosis,and suggest that this may reflect direct interaction with circulating blood monocytes. View Publication

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program,by reprogramming of a more differentiated cell type by oncogenic insults,or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5,K14,and vimentin),luminal (E-cadherin,K8,K18,or K19),and stem/progenitor (CD49f,CD29,CD44,and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways,such as Notch,Wnt,Hedgehog,and LIF,in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers,these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer. View Publication

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy with one of the worst outcomes among all cancers. PDA often recurs after initial treatment to result in patient death despite the use of chemotherapy or radiation therapy. PDA contains a subset of tumor-initiating cells capable of extensive self-renewal known as cancer stem cells (CSC),which may contribute to therapeutic resistance and metastasis. At present,conventional chemotherapy and radiotherapy are largely ineffective in depleting CSC pool,suggesting the need for novel therapies that specifically target the cancer-sustaining stem cells for tumor eradication and to improve the poor prognosis of PDA patients. In this study,we report that death receptor 5 (DR5) is enriched in pancreatic CSCs compared with the bulk of the tumor cells. Treating a collection of freshly generated patient-derived PDA xenografts with gemcitabine,the first-line chemotherapeutic agent for PDA,is initially effective in reducing tumor size,but largely ineffective in diminishing the CSC populations,and eventually culminated in tumor relapse. However,a combination of tigatuzumab,a fully humanized DR5 agonist monoclonal antibody,with gemcitabine proved to be more efficacious by providing a double hit to kill both CSCs and bulk tumor cells. The combination therapy produced remarkable reduction in pancreatic CSCs,tumor remissions,and significant improvements in time to tumor progression in a model that is considered more difficult to treat. These data provide the rationale to explore the DR5-directed therapies in combination with chemotherapy as a therapeutic option to improve the current standard of care for pancreatic cancer patients. View Publication

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state,and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics,these two reprogramming methods reset genomic methylation,an epigenetic modification of DNA that influences gene expression,leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin,which favours their differentiation along lineages related to the donor cell,while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming,or by treatment of iPSCs with chromatin-modifying drugs. In contrast,the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming,which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment. View Publication

Self-renewal and differentiation of hematopoietic stem cells (HSCs) are balanced by the concerted activities of the fibroblast growth factor (FGF),Wnt,and Notch pathways,which are tuned by enzyme-mediated remodeling of heparan sulfate proteoglycans (HSPGs). Sulfatase modifying factor 1 (SUMF1) activates the Sulf1 and Sulf2 sulfatases that remodel the HSPGs,and is mutated in patients with multiple sulfatase deficiency. Here,we show that the FGF signaling pathway is constitutively activated in Sumf1(-/-) HSCs and hematopoietic stem progenitor cells (HSPCs). These cells show increased p-extracellular signal-regulated kinase levels,which in turn promote beta-catenin accumulation. Constitutive activation of FGF signaling results in a block in erythroid differentiation at the chromatophilic erythroblast stage,and of B lymphocyte differentiation at the pro-B cell stage. A reduction in mature myeloid cells and an aberrant development of T lymphocytes are also seen. These defects are rescued in vivo by blocking the FGF pathway in Sumf1(-/-) mice. Transplantation of Sumf1(-/-) HSPCs into wild-type mice reconstituted the phenotype of the donors,suggesting a cell autonomous defect. These data indicate that Sumf1 controls HSPC differentiation and hematopoietic lineage development through FGF and Wnt signaling. View Publication

AIMS: Macrophage scavenger receptor A (SR-A) and class B scavenger receptor CD36 (CD36) contribute to foam cell formation and atherogenesis via uptake of modified lipoproteins. So far,the role of these scavenger receptors has been studied mainly using knockout models totally lacking these receptors. We studied the role of SR-A and CD36 in foam cell formation and atherogenesis by RNA interference (RNAi)-mediated silencing,which is a clinically feasible method to down-regulate the expression of these receptors. METHODS AND RESULTS: We constructed lentivirus vectors encoding short hairpin RNAs (shRNAs) against mouse SR-A and CD36. Decreased SR-A but not CD36 expression led to reduced foam cell formation caused by acetylated low-density lipoprotein (LDL) in mouse macrophages,whereas the uptake of oxidized LDL was not altered. More importantly,silencing of SR-A upregulates CD36 and vice versa. In LDL receptor-deficient apolipoprotein B100 (LDLR(-/-)ApoB(100/100)) mice kept on a western diet,silencing of either SR-A or CD36 in bone marrow cells led to a marked decrease (37.4 and 34.2%,respectively) in cross-sectional lesion area,whereas simultaneous silencing of both receptors was not effective. CONCLUSION: Our results suggest that silencing of either SR-A or CD36 alone reduces atherogenesis in mice. However,due to reciprocal upregulation,silencing of both SR-A and CD36 is not effective. View Publication

OBJECTIVE: The aim of this narrative review was to evaluate the role of cancer stem cells (CSCs) and sex steroids in the pathophysiology of human breast cancer. METHODS: A key-word search was performed using the Scopus database. Preference was given to studies using human cells and tissues. RESULTS: Long-term estrogen-progestin hormone therapy is known to increase breast cancer risk,although the mechanisms are poorly understood. In the last few years,it has become clear that many human breast cancers contain CSCs,which may be responsible for much of the tumor's malignant behavior. Very recently,the impact of estrogen,progesterone,and progestins on breast CSCs and their progeny has been studied and clarified. Most breast CSCs are estrogen receptor negative and progesterone receptor negative,although some intermediary progenitor forms have hormone receptors,especially progesterone receptor. Most mature human breast cancer cellsare estrogen receptor positive and can thus be stimulated by estrogen. Breast CSCs usually elaborate CD44+,CD24j/low and/or ALDEFLUOR+ cell markers and are lineage markers negative. One of the main roles of progesterone and progestin seems to be on certain breast cancer stem intermediate forms,inducing them to revert back to a more primitive breast CSC form. CONCLUSIONS: As the pathophysiology of human breast CSC is clarified,it is probable that this will lead to novel,effective breast cancer treatments and,perhaps,new breast cancer preventive agents. This research may also lead to safer hormone therapy regimens. View Publication

The longevity-promoting NAD+-dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4,TBX3,and PAX6,which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells. View Publication

AIMS: Endothelial progenitor cells (EPC) have been shown to repair pulmonary endothelium,although they can also migrate into the arterial intima and differentiate into smooth muscle-like (mesenchymal) cells contributing to intimal hyperplasia. The molecular mechanisms by which this process proceeds have not been fully elucidated. Here,we study whether genes involved in the endothelial-to-mesenchymal transition (EnMT) may contribute to the mesenchymal phenotype acquisition of EPC and we evaluate whether transforming growth factor β1 (TGFβ1) is involved in this process. METHODS AND RESULTS: Our results show that co-culture of EPC with smooth muscle cells (SMC) increases the expression of the mesenchymal cell markers α-smooth muscle actin,sm22-α,and myocardin,and decreases the expression of the endothelial cell marker CD31. In the same conditions,we also observed a concomitant increase in the gene expression of the EnMT-related transcription factors: slug,snail,zeb1,and endothelin-1. This indicates that mesenchymal phenotype acquisition occurred through an EnMT-like process. Inhibition of TGFβ receptor I (TGFβRI) downregulated snail gene expression,blocked the EnMT,and facilitated the differentiation of EPC to the endothelial cell lineage. Furthermore,TGFβRI inhibition decreased migration of EPC stimulated by SMC without affecting their functionality and adhesion capacity. CONCLUSION: These results indicate that EPC may differentiate into SMC-like cells through an EnMT-like process and that TGFβI plays an important role in the fate of EPC. View Publication

In recent years,human embryonic stem (hES) cells have become a promising cell source for regenerative medicine. Although hES cells have the ability for unlimited self-renewal,potential adverse effects of long-term cell culture upon hES cells must be investigated before therapeutic applications of hES cells can be realized. Here we investigated changes in molecular profiles associated with young (textless60 passages) and old (textgreater120 passages) cells of the H9 hES cell line as well as young (textless85 passages) and old (textgreater120 passages) cells of the PKU1 hES cell line. Our results show that morphology,stem cell markers,and telomerase activity do not differ significantly between young and old passage cells. Cells from both age groups were also shown to differentiate into derivatives of all 3 germ layers upon spontaneous differentiation in vitro. Interestingly,mitochondrial dysfunction was found to occur with prolonged culture. Old passage cells of both the H9 and PKU1 lines were characterized by higher mitochondrial membrane potential,larger mitochondrial morphology,and higher reactive oxygen species content than their younger counterparts. Teratomas derived from higher passage cells were also found to have an uneven preference for differentiation compared with tumors derived from younger cells. These findings suggest that prolonged culture of hES cells may negatively impact mitochondrial function and possibly affect long-term pluripotency. View Publication

Glioblastoma (GBM) is the most aggressive primary brain tumor and is resistant to all therapeutic regimens. Relapse occurs regularly and might be caused by a poorly characterized tumor stem cell (TSC) subpopulation escaping therapy. We suggest aldehyde dehydrogenase 1 (ALDH1) as a novel stem cell marker in human GBM. Using the neurosphere formation assay as a functional method to identify brain TSCs,we show that high protein levels of ALDH1 facilitate neurosphere formation in established GBM cell lines. Even single ALDH1 positive cells give rise to colonies and neurospheres. Consequently,the inhibition of ALDH1 in vitro decreases both the number of neurospheres and their size. Cell lines without expression of ALDH1 do not form tumor spheroids under the same culturing conditions. High levels of ALDH1 seem to keep tumor cells in an undifferentiated,stem cell-like state indicated by the low expression of beta-III-tubulin. In contrast,ALDH1 inhibition induces premature cellular differentiation and reduces clonogenic capacity. Primary cell cultures obtained from fresh tumor samples approve the established GBM cell line results. View Publication