技术资料

Stem or progenitor cell populations are often established in unique niche microenvironments that regulate cell fate decisions. Although niches have been shown to be critical for the normal development of several tissues,their role in the cardiovascular system is poorly understood. In this study,we characterized the cardiovascular progenitor cell (CPC) niche in developing human and mouse hearts,identifying signaling pathways and extracellular matrix (ECM) proteins that are crucial for CPC maintenance and expansion. We demonstrate that collagen IV (ColIV) and β-catenin-dependent signaling are essential for maintaining and expanding undifferentiated CPCs. Since niches are three-dimensional (3D) structures,we investigated the impact of a 3D microenvironment that mimics the in vivo niche ECM. Employing electrospinning technologies,3D in vitro niche substrates were bioengineered to serve as culture inserts. The three-dimensionality of these structures increased mouse embryonic stem cell differentiation into CPCs when compared to 2D control cultures,which was further enhanced by incorporation of ColIV into the substrates. Inhibiting p300-dependent β-catenin signals with the small molecule IQ1 facilitated further expansion of CPCs. Our study represents an innovative approach to bioengineer cardiac niches that can serve as unique 3D in vitro systems to facilitate CPC expansion and study CPC biology. View Publication

Sox6 belongs to the Sry (sex-determining region Y)-related high-mobility-group-box family of transcription factors,which control cell-fate specification of many cell types. Here,we explored the role of Sox6 in human erythropoiesis by its overexpression both in the erythroleukemic K562 cell line and in primary erythroid cultures from human cord blood CD34+ cells. Sox6 induced significant erythroid differentiation in both models. K562 cells underwent hemoglobinization and,despite their leukemic origin,died within 9 days after transduction; primary erythroid cultures accelerated their kinetics of erythroid maturation and increased the number of cells that reached the final enucleation step. Searching for direct Sox6 targets,we found SOCS3 (suppressor of cytokine signaling-3),a known mediator of cytokine response. Sox6 was bound in vitro and in vivo to an evolutionarily conserved regulatory SOCS3 element,which induced transcriptional activation. SOCS3 overexpression in K562 cells and in primary erythroid cells recapitulated the growth inhibition induced by Sox6,which demonstrates that SOCS3 is a relevant Sox6 effector. View Publication

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins,the widely expressed basic helix-loop-helix transcription factors,contribute to HSC and MPP activity,but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches,we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However,long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover,E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism,and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together,these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism,and demonstrate that E47 is not required for short-term myeloid differentiation. View Publication

A major goal of stem-cell research is to identify conditions that reliably regulate their differentiation into specific cell types. This goal is particularly important for human stem cells if they are to be used for in vivo transplantation or as a platform for drug development. Here we describe the establishment of procedures to direct the differentiation of human embryonic stem cells and human induced pluripotent stem cells into forebrain neurons that are capable of forming synaptic connections. In addition,HEK293T cells expressing Neuroligin (NLGN) 3 and NLGN4,but not those containing autism-associated mutations,are able to induce presynaptic differentiation in human induced pluripotent stem cell-derived neurons. We show that a mutant NLGN4 containing an in-frame deletion is unable to localize correctly to the cell surface when overexpressed and fails to enhance synapse formation in human induced pluripotent stem cell-derived neurons. These findings establish human pluripotent stem cell-derived neurons as a viable model for the study of synaptic differentiation and function under normal and disorder-associated conditions. View Publication

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies,but is absent on normal tissues,including hematopoietic progenitor cells,and may therefore be an appropriate candidate for T cell-mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity,PRAME-specific cytotoxic T lymphocytes (CTLs),we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal,PRAME-specific CTL lines and elicited high-avidity CTLs,with a high proportion of cells recognizing a previously uninvestigated HLA-A*02-restricted epitope,P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME(+) hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts,but also against leukemic progenitor cells as assessed by colony-forming-inhibition assays,which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors,indicating that this approach may be of value for immunotherapy of PRAME(+) hematologic malignancies. View Publication

BACKGROUND: Cancer stem cells (CSCs) have been shown to be a small stem cell-like cell population which appears to drive tumorigenesis,tumor recurrence and metastasis. Thus,identification and characterization of CSCs may be critical to defining effective anticancer therapies. In prostate cancer (PCa),the CD44(+) cell population appears to have stem cell-like properties including being tumorigenic. The enzyme aldehyde dehydrogenase (ALDH) has been found to identify hematopoietic stem cells and our aim was to determine the utility of ALDH activity and CD44 in identifying PCa stem cell-like cells in PCa cell lines. MATERIALS AND METHODS: LNCaP cells and PC-3 cells were sorted based on their expression of CD44 and ALDH activity. The cell populations were investigated using colony-forming assays,invasion assays,sphere formation experiments in a non-adherent environment and 3-D Matrigel matrix culture to observe the in vitro stem-cell like properties. Different sorted cell populations were injected subcutaneously into NOD/SCID mice to determine the corresponding tumorigenic capacities. RESULTS: ALDH(hi) CD44(+) cells exhibit a higher proliferative,clonogenic and metastatic capacity in vitro and demonstrate higher tumorigenicity capacity in vivo than did ALDH(lo) CD44(-) cells. The tumors recapitulated the population of the original cell line. However,ALDHlo CD44(-) cells were able to develop tumors,albeit with longer latency periods. CONCLUSION: ALDH activity and CD44 do not appear to identify PCa stem cells; however,they do indicate increased tumorigenic and metastatic potential,indicating their potential importance for further exploration. View Publication

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However,the well-known limitations of current reprogramming technologies include low efficiency,slow kinetics,and transgene integration and residual expression. In the present study,we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using non-integrating episomal vectors. The reprogramming described here is up to 100 times more efficient,occurs 1-3 weeks faster compared with the reprogramming of fibroblasts,and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR,indicating that their origin is non-B- or non-T-lymphoid cells. When cocultured on OP9,blood-derived iPSCs could be differentiated back to the blood cells,albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods. View Publication

AIMS Pifithrin α (PFTα),an inhibitor of the p53 protein,is regarded as a lead compound for cancer and neurodegenerative disease therapy. There is some evidence that this compound activates the aryl hydrocarbon receptor (AhR) in a complete independent way of the p53 inhibition and that it is easily converted to its condensation product pifithrin β (PFTβ). The aim of this study was to explore the ability of PFTα and of PFTβ to induce a variety of AhR mediated processes. MAIN METHODS Computational analysis using quantum chemical calculations and chemical analysis have been used to study the conformation of the compounds as well as the cyclization reaction. The AhR mediated processes of these compounds have been studied in a rainbow trout cell line (RTG-2) and in a rat hepatoma cell line (H4IIE). KEY FINDINGS PFTα molecule could not take a planar conformation required for AhR activation whereas PFTβ showed a conformation similar to those of the prototypical AhR ligand β-naphthoflavone. In both cell lines,PFTα and PFTβ provoked different responses related with AhR activation. However,when cyclization of PFTα to PFTβ was hampered by acetylation of the exocyclic nitrogen,all these responses were not observed. These results lead to the conclusion that the activation of the AhR is probably caused by PFTβ instead of PFTα. SIGNIFICANCE Since PFTα is a promising compound for the development of new pharmaceuticals inhibiting p53,the chemical instability of this compound as well as the capacity of its transformation product should be taken into account. View Publication

Epithelial-to-mesenchymal transition (EMT) is strongly associated with cancer progression,but its potential role during premalignant development has not been studied. Here,we show that a 4-week exposure of immortalized human bronchial epithelial cells (HBEC) to tobacco carcinogens can induce a persistent,irreversible,and multifaceted dedifferentiation program marked by EMT and the emergence of stem cell-like properties. EMT induction was epigenetically driven,initially by chromatin remodeling through H3K27me3 enrichment and later by ensuing DNA methylation to sustain silencing of tumor-suppressive microRNAs (miRNA),miR-200b,miR-200c,and miR-205,which were implicated in the dedifferentiation program in HBECs and also in primary lung tumors. Carcinogen-treated HBECs acquired stem cell-like features characterized by their ability to form spheroids with branching tubules and enrichment of the CD44(high)/CD24(low),CD133,and ALDH1 stem cell-like markers. miRNA overexpression studies indicated that regulation of the EMT,stem-like,and transformed phenotypes in HBECs were distinct events. Our findings extend present concepts of how EMT participates in cancer pathophysiology by showing that EMT induction can participate in cancer initiation to promote the clonal expansion of premalignant lung epithelial cells. View Publication

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However,despite an overall significant hematological and cytogenetic response,imatinib therapy may favor the emergence of drug-resistant clones,ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression,either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid,a clinically used drug. Furthermore,we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore,combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease. View Publication

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods,it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous,nonsense or splice variants,and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies,whereas the rest occurred during or after reprogramming. Thus,hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use. View Publication

PURPOSE: The combination of erlotinib and gemcitabine has shown a small but statistically significant survival advantage when compared with gemcitabine alone in patients with advanced pancreatic cancer. However,the overall survival rate with the erlotinib and gemcitabine combination is still low. In this study,we sought to identify gene targets that,when inhibited,would enhance the activity of epidermal growth factor receptor (EGFR)-targeted therapies in pancreatic cancer cells. EXPERIMENTAL DESIGN: A high-throughput RNA interference (RNAi) screen was carried out to identify candidate genes. Selected gene hits were further confirmed and mechanisms of action were further investigated using various assays. RESULTS: Six gene hits from siRNA screening were confirmed to significantly sensitize BxPC-3 pancreatic cancer cells to erlotinib. One of the hits,mitogen-activated protein kinase (MAPK) 1,was selected for further mechanistic studies. Combination treatments of erlotinib and two MAP kinase kinase (MEK) inhibitors,RDEA119 and AZD6244,showed significant synergistic effect for both combinations (RDEA119-erlotinib and AZD6244-erlotinib) compared with the corresponding single drug treatments in pancreatic cancer cell lines with wild-type KRAS (BxPC-3 and Hs 700T) but not in cell lines with mutant KRAS (MIA PaCa-2 and PANC-1). The enhanced antitumor activity of the combination treatment was further verified in the BxPC-3 and MIA PaCa-2 mouse xenograft model. Examination of the MAPK signaling pathway by Western blotting indicated effective inhibition of the EGFR signaling by the drug combination in KRAS wild-type cells but not in KRAS mutant cells. CONCLUSIONS: Overall,our results suggest that combination therapy of an EGFR and MEK inhibitors may have enhanced efficacy in patients with pancreatic cancer. View Publication