参考文献

Although practiced clinically for more than 40 years,the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative,StemRegenin 1 (SR1),that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy. View Publication

The aryl hydrocarbon receptor (AhR),a transcription factor known for mediating xenobiotic toxicity,is expressed in B cells,which are known targets for environmental pollutants. However,it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up-regulated upon B-cell receptor (BCR) engagement and IL-4 treatment. Addition of a natural ligand of AhR,FICZ,induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1,showing that the AhR pathway is functional in B cells. AhR-deficient (Ahr(-/-)) B cells proliferate less than AhR-sufficient (Ahr(+/+)) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr(-/-) B cells are outcompeted by Ahr(+/+) cells. Transcriptome comparison of AhR-deficient and AhR-sufficient B cells identified cyclin O (Ccno),a direct target of AhR,as a top candidate affected by AhR deficiency. View Publication

Interleukin (IL)-23 is thought to be critical in the pathogenesis of psoriasis,and anti-IL-23 monoclonal antibodies (mAbs) have been approved for the treatment of psoriasis. We speculated that an anti-IL-23 receptor mAb might have greater efficacy than an anti-IL-23 mAb in the treatment of local inflamed lesions with high IL-23 levels. We previously generated an anti-human IL-23 receptor mAb,AS2762900-00,which potently blocked IL-23-induced cell proliferation,regardless of the concentration of IL-23. Here,we evaluated the therapeutic potential of AS2762900-00 in the treatment of psoriasis. Compared with untreated control,AS2762900-00 significantly reduced the epidermal thickness of lesions in a clinically relevant psoriatic human skin xenograft model. The expression of inflammatory genes including genes downstream of IL-23 signaling in the lesion tended to be lower in the AS2762900-00 group than the untreated group,suggesting that the inhibitory effects of AS2762900-00 in the psoriatic human skin xenograft model might occur via blockade of IL-23 signaling pathways. Further,AS2762900-00 showed an inhibitory effect on signal transducer and activator of transcription 3 (STAT3) phosphorylation as a downstream signal of IL-23 receptor activation in whole blood from patients with psoriasis. We also confirmed that AS2762900-00 inhibited IL-23-induced STAT3 phosphorylation in a concentration-dependent manner using whole blood from healthy donors. These data suggest that AS2762900-00 is a promising drug candidate for the treatment of psoriasis. In addition,STAT3 phosphorylation in whole blood may be a useful biomarker for the evaluation of the pharmacodynamic effects of AS2762900-00 in healthy volunteers in clinical development. View Publication

BACKGROUND Embryonic stem (ES) cells are capable of self-renewal and differentiation into cellular derivatives of all 3 germ layers. In appropriate culture conditions,ES cells can differentiate into specialized cells,including cardiac myocytes,but the efficiency is typically low and the process is incompletely understood. METHODS AND RESULTS We evaluated a chemical library for its potential to induce cardiac differentiation of ES cells in the absence of embryoid body formation. Using ES cells stably transfected with cardiac-specific alpha-cardiac myosin heavy chain (MHC) promoter-driven enhanced green fluorescent protein (EGFP),880 compounds approved for human use were screened for their ability to induce cardiac differentiation. Treatment with ascorbic acid,also known as vitamin C,markedly increased the number of EGFP-positive cells,which displayed spontaneous and rhythmic contractile activity and stained positively for sarcomeric myosin and alpha-actinin. Furthermore,ascorbic acid induced the expression of cardiac genes,including GATA4,alpha-MHC,and beta-MHC in untransfected ES cells in a developmentally controlled manner. This effect of ascorbic acid on cardiac differentiation was not mimicked by the other antioxidants such as N-acetylcysteine,Tiron,or vitamin E. CONCLUSIONS Ascorbic acid induces cardiac differentiation in ES cells. This study demonstrates the potential for chemically modifying the cardiac differentiation program of ES cells. View Publication

The generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster,compromising the ability to generate entirely iPSC-derived adult mice ('all-iPSC mice'). Here,we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This approach allowed us to generate all-iPSC mice from mature B cells,which have until now failed to support the development of exclusively iPSC-derived postnatal animals. Our data show that transcription factor-mediated reprogramming can endow a defined,terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally,these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of the resultant iPSCs. View Publication

Fibroblast growth factor receptor 1 (Fgfr1) gene knockout impairs cardiomyocyte differentiation in murine embryonic stem cells (mESC). Here,various chemical compounds able to enhance cardiomyocyte differentiation in mESC [including dimethylsulfoxide,ascorbic acid (vitC),free radicals and reactive oxygen species] were tested for their ability to rescue the cardiomyogenic potential of Fgfr1(-/-) mESC. Among them,only the reduced form of vitC,l-ascorbic acid,was able to recover beating cell differentiation in Fgfr1(-/-) mESC. The appearance of contracting cells was paralleled by the expression of early and late cardiac gene markers,thus suggesting their identity as cardiomyocytes. In the attempt to elucidate the mechanism of action of vitC on Fgfr1(-/-) mESC,we analyzed several parameters related to the intracellular redox state,such as reactive oxygen species content,Nox4 expression,and superoxide dismutase activity. The results did not show any relationship between the antioxidant capacity of vitC and cardiomyocyte differentiation in Fgfr1(-/-) mESC. No correlation was found also for the ability of vitC to modulate the expression of pluripotency genes. Then,we tested the hypothesis that vitC was acting as a prolyl hydroxylase cofactor by maintaining iron in a reduced state. We first analyze hypoxia inducible factor (HIF)-1α mRNA and protein levels that were found to be slightly upregulated in Fgfr1(-/-) cells. We treated mESC with Fe(2+) or the HIF inhibitor CAY10585 during the first phases of the differentiation process and,similar to vitC,the two compounds were able to rescue cardiomyocyte formation in Fgfr1(-/-) mESC,thus implicating HIF-1α modulation in Fgfr1-dependent cardiomyogenesis. View Publication

Since the discovery of vitamin C,the number of its known biological functions is continually expanding. Both the names ascorbic acid and vitamin C reflect its antiscorbutic properties due to its role in the synthesis of collagen in connective tissues. Ascorbate acts as an electron-donor keeping iron in the ferrous state thereby maintaining the full activity of collagen hydroxylases; parallel reactions with a variety of dioxygenases affect the expression of a wide array of genes,for example via the HIF system,as well as via the epigenetic landscape of cells and tissues. In fact,all known physiological and biochemical functions of ascorbate are due to its action as an electron donor. The ability to donate one or two electrons makes AscH(-) an excellent reducing agent and antioxidant. Ascorbate readily undergoes pH-dependent autoxidation producing hydrogen peroxide (H(2)O(2)). In the presence of catalytic metals this oxidation is accelerated. In this review,we show that the chemical and biochemical nature of ascorbate contribute to its antioxidant as well as its prooxidant properties. Recent pharmacokinetic data indicate that intravenous (i.v.) administration of ascorbate bypasses the tight control of the gut producing highly elevated plasma levels; ascorbate at very high levels can act as prodrug to deliver a significant flux of H(2)O(2) to tumors. This new knowledge has rekindled interest and spurred new research into the clinical potential of pharmacological ascorbate. Knowledge and understanding of the mechanisms of action of pharmacological ascorbate bring a rationale to its use to treat disease especially the use of i.v. delivery of pharmacological ascorbate as an adjuvant in the treatment of cancer. View Publication

Vitamin C (ascorbic acid (AA)) is very popular for its antioxidant properties. Consequently,many other important aspects of this multifaceted molecule are often underestimated or even ignored. In the present paper,we have tried to bring to the foreground some of these aspects,including the peculiarities of the AA biosynthetic pathway in different organisms,the remarkable function of AA as a co-substrate of many important dioxygenases,the role of AA-regenerating enzymes and the known pathways of AA catabolism,as well as the intriguing function of AA in gene expression. View Publication

Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro,but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal,we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray,and tested their ability to perform mature hepatocyte functions (albumin and urea secretion,cytochrome activity). By these measures,ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. View Publication

Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia,the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment,although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features,stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines,non-malignant cells,3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays,flow cytometry,colony and spheroid formation assays,Western blot analysis,antibody protein arrays,electrophoretic mobility shift assays (EMSAs),immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability,self-renewal potential,and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo,and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression,inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly,aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells,chick embryos or mice. These results highlight aspirin as an effective,inexpensive and well-tolerated co-treatment to target inflammation,desmoplasia and CSC features PDA. View Publication