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Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently,differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately,differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether,our results provide a new platform for the study of kidney diseases and lineage commitment,and open new avenues for the future application of regenerative strategies in the clinic. View Publication

There is little information on the function of epididymal basal cells. These cells secrete prostaglandins,can metabolize radical oxygen species,and have apical projections that are components of the blood-epididymis barrier. The objective of this study was to develop a reproducible protocol to isolate rat epididymal basal cells and to characterize their function by gene expression profiling. Integrin-alpha6 was used to isolate a highly purified population of basal cells. Microarray analysis indicated that expression levels of 552 genes were enriched in basal cells relative to other cell types. Among these genes,45 were expressed at levels of 5-fold or greater. These highly expressed genes coded for proteins implicated in cell adhesion,cytoskeletal function,ion transport,cellular signaling,and epidermal function,and included proteases and antiproteases,signal transduction,and transcription factors. Several highly expressed genes have been reported in adult stem cells,suggesting that basal cells may represent an epididymal stem cell population. A basal cell culture was established that showed that these basal cells can differentiate in vitro from keratin (KRT) 5-positive cells to cells that express KRT8 and connexin 26,a marker of columnar cells. These data provide novel information on epididymal basal cell gene expression and suggest that these cells can act as adult stem cells. View Publication

BACKGROUND Adoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies,yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients,but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo,circumventing TLR-related toxicity. METHODS In this study we investigated how tumor-specific murine CD8+ T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG. RESULTS Herein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+ T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist,CpG,commonly used in the clinic,to bolster T cell-B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination,which are classically required for effective ACT therapy. CpG-conditioned CD8+ T cells acquired a unique proteomic signature hallmarked by an IL-2R$\alpha$highICOShighCD39low phenotype and an altered metabolic profile,all reliant on B cells transiently present in the culture. Likewise,human TILs benefitted from expansion with CpG ex vivo,as they also possessed the IL-2R$\alpha$highICOShighCD39low phenotype. CpG fostered the expansion of potent CD8+ T cells with the signature phenotype and antitumor ability via empowering a direct B-T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture. CONCLUSIONS Our results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+ T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors. View Publication

Interleukin-15 (IL-15) is essential for natural killer (NK) cell differentiation. In this study,we assessed whether the receptor tyrosine kinase Axl and its ligand,Gas6,are involved in IL-15-mediated human NK differentiation from CD34(+) hematopoietic progenitor cells (HPCs). Blocking the Axl-Gas6 interaction with a soluble Axl fusion protein (Axl-Fc) or the vitamin K inhibitor warfarin significantly diminished the absolute number and percentage of CD3(-)CD56(+) NK cells derived from human CD34(+) HPCs cultured in the presence of IL-15,probably resulting in part from reduced phosphorylation of STAT5. In addition,CD3(-)CD56(+) NK cells derived from culture of CD34(+) HPCs with IL-15 and Axl-Fc had a significantly diminished capacity to express interferon-gamma or its master regulator,T-BET. Culture of CD34(+) HPCs in the presence of c-Kit ligand and Axl-Fc resulted in a significant decrease in the frequency of NK precursor cells responding to IL-15,probably the result of reduced c-Kit phosphorylation. Collectively,our data suggest that the Axl/Gas6 pathway contributes to normal human NK-cell development,at least in part via its regulatory effects on both the IL-15 and c-Kit signaling pathways in CD34(+) HPCs,and to functional NK-cell maturation via an effect on the master regulatory transcription factor T-BET. View Publication

There is an enormous gap between the antiproliferative and in vivo antitumor efficacy of gemcitabine in cell line-based models and its clinical efficacy. This may be due to insensitiveness of the precursor,cancer stem cell (CSC) compartment to cytotoxic agents. The hedgehog pathway is associated with CSC signaling and control. We used a direct xenograft model of pancreatic cancer and a two-stage approach was used to test the hypotheses that targeting CSC could increase the efficacy of gemcitabine. Tumors from a gemcitabine-sensitive xenograft were treated with gemcitabine first,and randomized,after tumor regression to continuing treatment with gemcitabine,a hedgehog inhibitor alone or in combination with gemcitabine. We tested markers described as associated with CSC such as CD24,CD44,ALDH,nestin,and the hedgehog pathway. After induction with gemcitabine,treated tumor showed an enrichment in CSC markers such as ALDH and CD24. Subsequently,a release from gemcitabine prompted a repopulation of proliferating cells and a decrease in such markers to equilibrate from pretreatment levels. Combined treatment with gemcitabine and cyclopamine induced tumor regression and decrease in CSC markers and hedgehog signaling. Cytoplasmic CD24 and ALDH were inversely and strongly associated with growth and were expressed in a minority of cells that we propose constitute the CSC compartment. Hedgehog inhibitors as part of a dual compartment therapeutic approach were able to further reduce tumor growth and decreased both static and dynamic markers of CSC. Direct tumor xenografts are a valid platform to test multicompartment therapeutic approaches in pancreatic cancer. View Publication

PURPOSE: To evaluate the expression of stem cell-related markers at the cellular level in human breast tumors of different subtypes and histologic stage. EXPERIMENTAL DESIGN: We performed immunohistochemical analyses of 12 proteins [CD44,CD24,ALDH1,vimentin,osteonectin,EPCR,caveolin 1,connexin 43,cytokeratin 18 (CK18),MUC1,claudin 7,and GATA3] selected based on their differential expression in breast cancer cells with more differentiated and stem cell-like characteristics in 47 cases of invasive ductal carcinoma (IDC) only,135 cases of IDC with ductal carcinoma in situ (DCIS),35 cases of DCIS with microinvasion,and 58 cases of pure DCIS. We also analyzed 73 IDCs with adjacent DCIS to determine the differences in the expression of markers by histology within individual tumors. CD44+/CD24- and CD24-/CD24+ cells were detected using double immunohistochemistry. RESULTS: CD44 and EPCR expression was different among the four histologic groups and was lower in invasive compared with in situ tumors,especially in luminal A subtype. The expression of vimentin,osteonectin,connexin 43,ALDH1,CK18,GATA3,and MUC1 differed by tumor subtype in some histologic groups. ALDH1-positive cells were more frequent in basal-like and HER2+ than in luminal tumors. CD44+/CD24- cells were detected in 69% of all tumors with 100% of the basal-like and 52% of HER2+ tumors having some of these cells. CONCLUSIONS: Our findings suggest that in breast cancer,the frequency of tumor cells positive for stem cell-like and more differentiated cell markers varies according to tumor subtype and histologic stage. View Publication

Raman spectra often contain undesirable,randomly positioned,intense,narrow-bandwidth,positive,unidirectional spectral features generated when cosmic rays strike charge-coupled device cameras. These must be removed prior to analysis,but doing so manually is not feasible for large data sets. We developed a quick,simple,effective,semi-automated procedure to remove cosmic ray spikes from spectral data sets that contain large numbers of relatively homogenous spectra. Although some inhomogeneous spectral data sets can be accommodated—it requires replacing excessively modified spectra with the originals and removing their spikes with a median filter instead—caution is advised when processing such data sets. In addition,the technique is suitable for interpolating missing spectra or replacing aberrant spectra with good spectral estimates. The method is applied to baseline-flattened spectra and relies on fitting a third-order (or higher) polynomial through all the spectra at every wavenumber. Pixel intensities in excess of a threshold of 3× the noise standard deviation above the fit are reduced to the threshold level. Because only two parameters (with readily specified default values) might require further adjustment,the method is easily implemented for semi-automated processing of large spectral sets. View Publication

The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) is caused by a CAG-repeat expansion in the ATXN3 gene. In this study,induced pluripotent stem cell (iPSC) lines were established from two SCA3 patients. Dermal fibroblasts were reprogrammed using an integration-free method and the resulting SCA3 iPSCs were differentiated into neurons. These neuronal lines harbored the disease causing mutation,expressed comparable levels of several neuronal markers and responded to the neurotransmitters,glutamate/glycine,GABA and acetylcholine. Additionally,all neuronal cultures formed networks displaying synchronized spontaneous calcium oscillations within 28days of maturation,and expressed the mature neuronal markers NeuN and Synapsin 1 implying a relatively advanced state of maturity,although not comparable to that of the adult human brain. Interestingly,we were not able to recapitulate the glutamate-induced ataxin-3 aggregation shown in a previously published iPSC-derived SCA3 model. In conclusion,we have generated a panel of SCA3 patient iPSCs and a robust protocol to derive neurons of relatively advanced maturity,which could potentially be valuable for the study of SCA3 disease mechanisms. View Publication

Thiazolidinedione derivatives are antidiabetic agents that increase the insulin sensitivity of target tissues in animal models of non-insulin-dependent diabetes mellitus. In vitro,thiazolidinediones promote adipocyte differentiation of preadipocyte and mesenchymal stem cell lines; however,the molecular basis for this adipogenic effect has remained unclear. Here,we report that thiazolidinediones are potent and selective activators of peroxisome proliferator-activated receptor gamma (PPAR gamma),a member of the nuclear receptor superfamily recently shown to function in adipogenesis. The most potent of these agents,BRL49653,binds to PPAR gamma with a Kd of approximately 40 nM. Treatment of pluripotent C3H10T1/2 stem cells with BRL49653 results in efficient differentiation to adipocytes. These data are the first demonstration of a high affinity PPAR ligand and provide strong evidence that PPAR gamma is a molecular target for the adipogenic effects of thiazolidinediones. Furthermore,these data raise the intriguing possibility that PPAR gamma is a target for the therapeutic actions of this class of compounds. View Publication

Mitomycin C (MMC) is the prototype bioreductive DNA alkylating agent. To exploit its unique properties and maximize patient responses,different therapeutic approaches have been investigated. Recently,the focus has concentrated on monitoring the levels of the proteins metabolizing the drug and relating these to activity in a regimen referred to as enzyme-directed bioreductive drug development. To be successful,it is important to understand the enzymology of metabolic activation not only in cell lines but also in solid tumour models. A general mechanism of action for MMC has now emerged that is activated regardless of the source of reducing equivalents,comprising three competing pathways that give rise to unique reactive intermediates and different DNA adducts. Partitioning into the pathways is dictated by chemical considerations such as pH and drug concentration. DT-diaphorase stands out in this mechanism,since it is much less effective at metabolizing MMC at neutral pH. At least five different enzymes can catalyse MMC bioreduction in vitro,and as many activities may be present in solid tumours,including a series of novel mitochondrial reductases such as a cytochrome P450 reductase. Competition between reductases for MMC appears to be based solely on protein levels rather than enzyme kinetics. Consequentially,DT-diaphorase can occupy a central role in MMC metabolic activation since it is often highly overexpressed in cancer cells. Although a good correlation has been observed in cell lines between DT-diaphorase expression and aerobic cytotoxicity,this does not hold consistently in vivo for any single bioreductive enzyme,suggesting revision of the enzyme-directed hypothesis as originally formulated. View Publication

CD4+ T cells recognize antigens through their T cell receptors (TCRs); however,additional signals involving costimulatory receptors,for example,CD28,are required for proper T cell activation. Alternative costimulatory receptors have been proposed,including members of the Toll-like receptor (TLR) family,such as TLR5 and TLR2. To understand the molecular mechanism underlying a potential costimulatory role for TLR5,we generated detailed molecular maps and logical models for the TCR and TLR5 signaling pathways and a merged model for cross-interactions between the two pathways. Furthermore,we validated the resulting model by analyzing how T cells responded to the activation of these pathways alone or in combination,in terms of the activation of the transcriptional regulators CREB,AP-1 (c-Jun),and NF-kappaB (p65). Our merged model accurately predicted the experimental results,showing that the activation of TLR5 can play a similar role to that of CD28 activation with respect to AP-1,CREB,and NF-kappaB activation,thereby providing insights regarding the cross-regulation of these pathways in CD4+ T cells. View Publication