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Abnormalities in mucin-type glycoprotein expression have been documented in a variety of cancers,identifying these molecules as targets for immunologically based therapies and prognostic/diagnostic assays. We examined the expression of the membrane-bound MUC1 mucin in normal,histologically atypical,and neoplastic lung to determine its potential contribution to lung carcinogenesis. In vivo,intense MUC1 immunoreactivity was present in normal type II pneumocytes as well as in a range of atypical lesions derived from type II cells and textgreater60% of primary and metastatic non-small cell lung cancers. Expression was not associated with altered survival,although it was highly correlated with the adenocarcinoma histology. A carcinogenesis model using 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone-exposed hamsters revealed that MUC1 mRNA increased prior to the histological appearance of tumors. In vitro studies using MUC1 expressing non-small cell lung cancer cell lines revealed that differentiation away from a type II cell lineage was associated with dramatic down-regulation of MUC1. We propose that MUC1 is a powerful new marker for the type II pneumocyte cell lineage that allows us to follow the type II pneumocyte lineage during the process of lung carcinogenesis. View Publication

The RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway provides numerous opportunities for targeted oncology therapeutics. In particular,the MEK enzyme is attractive due to high selectivity for its target ERK and the central role that activated ERK plays in driving cell proliferation. The structural,pharmacologic,and pharmacokinetic properties of RDEA119/BAY 869766,an allosteric MEK inhibitor,are presented. RDEA119/BAY 869766 is selectively bound directly to an allosteric pocket in the MEK1/2 enzymes. This compound is highly efficacious at inhibiting cell proliferation in several tumor cell lines in vitro. In vivo,RDEA119/BAY 869766 exhibits potent activity in xenograft models of melanoma,colon,and epidermal carcinoma. RDEA119/BAY 869766 exhibits complete suppression of ERK phosphorylation at fully efficacious doses in mice. RDEA119/BAY 869766 shows a tissue selectivity that reduces its potential for central nervous system-related side effects. Using pharmacokinetic and pharmacodynamic data,we show that maintaining adequate MEK inhibition throughout the dosing interval is likely more important than achieving high peak levels because greater efficacy was achieved with more frequent but lower dosing. Based on its longer half-life in humans than in mice,RDEA119/BAY 869766 has the potential for use as a once- or twice-daily oral treatment for cancer. RDEA119/BAY 869766,an exquisitely selective,orally available MEK inhibitor,has been selected for clinical development because of its potency and favorable pharmacokinetic profile. View Publication

The dose response curve is the gold standard for measuring the effect of a drug treatment,but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns,but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose,calculate classical pharmacological parameters such as the EC50,and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib,nilotinib,dasatinib and PD0325901) across a six-logarithm dose range,using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets,dose-dependent effects on cellular processes were identified using automated pathway analysis,and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover,these methods are automated,robust to non-optimized assays,and could be applied to other sources of quantitative data. View Publication

Due to the unlimited proliferation capacity and the unique differentiation ability of pluripotent stem cells (PSCs),including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),large numbers of PSC-derived cell products are in demand for applications in drug screening,disease modeling,and especially cell therapy. In stem cell-based therapy,tracking transplanted cells with magnetic resonance imaging (MRI) has emerged as a powerful technique to reveal cell survival and distribution. This chapter illustrated the basic steps of labeling PSC-derived neural progenitors (NPs) with micron-sized particles of iron oxide (MPIO,0.86 $$m) for MRI analysis. The protocol described PSC expansion and differentiation into NPs,and the labeling of the derived cells either after replating on adherent surface or in suspension. The labeled cells can be analyzed using in vitro MRI analysis. The methods presented here can be easily adapted for cell labeling in cell processing facilities under current Good Manufacturing Practices (cGMP). The iron oxide-labeled NPs can be used for cellular monitoring of in vitro cultures and in vivo transplantation. View Publication

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript,we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore,we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages,further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. View Publication

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion within Huntingtin (Htt) protein. In the phenotypic screen we identified a class of quinazoline-derived compounds that delayed a progression of a motor phenotype in transgenic Drosophila HD flies. We found that the store-operated calcium (Ca(2+)) entry (SOC) pathway activity is enhanced in neuronal cells expressing mutant Htt and that the identified compounds inhibit SOC pathway in HD neurons. The same compounds exerted neuroprotective effects in glutamate-toxicity assays with YAC128 medium spiny neurons primary cultures. We demonstrated a key role of TRPC1 channels in supporting SOC pathway in HD neurons. We concluded that the TRPC1-mediated neuronal SOC pathway constitutes a novel target for HD treatment and that the identified compounds represent a novel class of therapeutic agents for treatment of HD and possibly other neurodegenerative disorders. View Publication

R2 elements,a class of non-long terminal repeat (non-LTR) retrotransposons,have the potential to be harnessed for transgene insertion. However,efforts to achieve this are limited by our understanding of the retrotransposon mechanisms. Here,we structurally and biochemically characterize R2 from Taeniopygia guttata (R2Tg). We show that R2Tg cleaves both strands of its ribosomal DNA target and binds a pseudoknotted RNA element within the R2 3′ UTR to initiate target-primed reverse transcription. Guided by these insights,we engineer and characterize an all-RNA system for transgene insertion. We substantially reduce the system’s size and insertion scars by eliminating unnecessary R2 sequences on the donor. We further improve the integration efficiency by chemically modifying the 5′ end of the donor RNA and optimizing delivery,creating a compact system that achieves over 80% integration efficiency in several human cell lines. This work expands the genome engineering toolbox and provides mechanistic insights that will facilitate future development of R2-mediated gene insertion tools. Subject terms: Transferases,Protein design,Genetic engineering View Publication

The immune-modulatory effects of mesenchymal stromal cells (MSCs) are widely used to treat inflammatory disorders,with indoleamine 2,4-dioxygenase-1 (IDO-1) playing a pivotal role in suppressing stimulated T-cell proliferation. Taking that three-dimensional (3D) cultures enhance MSCs’ anti-inflammatory properties compared with two-dimensional (2D) cultures,the differentially expressed miRNAs were examined. Thus,we identified hsa-miR-4662a-5p (miR-4662a) as a key inducer of IDO-1 via its suppression of bridging integrator-1 (BIN-1),a negative regulator of the IDO-1 gene. The IDO-1-inducing potential of miR-4662a was conserved across primary MSCs from various donors and sources but exhibited variability. Notably,iPSC-derived MSCs (iMSCs) demonstrated superior IDO-1 induction and immune-modulatory efficacy compared with their donor-matched primary MSCs. Accordingly,iMSCs expressing miR-4662a (4662a/iMSC) exhibited stronger suppressive effects on T-cell proliferation and more potent suppressive effects on graft-versus-host disease (GVHD),improving survival rates and reducing tissue damage in the liver and gut. Our results point to the therapeutic potential of standardized,off-the-shelf 4662a/iMSC as a robust immune-modulating cell therapy for GVHD. View Publication

IntroductionEmerging biotechnologies are increasingly being explored for food production,including the development of cell-cultivated meat. Conventional approaches typically rely on satellite cell (SC) biopsies,which present challenges in scalability. Bovine induced pluripotent stem cells (biPSCs) represent a promising alternative due to their capacity for self-renewal and developmental plasticity.MethodsThis study utilized both lentiviral (integrating) and episomal (non-integrating) reprogramming strategies to generate biPSCs suitable for myogenic differentiation. Bovine fetal fibroblasts (bFFs) were reprogrammed using episomal vectors pMaster K and pCXB-EBNA1,leading to the emergence of putative iPSC colonies 13 days post-nucleofection. A clonal line,bFF-iPSCs pMK,was selected for further analysis.ResultsThe bFF-iPSCs pMK line expressed key pluripotency markers including alkaline phosphatase (AP),OCT4,SOX2,and NANOG,and was stably maintained for over 33 passages,although episomal plasmids remained detectable. in vitro myogenic differentiation was assessed by comparing this line to a previously established lentiviral reprogrammed line (bFF-iPSCs mOSKM). Both lines exhibited downregulation of pluripotency markers and upregulation of the early myogenic marker PAX3. By day 30,the bFF-iPSCs pMK line formed elongated,multinucleated cells characteristic of myotubes and displayed a corresponding gene expression profile.DiscussionThese results provide new insights into bovine in vitro myogenesis and its application in cultured meat production. While promising,the study also highlights the difficulty in achieving complete myogenic differentiation,indicating a need for further optimization of differentiation protocols. Graphical abstract View Publication