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Vascularization of engineered human skin constructs is crucial for recapitulation of systemic drug delivery and for their long-term survival,functionality,and viable engraftment. In this study,the latest microfabrication techniques are used and a novel bioengineering approach is established to micropattern spatially controlled and perfusable vascular networks in 3D human skin equivalents using both primary and induced pluripotent stem cell (iPSC)-derived endothelial cells. Using 3D printing technology makes it possible to control the geometry of the micropatterned vascular networks. It is verified that vascularized human skin equivalents (vHSEs) can form a robust epidermis and establish an endothelial barrier function,which allows for the recapitulation of both topical and systemic delivery of drugs. In addition,the therapeutic potential of vHSEs for cutaneous wounds on immunodeficient mice is examined and it is demonstrated that vHSEs can both promote and guide neovascularization during wound healing. Overall,this innovative bioengineering approach can enable in vitro evaluation of topical and systemic drug delivery as well as improve the potential of engineered skin constructs to be used as a potential therapeutic option for the treatment of cutaneous wounds. View Publication

Helper-dependent adenoviral vectors (HDAd) that express certain transgene products are impossible to produce because the transgene product is toxic to the producer cells,especially when made in large amounts during vector production. Downregulating transgene expression from the HDAd during vector production is a way to solve this problem. In this report,we show that this can be accomplished by inserting the target sequence for the adenoviral VA RNAI into the 3' untranslated region of the expression cassette in the HDAd. Thus during vector production,when the producer cells are coinfected with both the helper virus (HV) and the HDAd,the VA RNAI produced by the HV will target the transgene mRNA from the HDAd via the endogenous cellular RNAi pathway. Once the HDAd is produced and purified,transduction of the target cells results in unimpeded transgene expression because of the absence of HV. This simple and universal strategy permits for the robust production of otherwise recalcitrant HDAds. View Publication

Mechanisms determining intrinsic differentiation bias inherent to human pluripotent stem cells (hPSCs) toward cardiogenic fate remain elusive. We evaluated the interplay between ErbB4 and EGFR in determining cardiac differentiation in vitro as these receptor tyrosine kinases (RTKs) are key to heart and brain development in vivo. Our results demonstrate that during cardiac differentiation,cell fate biases exist in hPSCs due to cardiac/neuroectoderm divergence post cardiac mesoderm stage. Stage-specific up-regulation of EGFR in concert with persistent Wnt3a signaling post cardiac mesoderm favors commitment towards neural progenitor cells (NPCs). Inhibition of EGFR abrogates these effects with enhanced (textgreater2-fold) cardiac differentiation efficiencies by increasing proliferation of Nkx2-5 expressing cardiac progenitors while reducing proliferation of Sox2 expressing NPCs. Forced overexpression of ErbB4 rescued cardiac commitment by augmenting Wnt11 signaling. Convergence between EGFR/ErbB4 and canonical/non-canonical Wnt signaling determines cardiogenic fate in hPSCs. This article is protected by copyright. All rights reserved. View Publication

Dietary microRNAs (miRNAs) modulation could be important for health and wellbeing. Part of the healthful activities of polyphenols might be due to a modulation of miRNAs' expression. Among the most biologically active polyphenols,hydroxytyrosol (HT) has never been studied for its actions on miRNAs. We investigated whether HT could modulate the expression of miRNAs in vivo. We performed an unbiased intestinal miRNA screening in mice supplemented (for 8 weeks) with nutritionally relevant amounts of HT. HT modulated the expression of several miRNAs. Analysis of other tissues revealed consistent HT-induced modulation of only few miRNAs. Also,HT administration increased triglycerides levels. Acute treatment with HT and in vitro experiments provided mechanistic insights. The HT-induced expression of one miRNA was confirmed in healthy volunteers supplemented with HT in a randomized,double-blind and placebo-controlled trial. HT consumption affects specific miRNAs' expression in rodents and humans. Our findings suggest that the modulation of miRNAs' action through HT consumption might partially explain its healthful activities and might be pharmanutritionally exploited in current therapies targeting endogenous miRNAs. However,the effects of HT on triglycerides warrant further investigations. View Publication

In Wilson disease (WD),functional loss of ATPase copper-transporting $$ (ATP7B) impairs biliary copper excretion,leading to excessive copper accumulation in the liver and fulminant hepatitis. Current US Food and Drug Administration- and European Medicines Agency-approved pharmacological treatments usually fail to restore copper homeostasis in patients with WD who have progressed to acute liver failure,leaving liver transplantation as the only viable treatment option. Here,we investigated the therapeutic utility of methanobactin (MB),a peptide produced by Methylosinus trichosporium OB3b,which has an exceptionally high affinity for copper. We demonstrated that ATP7B-deficient rats recapitulate WD-associated phenotypes,including hepatic copper accumulation,liver damage,and mitochondrial impairment. Short-term treatment of these rats with MB efficiently reversed mitochondrial impairment and liver damage in the acute stages of liver copper accumulation compared with that seen in untreated ATP7B-deficient rats. This beneficial effect was associated with depletion of copper from hepatocyte mitochondria. Moreover,MB treatment prevented hepatocyte death,subsequent liver failure,and death in the rodent model. These results suggest that MB has potential as a therapeutic agent for the treatment of acute WD. View Publication

Neural rosettes derived from human induced pluripotent stem cells (iPSCs) have been claimed to be a highly robust in vitro cellular model for biomedical application. They are able to propagate in vitro in the presence of mitogens,including basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). However,these two mitogens are also involved in anterior-posterior patterning in a gradient dependent manner along the neural tube axis. Here,we compared the regional identity of neural rosette cells and specific neural subtypes of their progeny propagated with low and high concentrations of bFGF and EGF. We observed that low concentrations of bFGF and EGF in the culturing system were able to induce forebrain identity of the neural rosettes and promote subsequent cortical neuronal differentiation. On the contrary,high concentrations of these mitogens stimulate a mid-hindbrain fate of the neural rosettes,resulting in subsequent cholinergic neuron differentiation. Thus,our results indicate that different concentrations of bFGF and EGF supplemented during propagation of neural rosettes are involved in altering the identity of the resultant neural cells. View Publication

Organogenesis of the trachea and lungs requires a complex series of mesoderm-endoderm interactions mediated by WNT,BMP,retinoic acid (RA),and hedgehog (Hh),but how these pathways interact in a gene regulatory network is less clear. Using Xenopus embryology,mouse genetics,and human ES cell cultures,we identified a conserved signaling cascade that initiates respiratory lineage specification. We show that RA has multiple roles; first RA pre-patterns the lateral plate mesoderm and then it promotes Hh ligand expression in the foregut endoderm. Hh subsequently signals back to the pre-patterned mesoderm to promote expression of the lung-inducing ligands Wnt2/2b and Bmp4. Finally,RA regulates the competence of the endoderm to activate the Nkx2-1+ respiratory program in response to these mesodermal WNT and BMP signals. These data provide insights into early lung development and a paradigm for how mesenchymal signals are coordinated with epithelial competence during organogenesis. View Publication

Graphical Abstract Highlights d SMAD activity is active in suprabasal cells but is weaker in basal epithelial cells d SMAD signaling activity correlates with mucociliary differentiation in the airway d Dual TGFb/BMP inhibition prevents spontaneous differentiation in culture d Dual TGFb/BMP inhibition allows prolonged culture of diverse epithelial basal cells Correspondence jrajagopal@partners.org In Brief Mou et al. show that small-molecule-mediated SMAD signaling inhibition allows prolonged feeder-free culture of diverse functional epithelial basal stem cells in a 2D format. This methodology provides a facile patient-specific epithelial disease modeling platform,as shown by the expansion of airway epithelium from non-invasively obtained specimens from cystic fibrosis patients. View Publication

Human somatic stem cells such as mesenchymal stem cells (hMSCs) have the capacity to differentiate into mesenchymal tissue lineages and to alter immune regulatory functions. As such,they hold promise for use in stem cell-based therapies. However,no method is currently available to evaluate the actual differentiation capacity of hMSCs prior to cell transplantation. Previously,we performed a comprehensive glycan profiling of adipose-derived hMSCs using high-density lectin microarray and demonstrated that $$2-6-sialylation is a marker of the differentiation potential of these cells. Nevertheless,no information was available about the structural details of these of $$2-6-sialylated glycans. Here we used high performance liquid chromatography (HPLC) analysis combined with mass spectrometry (MS) to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from early (with differentiation ability) and late (without differentiation ability) passages of adipose tissue-derived hMSCs. Findings in these cells were compared with those from human induced pluripotent stem cells (hiPSCs),human dermal fibroblasts (hFibs) and cartilage tissue-derived chondrocytes. A higher percentage of $$2-6-sialylated N-glycans was detected in early passage cells (24-28 % of sialylated N-glycans) compared with late passage cells (13-15 %). A major $$2-6-sialylated N-glycan structure detected in adipose-derived hMSCs was that of mono-sialylated biantennary N-glycan. Similar results were obtained for the cartilage tissue-derived chondrocytes,Yub621c (28 % for passage 7 and 5 % for passage 28). In contrast,no significant differences were observed between early and late passage hMSCs with respect to $$2-6-sialylated O-glycan percentages. These results demonstrate that levels of $$2-6-sialylated N-glycans,but not O-glycans,could be used as markers of the differential potential of hMSCs. View Publication

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins,however,lack small-molecule ligands,and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued,covalent fragments provide an alternative route to small-molecule probes,including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for textgreater700 cysteines found in both druggable proteins and proteins deficient in chemical probes,including transcription factors,adaptor/scaffolding proteins,and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells,showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems. View Publication

BACKGOUND The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson's disease (PD). METHODS The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD,using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study,patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson's Disease Rating Scale (UPDRS) to measure clinical symptoms. RESULTS The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC,consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; ptextless0.05 for all values) in the PD group treated with NAC,and no measurable changes in the control group. UPDRS scores were also significantly improved in the NAC group (mean improvement of 12.9%,p = 0.01). CONCLUSIONS The results of this preliminary study demonstrate for the first time a potential direct effect of NAC on the dopamine system in PD patients,and this observation may be associated with positive clinical effects. A large-scale clinical trial to test the therapeutic efficacy of NAC in this population and to better elucidate the mechanism of action is warranted. TRIAL REGISTRATION ClinicalTrials.gov NCT02445651. View Publication

Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body,including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system,provides a platform for the screening of chemical libraries that affect these processes,and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However,defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner,with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells,exhibit spontaneous electrical activity,and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4-5 months,even without astrocyte feeder layers. View Publication