参考文献

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are defined as pluripotent in view of their self-renewal ability and potential to differentiate to cells of all three germ layers. Recent studies have indicated that microRNAs (miRNAs) play an important role in the maintenance of pluripotency and cell cycle regulation. We used a microarray based approach to identify miRNAs that were enriched in hESCs when compared to differentiated cells and at the same time showed significant expression changes between different phases of cell cycle. We identified 34 candidate miRNAs and performed functional studies on one of these,miR-1305,which showed the highest expression change during cell cycle transition. Overexpression of miR-1305 induced differentiation of pluripotent stem cells,increased cell apoptosis and sped up G1/S transition,while its downregulation facilitated the maintenance of pluripotency and increased cell survival. Using target prediction software and luciferase based reporter assays we identified POLR3G as a downstream target by which miR-1305 regulates the fine balance between maintenance of pluripotency and onset of differentiation. Overexpression of POLR3G rescued pluripotent stem cell differentiation induced by miR-1305 overexpression. In contrast,knock-down of POLR3G expression abolished the miR-1305-knockdown mediated enhancement of pluripotency,thus validating its role as miR-1305 target in human pluripotent stem cells. Together our data point to an important role for miR-1305 as a novel regulator of pluripotency,cell survival and cell cycle and uncovers new mechanisms and networks by which these processes are intertwined in human pluripotent stem cells. This article is protected by copyright. All rights reserved. View Publication

Derivation of functional vascular smooth muscle cells (VSMCs) from human induced pluripotent stem cells (hiPSCs) to generate tissue-engineered blood vessels (TEBVs) holds great potential in treating patients with vascular diseases. Herein,hiPSCs were differentiated into alpha-smooth muscle actin ($$-SMA) and calponin-positive VSMCs,which were seeded onto polymer scaffolds in bioreactors for vascular tissue growth. A functional TEBV with abundant collagenous matrix and sound mechanics resulted,which contained cells largely positive for $$-SMA and smooth muscle myosin heavy chain (SM-MHC). Moreover,when hiPSC-derived TEBV segments were implanted into nude rats as abdominal aorta interposition grafts,they remained unruptured and patent with active vascular remodeling,and showed no evidence of teratoma formation during a 2-week proof-of-principle study. Our studies represent the development of the first implantable TEBVs based on hiPSCs,and pave the way for developing autologous or allogeneic grafts for clinical use in patients with vascular disease. View Publication

HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP,apolipoprotein B mRNA-editing,enzyme-catalytic,polypeptide-like 3 (APOBEC3) cytidine deaminases,and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase,which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA,and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr,found in all primate lentiviruses,and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx,hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions,but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here,we identify HLTF,a postreplication DNA repair helicase,as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81,which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus,separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast,we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery,suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host. View Publication

Haplo-cord stem cell transplantation combines the infusion of CD34 selected hematopoietic progenitors from a haplo-identical donor with an umbilical cord blood (UCB) graft from an unrelated donor and allows faster count recovery,with low rates of disease recurrence and chronic graft-versus-host disease (GVHD). But the contribution of the umbilical cord blood graft to long-term transplant outcome remains unclear. We analyzed 39 recipients of haplo-cord transplants with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS),engrafted and in remission at 2 months. Median age was 66 (18-72) and all had intermediate,high,or very-high risk disease. Less than 20% UCB chimerism in the CD33 lineage was associated with an increased rate of disease recurrence (54% versus 11% p textless 0.0001) and decrease in one year progression-free (20% versus 55%,p = 0.004) and overall survival (30% versus 62%,p = 0.02). Less than 100% UCB chimerism in the CD3 lineage was associated with increase rate of disease recurrence (46% versus 12%,p = 0.007). Persistent haplo-chimerism in the CD3 lineage was associated with an increased rate of disease recurrence (40% versus 15%,p = 0.009) Chimerism did not predict for treatment related mortality. The cumulative incidence of acute GVHD by day 100 was 43%. The cumulative incidence of moderate/severe chronic GVHD was only 5%. Engraftment of the umbilical cord blood grafts provides powerful graft-versus-leukemia (GVL) effects which protect against disease recurrence and is associated with low risk of chronic GVHD. Engraftment of CD34 selected haplo-identical cells can lead to rapid development of circulating T-cells,but when these cells dominate,GVL-effects are limited and rates of disease recurrence are high. View Publication

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