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Huntington’s disease (HD) is the best-known example of a neurodegenerative disorder caused by the expansion of a glutamine-encoding CAG repeat in the causative gene. Growing evidence indicates that somatic CAG expansions play a key role in disease progression,providing a strong rationale for therapeutic strategies directly targeting the repeat tract. However,achieving sufficient efficacy while maintaining allele selectivity and minimizing off-target effects remains a major challenge. Here,we developed allele-selective,CAG-targeting artificial microRNA (amiRNA) molecules that exhibit significantly reduced off-target risk. This was achieved by introducing specific substitutions at selected positions within the guide strand. These molecules effectively downregulated polyglutamine (polyQ) proteins in cellular models of HD,spinocerebellar ataxias types 1 and 3,and dentatorubral pallidoluysian atrophy. The most promising candidate,amiR136-13A,reduced mutant huntingtin levels in different brain regions of the HD mouse model and did not induce toxicity up to 28 weeks following a single administration of an AAV5 vector. Transcriptomic profiling of human HD neural stem cells treated with amiR136-13A revealed minor changes in gene expression. Moreover,amiR136-13A reduced the level of HTT1a,a short pathogenic isoform of huntingtin. Collectively,these findings identify amiR136-13A as a potent,selective,and safe therapeutic candidate for HD and potentially other polyQ disorders. View Publication

OBJECTIVE The differentiation program for human thyroid follicular cells (TFCs) relies on the interplay between sequence-specific transcription factors and transcriptional co-regulators. Transcriptional co-activator with PDZ-binding motif (TAZ) is a co-activator that regulates several transcription factors,including PAX8 and NKX2-1,which play a central role in thyroid-specific gene transcription. TAZ and PAX8/NKX2-1 are co-expressed in the nuclei of thyroid cells,and TAZ interacts directly with both PAX8 and NKX2-1,leading to their enhanced transcriptional activity on the thyroglobulin (TG) promoter and additional genes. METHODS The use of a small molecule,ethacridine,recently identified as a TAZ activator,in the differentiation of thyroid cells from human embryonic stem (hES) cells was studied. First,endodermal cells were derived from hES cells using Activin A,followed by induction of differentiation into thyroid cells directed by ethacridine and thyrotropin (TSH). RESULTS The expression of TAZ was increased in the Activin A-derived endodermal cells by ethacridine in a dose-dependent manner and followed by increases in PAX8 and NKX2-1 when assessed by both quantitative polymerase chain reaction and immunostaining. Following further differentiation with the combination of ethacridine and TSH,the thyroid-specific genes TG,TPO,TSHR,and NIS were all induced in the differentiated hES cells. When these cells were cultured with extracellular matrix-coated dishes,thyroid follicle formation and abundant TG protein expression were observed. Furthermore,such hES cell-derived thyroid follicles showed a marked TSH-induced and dose-dependent increase in radioiodine uptake and protein-bound iodine accumulation. CONCLUSION These data show that fully functional human thyroid cells can be derived from hES cells using ethacridine,a TAZ activator,which induces thyroid-specific gene expression and promotes thyroid cell differentiation from the hES cells. These studies again demonstrate the importance of transcriptional regulation in thyroid cell development. This approach also yields functional human thyrocytes,without any gene transfection or complex culture conditions,by directly manipulating the transcriptional machinery without interfering with intermediate signaling events. View Publication

The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study,we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM),Flk-1,tie-1,tie-2,vascular endothelial (VE) cadherin,MECA-32,and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4,whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1,PECAM,VE-cadherin,MECA-32,and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin,interleukin-6,fibroblast growth factor 2,and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis. View Publication

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However,most of the studies used skin fibroblasts as the starting population for reprogramming,which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore,iPS cells can be readily derived from adult hASCs in a feeder-free condition,thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion,are easy to maintain in culture,and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells. View Publication

Motor neuron diseases (MNDs) are a group of neurological disorders that selectively affect motor neurons. There are currently no cures or efficacious treatments for these diseases. In recent years,significant developments in stem cell research have been applied to MNDs,particularly regarding neuroprotection and cell replacement. However,a consistent source of motor neurons for cell replacement is required. Human embryonic stem cells (hESCs) could provide an inexhaustible supply of differentiated cell types,including motor neurons that could be used for MND therapies. Recently,it has been demonstrated that induced pluripotent stem (iPS) cells may serve as an alternative source of motor neurons,since they share ES characteristics,self-renewal,and the potential to differentiate into any somatic cell type. In this review,we discuss several reproducible methods by which hESCs or iPS cells are efficiently isolated and differentiated into functional motor neurons,and possible clinical applications. View Publication

Human embryonic stem cells (hESCs) have the potential to generate multiple cell types and hold promise for future therapeutic applications. Although undifferentiated hESCs can proliferate indefinitely,hESC derivatives significantly downregulate telomerase and have limited replication potential. In this study we examine whether the replicative lifespan of hESC derivatives can be extended by ectopic expression of human telomerase reverse transcriptase (hTERT),the catalytic component of the telomerase complex. To this end,we have derived HEF1 cells,a fibroblast-like cell type,differentiated from hESCs. Infection of HEF1 cells with a retrovirus expressing hTERT extends their replicative capacity,resulting in immortal human HEF1-hTERT cells. HEF1-hTERT cells can be used to produce conditioned medium (CM) capable of supporting hESC growth under feeder-free conditions. Cultures maintained in HEF1-CM show characteristics similar to mouse embryonic fibroblast CM control cultures,including morphology,surface marker and transcription factor expression,telomerase activity,differentiation,and karyotypic stability. In addition,HEF1-hTERT cells have the capacity to differentiate into cells of the osteogenic lineage. These results suggest that immortalized cell lines can be generated from hESCs and that cells derived from hESCs can be used to support their own growth,creating a genotypically homogeneous system for the culture of hESCs. View Publication

Cardiovascular progenitor cells (CVPCs) derived from human embryonic stem cells and human induced pluripotent stem cells represent an invaluable potential source for the study of early embryonic cardiovascular development and stem cell-based therapies for congenital and acquired heart diseases. To fully realize their values,it is essential to establish an efficient and stable differentiation system for the induction of these pluripotent stem cells (PSCs) into the CVPCs and robustly expand them in culture,and then further differentiate these CVPCs into multiple cardiovascular cell types. Here we describe the protocols for efficient derivation,expansion,and differentiation of CVPCs from hPSCs in a chemically defined medium under feeder- and serum-free culture conditions. View Publication

Peripheral blood is the easy-to-access,minimally invasive,and the most abundant cell source to use for cell reprogramming. The episomal vector is among the best approaches for generating integration-free induced pluripotent stem (iPS) cells due to its simplicity and affordability. Here we describe the detailed protocol for the efficient generation of integration-free iPS cells from peripheral blood mononuclear cells. With this optimized protocol,one can readily generate hundreds of iPS cell colonies from 1 ml of peripheral blood. View Publication

BACKGROUND Neuronal stem cells (NSCs) are promising for neurointestinal disease therapy. Although NSCs have been isolated from intestinal musclularis,their presence in mucosa has not been well described. Mucosa-derived NSCs are accessible endoscopically and could be used autologously. Brain-derived Nestin-positive NSCs are important in endogenous repair and plasticity. The aim was to isolate and characterize mucosa-derived NSCs,determine their relationship to Nestin-expressing cells and to demonstrate their capacity to produce neuroglial networks in vitro and in vivo. METHODS Neurospheres were generated from periventricular brain,colonic muscularis (Musc),and mucosa-submucosa (MSM) of mice expressing green fluorescent protein (GFP) controlled by the Nestin promoter (Nestin-GFP). Neuronal stem cells were also grown as adherent colonies from intestinal mucosal organoids. Their differentiation potential was assessed using immunohistochemistry using glial and neuronal markers. Brain and gut-derived neurospheres were transplanted into explants of chick embryonic aneural hindgut to determine their fate. KEY RESULTS Musc- and MSM-derived neurospheres expressed Nestin and gave rise to cells of neuronal,glial,and mesenchymal lineage. Although Nestin expression in tissue was mostly limited to glia co-labelled with glial fibrillary acid protein (GFAP),neurosphere-derived neurons and glia both expressed Nestin in vitro,suggesting that Nestin+/GFAP+ glial cells may give rise to new neurons. Moreover,following transplantation into aneural colon,brain- and gut-derived NSCs were able to differentiate into neurons. CONCLUSIONS & INFERENCES Nestin-expressing intestinal NSCs cells give rise to neurospheres,differentiate into neuronal,glial,and mesenchymal lineages in vitro,generate neurons in vivo and can be isolated from mucosa. Further studies are needed for exploring their potential for treating neuropathies. View Publication

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood,thymus,and spleen of IFN-beta-/- mice,activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production,relative to IFN-beta+/+ mice. Notably,constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages,respectively,of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice,associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1,IgM,and CD23 expression. Circulating IgM-,Mac-1-,and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice,shown by the reduction of colony-forming units,granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether,our data suggest that,in addition to the direct growth-inhibitory effects on tumor cells,IFN-beta is required during different stages of maturation in the development of the immune system. View Publication

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue,but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus,fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies,but that of BOECs was lower. In terms of invasiveness of biopsy sampling,biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs,but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative. View Publication

Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day,7-stage protocol. Cultures were ˜90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17,but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine,but not glucose in perifusion studies. Compared to adult human islets,hESC-derived cells expressed ˜10-fold higher levels of glucose transporter 1 (GLUT1) mRNA,but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However,GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells,whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays,hESC-derived cells displayed mild potassium channel activity,but no responsiveness to glucose,metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells,suggesting a lack of functional KATP channels at the cell surface. In addition,KATP channel subunit transcript levels were not at a 1:1 ratio,as would be expected (SUR1 levels were ˜5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells,and along with lack of GLUT1,may explain the absence of glucose-stimulated insulin secretion.?? 2013 Elsevier B.V. View Publication