搜索结果: 'gmp t cell activator'

DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway,which promotes error-free repair of DNA double-strand breaks,is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus,cells from Fanconi anemia patients,which lack this critical pathway,fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study,we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6,but not E7,recovers FA iPSC colony formation and,furthermore,that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase,NANOG,and Tra-1-60,indicating that they were fully reprogrammed into pluripotent cells. However,FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression,whereas their FA-complemented counterparts grew efficiently. Thus,we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. IMPORTANCE A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53. View Publication

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function,little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet,understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore,we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs,from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties,including resting membrane potential,action potential,sodium and potassium channel currents,somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons,the resting membrane potential became more negative,the expression of voltage-gated sodium channels increased,the membrane became capable of generating action potentials following adequate depolarization and,at day 48-55,50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step,of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology,as electrophysiological properties of iPSC-derived neurons mature over time. View Publication

Mesenchymal stem cells (MSCs) may be used as powerful tools for the repair and regeneration of damaged tissues. However,isolating tissue specific-derived MSCs may cause pain and increased infection rates in patients,and repetitive isolations may be required. To overcome these difficulties,we have examined alternative methods for MSC production. Here,we show that induced pluripotent stem cells (iPSCs) may be differentiated into mesenchymal stem cells (iMSCs) following exposure to SB431542. Purified iMSCs were administered to mdx mice to study skeletal muscle regeneration in a murine model of muscular dystrophy. Purified iMSCs displayed fibroblast-like morphology,formed three-dimensional spheroid structures,and expressed characteristic mesenchymal stem cell surface markers such as CD29,CD33,CD73,CD90,and CD105. Moreover,iMSCs were capable of differentiating into adipogenic,osteogenic,and chondrogenic lineages. Transplanting iMSC cells to tibialis anterior skeletal muscle tissue in mdx mice lowered oxidative damage as evidenced by a reduction in nitrotyrosine levels,and normal dystrophin expression levels were restored. This study demonstrates the therapeutic potential of purified iMSCs in skeletal muscle regeneration in mdx mice,and suggests that iPSCs are a viable alternate source for deriving MSCs as needed. textcopyright 2014 Elsevier Inc. View Publication

Differentiation of neural lineages from human pluripotent stem cells (hPSCs) raises the hope of generating functional cells for the treatment of neural diseases. However,current protocols for differentiating hPSCs into neural lineages remain inefficient and largely variable between different hPSC lines. We report that microRNA 376c (miR-376c) significantly enhanced neural differentiation of hPSCs in a defined condition by suppressing SMAD4,the co-SMAD for TGF-β signaling. Downstream,SMAD4 directly bound and suppressed PAX6,the critical neural lineage specification factor. Interestingly,we also found that SMAD4 binds and suppresses miR-376c clusters in undifferentiated hESCs. In summary,our findings revealed a reciprocal antagonism between miR-376c and SMAD signaling that regulates cell fate during human neural differentiation.-Liu,J.,Wang,L.,Su,Z.,Wu,W.,Cai,X.,Li,D.,Hou,J.,Pei,D.,Pan,G. A reciprocal antagonism between miR-376c and TGF-β signaling regulates neural differentiation of hPSCs. View Publication

Trisomy 21 is the most common chromosomal abnormality and is associated primarily with cardiovascular,hematological,and neurological complications. A robust patient-derived cellular model is necessary to investigate the pathophysiology of the syndrome because current animal models are limited and access to tissues from affected individuals is ethically challenging. We aimed to derive induced pluripotent stem cells (iPSCs) from trisomy 21 human mid-trimester amniotic fluid stem cells (AFSCs) and describe their hematopoietic and neurological characteristics. Human AFSCs collected from women undergoing prenatal diagnosis were selected for c-KIT(+) and transduced with a Cre-lox-inducible polycistronic lentiviral vector encoding SOX2,OCT4,KLF-4,and c-MYC (50,000 cells at a multiplicity of infection (MOI) 1-5 for 72 h). The embryonic stem cell (ESC)-like properties of the AFSC-derived iPSCs were established in vitro by embryoid body formation and in vivo by teratoma formation in RAG2(-/-),$\$-chain(-/-),C2(-/-) immunodeficient mice. Reprogrammed cells retained their cytogenetic signatures and differentiated into specialized hematopoietic and neural precursors detected by morphological assessment,immunostaining,and RT-PCR. Additionally,the iPSCs expressed all pluripotency markers upon multiple rounds of freeze-thawing. These findings are important in establishing a patient-specific cellular platform of trisomy 21 to study the pathophysiology of the aneuploidy and for future drug discovery. View Publication

Differentiation of human stem cells to hepatocytes is crucial for industrial applications as well as to develop new therapeutic strategies for liver disease. The protocol described here,using sequentially growth factors known to play a role in liver embryonic development,efficiently differentiates human embryonic stem cells (hESC) as well as human-induced pluripotent stem cells (hiPSC) to hepatocytes by directing them through defined embryonic intermediates,namely,mesendoderm/definitive endoderm and hepatoblast and hepatocyte phenotype. After 28 days,the final differentiated progeny is a mixture of cells,comprising cells with characteristics of hepatoblasts and a smaller cell fraction with morphological and phenotypical features of mature hepatocytes. An extensive functional characterization of the stem cell progeny should be used to confirm that differentiated cells display functional characteristics of mature hepatocytes including albumin secretion,glycogen storage,and several detoxifying functions such as urea production,bilirubin conjugation,glutathione S-transferase activity,cytochrome activity and drug transporter activity. View Publication

Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast,induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs,which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology,immunophenotype,in vitro differentiation potential,and gene expression profiles as primary MSCs. However,iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics,whereas tissue-specific,senescence-associated,and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion,but they remained rejuvenated with regard to age-related DNAm. Overall,iPS-MSCs are similar to MSCs,but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns - particularly of DNAm patterns associated with tissue type and aging. View Publication

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson's disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development,A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here,we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons,which mimics embryonic DA neuron development. In our protocol,we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method,and then convert the FP cells to A9 DA neurons,which could be maintained in vitro for several months. This efficient,repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients,in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD. View Publication

Embryonic stem (ES) cells are characterized by pluripotency,defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade,great progress has been made on the cell culture conditions,transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions,responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein-tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells,and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal,while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation,the role of this kinase family in human ES cells is largely unknown. Here,we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome,Fyn,c-Yes,c-Src,Lyn,Lck and Hck were expressed in H1,H7 and H9 hES cells,while Fgr,Blk,Srm,Brk,and Frk transcripts were not detected. Of these,c-Yes,Lyn,and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs,while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast,Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation,cultures were treated with inhibitors specific for the Src kinase family. Remarkably,human ES cells maintained in the presence of the potent Src-family kinase inhibitor A-419259 retained the morphology of domed,pluripotent colonies and continued to express the self-renewal marker TRA-1-60 despite culture under differentiation conditions. Taken together,these observations support a role for Src-family kinase signaling in the regulation of human ES cell fate,and suggest that the activities of individual Src-family members are required for the initiation of the differentiation program. View Publication

For years,our ability to study pathological changes in neurological diseases has been hampered by the lack of relevant models until the recent groundbreaking work from Yamanaka's group showing that it is feasible to generate induced pluripotent stem cells (iPSCs) from human somatic cells and to redirect the fate of these iPSCs into differentiated cells. In particular,much interest has focused on the ability to differentiate human iPSCs into neuronal progenitors and functional neurons for relevance to a large number of pathologies including mental retardation and behavioral or degenerative syndromes. Current differentiation protocols are time-consuming and generate limited amounts of cells,hindering use on a large scale. We describe a feeder-free method relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. Four days after induction,expression of markers of the neurectoderm lineage is detectable. Between 4 and 7 days,neuronal precursors can be expanded,frozen,and thawed without loss of proliferation and differentiation capacities or further differentiated. Terminal differentiation into the different subtypes of mature neurons found in the human brain were observed. At 6-35 days after induction,cells express typical voltage-gated and ionotrophic receptors for GABA,glycine,and acetylcholine. This specific and efficient single-step strategy in a chemically defined medium allows the production of mature neurons in 20-40 days with multiple applications,especially for modeling human pathologies. View Publication

The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report,human mesenchymal cells (hMSCs) generated from the human ES cell line H9,were reprogrammed back to induced pluripotent state using Oct-4,Sox2,Nanog,and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes,lipid droplets,glycogen,scarce reticulum) and nuclear levels (features of nuclear plasticity,presence of euchromatin,reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal-epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. View Publication

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover,we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining,transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling. View Publication