参考文献

Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population,and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular,disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors,aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits,we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as globulomers" View Publication

Generation of functional cells from human pluripotent stem cells (PSCs) through in vitro differentiation is a promising approach for drug screening and cell therapy. However,the observed large and unavoidable variation in the differentiation potential of different human embryonic stem cell (hESC)/induced PSC (iPSC) lines makes the selection of an appropriate cell line for the differentiation of a particular cell lineage difficult. Here,we report identification of WNT3 as a biomarker capable of predicting definitive endoderm (DE) differentiation potential of hESCs. We show that the mRNA level of WNT3 in hESCs correlates with their DE differentiation efficiency. In addition,manipulations of hESCs through WNT3 knockdown or overexpression can respectively inhibit or promote DE differentiation in a WNT3 level-dependent manner. Finally,analysis of several hESC lines based on their WNT3 expression levels allowed accurate prediction of their DE differentiation potential. Collectively,our study supports the notion that WNT3 can serve as a biomarker for predicting DE differentiation potential of hESCs. ?? 2013 The Authors. View Publication

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has provided huge insight into the pathways,mechanisms and transcription factors that control differentiation. Here we use high-throughput RT-PCR technology to take a snapshot of splicing changes in the full spectrum of high- and low-expressed genes during induction of fibroblasts,from several donors,into iPSCs and their subsequent redifferentiation. We uncover a programme of concerted alternative splicing changes involved in late mesoderm differentiation and controlled by key splicing regulators MBNL1 and RBFOX2. These critical splicing adjustments arise early in vertebrate evolution and remain fixed in at least 10 genes (including PLOD2,CLSTN1,ATP2A1,PALM,ITGA6,KIF13A,FMNL3,PPIP5K1,MARK2 and FNIP1),implying that vertebrates require alternative splicing to fully implement the instructions of transcriptional control networks. View Publication

Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for malignant and nonmalignant hematologic diseases and metabolic disorders. Although successful,hematopoietic transplantation can be hindered by inadequate stem cell number or poor engrafting efficiency. To overcome these deficits,we and others have previously reported the HSC-enhancing ability of a short-term exposure of prostaglandin E2 (PGE2); this strategy has now progressed to phase 1 clinical trials in double cord blood transplantation. To further analyze the short- and long-term effects of HSC exposure to PGE2,we followed the repopulation kinetics of PGE2-treated hematopoietic grafts through 5 serial transplantations and compared inherent long-term competitiveness in a HSC head-to-head secondary transplantation model. Treatment with PGE2 did not result in a long-term increase in HSC competitiveness,lineage bias,or enhanced proliferative potential,demonstrating that pulse exposure to PGE2 results in transient increases in HSC homing and engraftment potential. View Publication

BACKGROUND: Niemann-Pick type C1 disease (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. In this lysosomal storage disorder the intracellular transport and sequestration of several lipids like cholesterol is severely impaired,resulting in an accumulation of lipids in late endosomes and lysosomes. The neurological manifestation of the disease is caused by dysfunction and cell death in the central nervous system. Several animal models were used to analyze the impaired pathways. However,the underlying pathogenic mechanisms are still not completely understood and the genetic variability in humans cannot be reflected in these models. Therefore,a human model using patient-specific induced pluripotent stem cells provides a promising approach. METHODS: We reprogrammed human fibroblasts from a NPC1 patient and a healthy control by retroviral transduction with Oct4,Klf4,Sox2 and c-Myc. The obtained human induced pluripotent stem cells (hiPSCs) were characterized by immunocytochemical analyses. Neural progenitor cells were generated and patch clamp recordings were performed for a functional analysis of derived neuronal cells. Filipin stainings and the Amplex Red assay were used to demonstrate and quantify cholesterol accumulation. RESULTS: The hiPSCs expressed different stem cell markers,e.g. Nanog,Tra-1-81 and SSEA4. Using the embryoid body assay,the cells were differentiated in cells of all three germ layers and induced teratoma in immunodeficient mice,demonstrating their pluripotency. In addition,neural progenitor cells were derived and differentiated into functional neuronal cells. Patch clamp recordings revealed voltage dependent channels,spontaneous action potentials and postsynaptic currents. The accumulation of cholesterol in different tissues is the main hallmark of NPC1. In this study we found an accumulation of cholesterol in fibroblasts of a NPC1 patient,derived hiPSCs,and neural progenitor cells,but not in cells derived from fibroblasts of a healthy individual. These findings were quantified by the Amplex Red assay,demonstrating a significantly elevated cholesterol level in cells derived from fibroblasts of a NPC1 patient. CONCLUSIONS: We generated a neuronal model based on induced pluripotent stem cells derived from patient fibroblasts,providing a human in vitro model to study the pathogenic mechanisms of NPC1 disease. View Publication

Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however,little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in most human breast cancers. In studies of breast cancer cells stably silenced for MUC1 or overexpressing the oncogenic MUC1-C subunit,we demonstrate that MUC1-C is sufficient for induction of MEK → ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn,MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor. The results further show that MUC1-C and C/EBPβ form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. MUC1-C-induced up-regulation of ALDH1A1 expression is associated with increases in ALDH activity and is detectable in stem-like cells when expanded as mammospheres. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK→C/EBPβ→ALDH1A1 pathway,and (ii) promotes the induction of ALDH activity in breast cancer cells. View Publication

Pluripotent stem cells have the potential to become most of the cell types that make up an organism. However,the signals that trigger these cells to turn into neurons rather than lung cells or muscle cells,for example,are not fully understood. Proteins called growth factors are known to have a role in this process,as are transcription factors,but it is not clear if other factors are also involved. In an attempt to identify additional mechanisms that could contribute to the formation of neurons,Sun et al. screened more than 2,000 small molecules for their ability to transform mouse pluripotent stem cells into neurons in cell culture. Surprisingly,they found that a compound called selamectin,which is used to treat parasitic flatworm infections,also triggered stem cells to turn into neurons. Selamectin works by blocking a particular type of ion channel in flatworms,but this ion channel is not found in vertebrates,which means that selamectin must be promoting the formation of neurons in mice via a different mechanism. Given that a drug related to selamectin is known to act on a subtype of receptors for the neurotransmitter GABA,Sun et al. wondered whether these receptors—known as GABAA receptors—might also underlie the effects of selamectin. Consistent with this idea,drugs that increased GABAA activity stimulated the formation of neurons,whereas drugs that reduced GABAA function blocked the effects of selamectin. In addition,Sun et al. showed that selamectin triggers human embryonic stem cells to become neurons,and that it also promotes the formation of new neurons in developing zebrafish in vivo. As well as revealing an additional mechanism for the formation of neurons from stem cells,the screening technique introduced by Sun et al. could help to identify further pro-neuronal molecules,which could aid the treatment of neurodevelopmental and neurodegenerative disorders. DOI: [http://dx.doi.org/10.7554/eLife.00508.002][1] [1]: /lookup/doi/10.7554/eLife.00508.002 View Publication

HIV-1 infected cells are eliminated in infected individuals by a variety of cellular mechanisms,the best characterized of which are cytotoxic T cell and NK cell-mediated killing. An additional antiviral mechanism is antibody-dependent cellular cytotoxicity. Here we use primary CD4(+) T cells infected with the BaL clone of HIV-1 as target cells and autologous NK cells,monocytes,and neutrophils as effector cells,to quantify the cytotoxicity mediated by the different effectors. This was carried out in the presence or absence of HIV-1-specific antiserum to assess antibody-dependent cellular cytotoxicity. We show that at the same effector to target ratio,NK cells and monocytes mediate similar levels of both antibody-dependent and antibody-independent killing of HIV-1-infected T cells. Neutrophils mediated significant antibody-dependent killing of targets,but were less effective than monocytes or NK cells. These data have implications for acquisition and control of HIV-1 in natural infection and in the context of vaccination. View Publication

Oct4 is critical to maintain the pluripotency of human embryonic stem cells (hESCs); however,the underlying mechanism remains to be fully understood. Here,we report that silencing of Oct4 in hESCs leads to the activation of tumor suppressor p53,inducing the differentiation of hESCs since acute disruption of p53 in p53 conditional knockout (p53CKO) hESCs prevents the differentiation of hESCs after Oct4 depletion. We further discovered that the silencing of Oct4 significantly reduces the expression of Sirt1,a deacetylase known to inhibit p53 activity and the differentiation of ESCs,leading to increased acetylation of p53 at lysine 120 and 164. The importance of Sirt1 in mediating Oct4-dependent pluripotency is revealed by the finding that the ectopic expression of Sirt1 in Oct4-silenced hESCs prevents p53 activation and hESC differentiation. In addition,using knock-in approach,we revealed that the acetylation of p53 at lysine 120 and 164 is required for both stabilization and activity of p53 in hESCs. In summary,our findings reveal a novel role of Oct4 in maintaining the pluripotency of hESCs by suppressing pathways that induce differentiation. Considering that p53 suppresses pluripotency after DNA damage response in ESCs,our findings further underscore the stringent mechanism to coordinate DNA damage response pathways and pluripotency pathways in order to maintain the pluripotency and genomic stability of hESCs. View Publication

RATIONALE: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction.backslashnbackslashnOBJECTIVE: To test the hypothesis that a short-course,dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents.backslashnbackslashnMETHODS AND RESULTS: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein,and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury,as assessed by magnetic resonance imaging. Mechanistically,costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile.backslashnbackslashnCONCLUSIONS: Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment,costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism. View Publication