技术资料

Frontotemporal dementia (FTD) and other tauopathies characterized by focal brain neurodegeneration and pathological accumulation of proteins are commonly associated with tau mutations. However,the mechanism of neuronal loss is not fully understood. To identify molecular events associated with tauopathy,we studied induced pluripotent stem cell (iPSC)-derived neurons from individuals carrying the tau-A152T variant. We highlight the potential of in-depth phenotyping of human neuronal cell models for pre-clinical studies and identification of modulators of endogenous tau toxicity. Through a panel of biochemical and cellular assays,A152T neurons showed accumulation,redistribution,and decreased solubility of tau. Upregulation of tau was coupled to enhanced stress-inducible markers and cell vulnerability to proteotoxic,excitotoxic,and mitochondrial stressors,which was rescued upon CRISPR/Cas9-mediated targeting of tau or by pharmacological activation of autophagy. Our findings unmask tau-mediated perturbations of specific pathways associated with neuronal vulnerability,revealing potential early disease biomarkers and therapeutic targets for FTD and other tauopathies. View Publication

How mutations in the microtubule-associated protein tau (MAPT) gene cause frontotemporal dementia (FTD) remains poorly understood. We generated and characterized multiple induced pluripotent stem cell (iPSC) lines from patients with MAPT IVS10+16 and tau-A152T mutations and a control subject. In cortical neurons differentiated from these and other published iPSC lines,we found that MAPT mutations do not affect neuronal differentiation but increase the 4R/3R tau ratio. Patient neurons had significantly higher levels of MMP-9 and MMP-2 and were more sensitive to stress-induced cell death. Inhibitors of MMP-9/MMP-2 protected patient neurons from stress-induced cell death and recombinant MMP-9/MMP-2 were sufficient to decrease neuronal survival. In tau-A152T neurons,inhibition of the ERK pathway decreased MMP-9 expression. Moreover,ectopic expression of 4R but not 3R tau-A152T in HEK293 cells increased MMP-9 expression and ERK phosphorylation. These findings provide insights into the molecular pathogenesis of FTD and suggest a potential therapeutic target for FTD with MAPT mutations. View Publication

The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) is caused by a CAG-repeat expansion in the ATXN3 gene. In this study,induced pluripotent stem cell (iPSC) lines were established from two SCA3 patients. Dermal fibroblasts were reprogrammed using an integration-free method and the resulting SCA3 iPSCs were differentiated into neurons. These neuronal lines harbored the disease causing mutation,expressed comparable levels of several neuronal markers and responded to the neurotransmitters,glutamate/glycine,GABA and acetylcholine. Additionally,all neuronal cultures formed networks displaying synchronized spontaneous calcium oscillations within 28days of maturation,and expressed the mature neuronal markers NeuN and Synapsin 1 implying a relatively advanced state of maturity,although not comparable to that of the adult human brain. Interestingly,we were not able to recapitulate the glutamate-induced ataxin-3 aggregation shown in a previously published iPSC-derived SCA3 model. In conclusion,we have generated a panel of SCA3 patient iPSCs and a robust protocol to derive neurons of relatively advanced maturity,which could potentially be valuable for the study of SCA3 disease mechanisms. View Publication

Various systems for differentiating hematopoietic cells from human pluripotent stem cells (PSCs) have been developed,although none have been fully optimized. In this report,we describe the development of a novel three-dimensional system for differentiating hematopoietic cells from PSCs using collagen sponges (CSs) reinforced with poly(ethylene terephthalate) fibers as a scaffold. PSCs seeded onto CSs were differentiated in a stepwise manner with appropriate cytokines under serum-free and feeder-free conditions. This process yielded several lineages of floating hematopoietic cells repeatedly for more than 1 month. On immunohistochemical staining,we detected CD34+ cells and CD45+ cells in the surface and cavities of the CS. Taking advantage of the portability of this system,we were able to culture multiple CSs together floating in medium,making it possible to harvest large numbers of hematopoietic cells repeatedly. Given these findings,we suggest that this novel three-dimensional culture system may be useful in the large-scale culture of PSC-derived hematopoietic cells. View Publication

Utilizing a fast 31P magnetic resonance spectroscopy (MRS) 2-dimensional chemical shift imaging (2D-CSI) method,this study examined the heterogeneity of creatine kinase (CK) forward flux rate of hearts with postinfarction left ventricular (LV) remodeling. Immunosuppressed Yorkshire pigs were assigned to 4 groups: 1) A sham-operated normal group (SHAM,n = 6); 2) A 60 minutes distal left anterior descending coronary artery ligation and reperfusion (MI,n = 6); 3) Open patch group; ligation injury plus open fibrin patch over the site of injury (Patch,n = 6); and 4) Cell group,hiPSCs-cardiomyocytes,-endothelial cells,and -smooth muscle cells (2 million,each) were injected into the injured myocardium pass through a fibrin patch (Cell+Patch,n = 5). At 4 weeks,the creatine phosphate (PCr)/ATP ratio,CK forward flux rate (Flux PCr→ATP),and k constant of CK forward flux rate (kPCr→ATP) were severely decreased at border zone myocardium (BZ) adjacent to MI. Cell treatment results in significantly increase of PCr/ATP ratio and improve the value of kPCr→ATP and Flux PCr→ATP in BZ myocardium. Moreover,the BZ myocardial CK total activity and protein expression of CK mitochondria isozyme and CK myocardial isozyme were significantly reduced,but recovered in response to cell treatment. Thus,cell therapy results in improvement of BZ bioenergetic abnormality in hearts with postinfarction LV remodeling,which is accompanied by significantly improvements in BZ CK activity and CK isozyme expression. The fast 2D 31P MR CSI mapping can reliably measure the heterogeneity of bioenergetics in hearts with post infarction LV remodeling. View Publication

Under steady-state conditions,aged neutrophils are removed from the circulation in bone marrow,liver,and spleen thereby maintaining myeloid cell homeostasis. The fate of these aged immune cells under inflammatory conditions,however,remains largely obscure. Here,we demonstrate that in the acute inflammatory response during endotoxemia aged neutrophils cease returning to the bone marrow and instead rapidly migrate to the site of inflammation. Having arrived in inflamed tissue,aged neutrophils were found to exhibit a higher phagocytic activity as compared to the subsequently recruited non-aged neutrophils. This distinct behavior of aged neutrophils under inflammatory conditions is dependent on specific age-related changes in their molecular repertoire that enable these 'experienced' immune cells to instantly translate inflammatory signals into immune responses. In particular,aged neutrophils engage toll-like receptor-4- and p38 mitogen-activated protein kinases-dependent pathways to induce conformational changes in β2 integrins which allow these phagocytes to effectively accomplish their mission in the front line of the inflammatory response. Hence,ageing in the circulation might represent a critical process for neutrophils that enables these immune cells to properly unfold their functional properties for host defense. View Publication

The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine,pharmacological drug screening,and toxicity testing. However,full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study,we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 10(6) hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A,Wnt3a,and sodium butyrate to the culture medium. For further maturation,hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP),a marker for DE,was significantly (p textless 0.05) higher in 2D cultures,while secretion of albumin,a typical characteristic for mature hepatocytes,was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2,CYP2B6,and CYP3A4 in both groups,although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p textless 0.05) higher in 3D bioreactors compared with 2D cultures,which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin,cytokeratin 18 (CK18),and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition,cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures. View Publication

Human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs),dental pulp stem cells (hDPSCs) and bone marrow MSCs (hBMSCs) are exciting cell sources in regenerative medicine. However,there has been no report comparing hDPSCs,hBMSCs and hiPSC-MSCs for bone engineering in an injectable calcium phosphate cement (CPC) scaffold. The objectives of this study were to: (1) develop a novel injectable CPC containing hydrogel fibers encapsulating stem cells for bone engineering,and (2) compare cell viability,proliferation and osteogenic differentiation of hDPSCs,hiPSC-MSCs from bone marrow (BM-hiPSC-MSCs) and from foreskin (FS-hiPSC-MSCs),and hBMSCs in CPC for the first time. The results showed that the injection did not harm cell viability. The porosity of injectable CPC was 62%. All four types of cells proliferated and differentiated down the osteogenic lineage inside hydrogel fibers in CPC. hDPSCs,BM-hiPSC-MSCs,and hBMSCs exhibited high alkaline phosphatase,runt-related transcription factor,collagen I,and osteocalcin gene expressions. Cell-synthesized minerals increased with time (ptextless0.05),with no significant difference among hDPSCs,BM-hiPSC-MSCs and hBMSCs (ptextgreater0.1). Mineralization by hDPSCs,BM-hiPSC-MSCs,and hBMSCs inside CPC at 14d was 14-fold that at 1d. FS-hiPSC-MSCs were inferior in osteogenic differentiation compared to the other cells. In conclusion,hDPSCs,BM-hiPSC-MSCs and hBMSCs are similarly and highly promising for bone tissue engineering; however,FS-hiPSC-MSCs were relatively inferior in osteogenesis. The novel injectable CPC with cell-encapsulating hydrogel fibers may enhance bone regeneration in dental,craniofacial and orthopedic applications. View Publication

Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology,including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells-based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well-known mechanisms that are activated by chemically-induced oxidative stress is the Nrf2 signaling pathway. Therefore,in the current study,we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)-derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone-induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM),as shown by Nrf2 nuclear translocation and the up-regulation of the Nrf2-downstream antioxidant enzymes,NQO1 and SRXN1. Interestingly,exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover,rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH(+)) cells was observed. The obtained results suggest that hiPSC-derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation. View Publication

Friedreich's ataxia (FRDA) is the most common autosomal recessive ataxia. This severe neurodegenerative disease is caused by an expansion of guanine-adenine-adenine (GAA) repeats located in the first intron of the frataxin (FXN) gene,which represses its transcription. Although transcriptional silencing is associated with heterochromatin-like changes in the vicinity of the expanded GAAs,the exact mechanism and pathways involved in transcriptional inhibition are largely unknown. As major remodeling of the epigenome is associated with somatic cell reprogramming,modulating chromatin modification pathways during the cellular transition from a somatic to a pluripotent state is likely to generate permanent changes to the epigenetic landscape. We hypothesize that the epigenetic modifications in the vicinity of the GAA repeats can be reversed by pharmacological modulation during somatic cell reprogramming. We reprogrammed FRDA fibroblasts into induced pluripotent stem cells (iPSCs) in the presence of various small molecules that target DNA methylation and histone acetylation and methylation. Treatment of FRDA iPSCs with two compounds,sodium butyrate (NaB) and Parnate,led to an increase in FXN expression and correction of repressive marks at the FXN locus,which persisted for several passages. However,prolonged culture of the epigenetically modified FRDA iPSCs led to progressive expansions of the GAA repeats and a corresponding decrease in FXN expression. Furthermore,we uncovered that differentiation of these iPSCs into neurons also results in resilencing of the FXN gene. Taken together,these results demonstrate that transcriptional repression caused by long GAA repeat tracts can be partially or transiently reversed by altering particular epigenetic modifications,thus revealing possibilities for detailed analyses of silencing mechanism and development of new therapeutic approaches for FRDA. View Publication