技术资料

The fact that Parkinson's disease (PD) can arise from numerous genetic mutations suggests a unifying molecular pathology underlying the various genetic backgrounds. To address this hypothesis,we took an integrated approach utilizing in vitro disease modeling and comprehensive transcriptome profiling to advance our understanding of PD progression and the concordant downstream signaling pathways across divergent genetic predispositions. To model PD in vitro,we generated neurons harboring disease-causing mutations from patient-specific,induced pluripotent stem cells (iPSCs). We observed signs of degeneration in midbrain dopaminergic neurons,reflecting the cardinal feature of PD. Gene expression signatures of PD neurons provided molecular insights into disease phenotypes observed in vitro,including oxidative stress vulnerability and altered neuronal activity. Notably,PD neurons show that elevated RBFOX1,a gene previously linked to neurodevelopmental diseases,underlies a pattern of alternative RNA-processing associated with PD-specific phenotypes. View Publication

The vascularization of tissue-engineered bone is a prerequisite step for the successful repair of bone defects. Hypoxia inducible factor-1$$ (HIF-1$$) plays an essential role in angiogenesis-osteogenesis coupling during bone regeneration and can activate the expression of angiogenic factors in mesenchymal stem cells (MSCs). Dimethyloxaloylglycine (DMOG) is an angiogenic small molecule that can inhibit prolyl hydroxylase (PHD) enzymes and thus regulate the stability of HIF-1$$ in cells at normal oxygen tension. Human induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) are promising alternatives for stem cell therapy. In this study,we evaluated the effect of DMOG on promoting hiPSC-MSCs angiogenesis in tissue-engineered bone and simultaneously explored the underlying mechanisms in vitro. The effectiveness of DMOG in improving the expression of HIF-1$$ and its downstream angiogenic genes in hiPSC-MSCs demonstrated that DMOG significantly enhanced the gene and protein expression profiles of angiogenic-related factors in hiPSC-MSCs by sustaining the expression of HIF-1$$. Further analysis showed that DMOG-stimulated hiPSC-MSCs angiogenesis was associated with the phosphorylation of protein kinase B (Akt) and with an increase in VEGF production. The effects could be blocked by the addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In a critical-sized calvarial defect model in rats,DMOG-treated hiPSC-MSCs showed markedly improved angiogenic capacity in the tissue-engineered bone,leading to bone regeneration. Collectively,the results indicate that DMOG,via activation of the PI3K/Akt pathway,promotes the angiogenesis of hiPSC-MSCs in tissue-engineered bone for bone defect repair and that DMOG-treated hiPSC-MSCs can be exploited as a potential therapeutic tool in bone regeneration. View Publication

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general,but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies,however,is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is,however,limited and thereby further optimization in terms of time,efficiency,and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts,at least in part,in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1,Inhba and Grem1. Hence,we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. View Publication

Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic,nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors,allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this,gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents. View Publication

Heterozygous loss-of-function mutations in GRIN2B,a subunit of the NMDA receptor,cause intellectual disability and language impairment. We developed clonal models of GRIN2B deletion and loss-of-function mutations in a region coding for the glutamate binding domain in human cells and generated neurons from a patient harboring a missense mutation in the same domain. Transcriptome analysis revealed extensive increases in genes associated with cell proliferation and decreases in genes associated with neuron differentiation,a result supported by extensive protein analyses. Using electrophysiology and calcium imaging,we demonstrate that NMDA receptors are present on neural progenitor cells and that human mutations in GRIN2B can impair calcium influx and membrane depolarization even in a presumed undifferentiated cell state,highlighting an important role for non-synaptic NMDA receptors. It may be this function,in part,which underlies the neurological disease observed in patients with GRIN2B mutations. View Publication

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here,we describe a new recessive neurodevelopmental syndrome with global developmental delay,seizures and intellectual disability. Using linkage analysis and exome sequencing,we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation,p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically,the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo,CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43,the ortholog of human CAMK2A. In vitro,neurons derived from patient iPSCs displayed profound synaptic defects. Together,our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders. View Publication

BACKGROUND Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia,although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer's disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD. METHODS We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling,insulin regulation of the major AD proteins Abeta$ and/or tau,and/or calcium fluxes is altered by the PSEN2 N141I mutation. RESULTS We report herein that wildtype,PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Abeta$42/40 ratio. Unexpectedly,we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux,which could be prevented by chronic exposure of BFCNs to insulin. CONCLUSIONS Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Abeta$42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence,our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD. View Publication

Methylation of cytosine is a DNA modification associated with gene repression. Recently,a novel cytosine modification,5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems,and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage,where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC,which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts,and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs,5-hmC is strongly enriched in bone marrow and brain,wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation,as has been reported previously,but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages,high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge,5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific. View Publication

Gastric cancer (GC) is one of the most common human cancers. Spheroid colony formation is an effective model for characterization of cancer stem cells. However,gene expression profiles of spheroid colonies obtained from GC cells have not been examined. We performed microarray analyses by Human Genome U133 Plus 2.0 Array in spheroid body-forming and parental cells from MKN-45 and MKN-74 GC cell lines. Kinesin family member C1 (KIFC1) was expressed textgreater2-fold higher in spheroid body-forming cells than in parental cells in both GC lines. Both the number and size of spheres from MKN-45 cells were significantly reduced upon KIFC1 siRNA-transfection compared with negative control siRNA-transfection. Immunohistochemical analysis of 114 GC tissue samples revealed that 42 (37%) of GC cases were positive for KIFC1 expression. GC cases positive for KIFC1 were found more frequently in stage III/IV cases than in stage I/II cases. GC cases positive for KIFC1 were found more frequently in intestinal type GC cases than in diffuse type GC cases. Furthermore,KIFC1-positive GC cases showed high Ki-67 labeling index. Kaplan-Meier analysis demonstrated that KIFC1 expression was not associated with survival. We found positive expression of KIFC1 in CD44‑positive GC and aldehyde dehydrogenase 1 (ALDH1)-positive GC cells. Our results showed that KIFC1 is overexpressed in GC. Since knockdown of KIFC1 inhibited sphere formation,KIFC1 likely plays an important role in cancer stem cells. View Publication

Abstract Purpose: The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic,with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells,but previous studies have shown variability in iPSC propensity to differentiate into RPE. Methods: This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid,directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE,which were characterized with respect to global gene expression,expression of RPE markers,and cellular function. Results: We found that all 5 iPSC lines (iPSC-1,iPSC-2,iPSC-3,iPSC-4,and iPSC-12) generated RPE using the directed differentiation protocol; however,2 of the 5 iPSC lines (iPSC-4 and iPSC-... View Publication

Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands,targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features,and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall,the approach described here allows for the rapid generation of human cardiac grafts from hPSCs,in a total of 24 days,providing a suitable platform for cardiac engineering and disease modeling in the human setting. View Publication

PURPOSE To model keratoconus (KC) using induced pluripotent stem cells (iPSC) generated from fibroblasts of both KC and normal human corneal stroma by a viral method. METHODS Both normal and KC corneal fibroblasts from four human donors were reprogramed directly by delivering reprogramming factors in a single virus using 2A self-cleaving" peptides� View Publication