技术资料

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

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However,primary hepatocyte scarcity,cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies,HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology,such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study,the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions,cytochrome P450 drug metabolism and serum protein secretion,in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug,primarily metabolised by Cyp2D6. In addition to metabolic capacity,fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker,alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype. View Publication

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease that leads to a profound loss of life quality and premature death. Around 10% of the cases are inherited and ALS8 is an autosomal dominant form of familial ALS caused by mutations in the vamp-associated protein B/C (VAPB) gene. The VAPB protein is involved in many cellular processes and it likely contributes to the pathogenesis of other forms of ALS besides ALS8. A number of successful drug tests in ALS animal models could not be translated to humans underscoring the need for novel approaches. The induced pluripotent stem cells (iPSC) technology brings new hope,since it can be used to model and investigate diseases in vitro. Here we present an additional tool to study ALS based on ALS8-iPSC. Fibroblasts from ALS8 patients and their non-carrier siblings were successfully reprogrammed to a pluripotent state and differentiated into motor neurons. We show for the first time that VAPB protein levels are reduced in ALS8-derived motor neurons but,in contrast to over-expression systems,cytoplasmic aggregates could not be identified. Our results suggest that optimal levels of VAPB may play a central role in the pathogenesis of ALS8,in agreement with the observed reduction of VAPB in sporadic ALS. View Publication

A microfluidic human pluripotent stem cell (hPSC) array has been developed for robust and reproducible hPSC culture methods to assess chemically defined serum- and feeder-free culture conditions. This microfluidic platform,combined with image cytometry,enables the systematic analysis of multiple simultaneously detected marker expression in individual cells,for screening of various chemically defined media across hPSC lines,and the study of phenotypic responses. 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

AIMS: Generation of human induced pluripotent stem cell (hiPSC) lines by reprogramming of fibroblast cells with virus-free methods offers unique opportunities for translational cardiovascular medicine. The aim of the study was to reprogramme fibroblast cells to hiPSCs and to study cardiomyogenic properties and ion channel characteristics of the virus-free hiPSC-derived cardiomyocytes. METHODS AND RESULTS: The hiPSCs generated by episomal vectors generated teratomas in severe combined immunodeficient mice,readily formed embryoid bodies,and differentiated into cardiomyocytes with comparable efficiency to human embryonic stem cells. Temporal gene expression of these hiPSCs indicated that differentiation of cardiomyocytes was initiated by increasing expression of cardio/mesodermal markers followed by cardiac-specific transcription factors,structural,and ion channel genes. Furthermore,the cardiomyocytes showed characteristic cross-striations of sarcomeric proteins and expressed calcium-handling and ion channel proteins,confirming their cardiac ontogeny. Microelectrode array recordings established the electrotonic development of a functional syncytium that responded predictably to pharmacologically active drugs. The cardiomyocytes showed a chronotropic dose-response (0.1-10 µM) to isoprenaline and Bay K 8644. Furthermore,carbamycholine (5 µM) suppressed the response to isoprenaline,while verapamil (2.5 µM) blocked Bay K 8644-induced inotropic activity. Moreover,verapamil (1 µM) reduced the corrected field potential duration by 45%,tetrodotoxin (10 µM) shortened the minimal field potential by 40%,and E-4031 (50 nM) prolonged field repolarization. CONCLUSION: Virus-free hiPSCs differentiate efficiently into cardiomyocytes with cardiac-specific molecular,structural,and functional properties that recapitulate the developmental ontogeny of cardiogenesis. These results,coupled with the potential to generate patient-specific hiPSC lines,hold great promise for the development of an in vitro platform for drug pharmacogenomics,disease modelling,and regenerative medicine. View Publication

Evi1 (ecotropic viral integration site 1) is essential for proliferation of hematopoietic stem cells and implicated in the development of myeloid disorders. Particularly,high Evi1 expression defines one of the largest clusters in acute myeloid leukemia and is significantly associated with extremely poor prognosis. However,mechanistic basis of Evi1-mediated leukemogenesis has not been fully elucidated. Here,we show that Evi1 directly represses phosphatase and tensin homologue deleted on chromosome 10 (PTEN) transcription in the murine bone marrow,which leads to activation of AKT/mammalian target of rapamycin (mTOR) signaling. In a murine bone marrow transplantation model,Evi1 leukemia showed modestly increased sensitivity to an mTOR inhibitor rapamycin. Furthermore,we found that Evi1 binds to several polycomb group proteins and recruits polycomb repressive complexes for PTEN down-regulation,which shows a novel epigenetic mechanism of AKT/mTOR activation in leukemia. Expression analyses and ChIPassays with human samples indicate that our findings in mice models are recapitulated in human leukemic cells. Dependence of Evi1-expressing leukemic cells on AKT/mTOR signaling provides the first example of targeted therapeutic modalities that suppress the leukemogenic activity of Evi1. The PTEN/AKT/mTOR signaling pathway and the Evi1-polycomb interaction can be promising therapeutic targets for leukemia with activated Evi1. View Publication

Since the derivation of the first human embryonic stem cell (hESC) lines by Thomson and coworkers in 1998,more than 1,200 different hESC lines have been established worldwide. Nevertheless,there is still a recognized interest in the establishment of new lines of hESC,particularly from HLA types and ethnic groups currently underrepresented among the available lines. The methodology of hESC derivation has evolved significantly since 1998,when human LIF (hLIF) was used for maintenance of pluripotency. However,there are a number of different strategies for the several steps involved in establishing a new line of hESC. Here we make a survey of the most relevant parameters used between 1998 and 2010 for the derivation of the 375 hESC lines deposited in two international stem cell registries,and able to form teratomas in immunocompromised mice. Although we identify some trends in the methodology for establishing hESC lines,our data reveal a much greater heterogeneity of strategies than what is used for derivation of murine ESC lines,indicating that optimum conditions have not been consolidated yet,and thus,hESC establishment is still an evolving field of research. View Publication

We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR™1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel™-coated substrates. Mechanistically,it was resolved that bovine serum albumin (BSA) in the mTeSR™1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g.,peptides,proteins,or Matrigel™) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion. View Publication

Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene,whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons,but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types,including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation,affected by early onset seizure variant and X-linked epileptic encephalopathy,respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore,the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro. View Publication