技术资料

GABAergic interneurons regulate cortical neural networks by providing inhibitory inputs,and their malfunction,resulting in failure to intricately regulate neural circuit balance,is implicated in brain diseases such as Schizophrenia,Autism,and Epilepsy. During early development,GABAergic interneuron progenitors arise from the ventral telencephalic area such as medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) by the actions of secreted signaling molecules from nearby organizers,and migrate to their target sites where they form local synaptic connections. In this study,using combinatorial and temporal modulation of developmentally relevant dorsoventral and rostrocaudal signaling pathways (SHH,Wnt,and FGF8),we efficiently generated MGE cells from multiple human pluripotent stem cells. Most importantly,modulation of FGF8/FGF19 signaling efficiently directed MGE versus CGE differentiation. Human MGE cells spontaneously differentiated into Lhx6-expressing GABAergic interneurons and showed migratory properties. These human MGE-derived neurons generated GABA,fired action potentials,and displayed robust GABAergic postsynaptic activity. Transplantation into rodent brains results in well-contained neural grafts enriched with GABAergic interneurons that migrate in the host and mature to express somatostatin or parvalbumin. Thus,we propose that signaling modulation recapitulating normal developmental patterns efficiently generate human GABAergic interneurons. This strategy represents a novel tool in regenerative medicine,developmental studies,disease modeling,bioassay,and drug screening. View Publication

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However,techniques to generate cell type-specific lineage reporters,as well as reliable tools to disrupt,repair or overexpress genes by gene targeting,are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First,using ZFNs specific for the OCT4 (POU5F1) locus,we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second,we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally,we targeted the PITX3 gene,demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. View Publication

Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However,marker-free genome editing using standard protocols remains inefficient,yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing,so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA),donor DNA and piggyBac transposase resulted in efficient,targeted genome editing and concurrent scarless transgene excision. Using this approach,in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%. View Publication

Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. We investigated the ability of two commercially available electroporation systems,the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However,the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency,producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally,we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. Our results indicate that these electroporation methods provide a reliable,efficient,and high-throughput approach to the genetic manipulation of hESC. View Publication

Human macrophages are specialised hosts for HIV-1,dengue virus,Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens,because available myeloid cell lines are,by definition,not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined,feeder- and serum-free culture conditions and produces very consistent,pure,high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively,up to ∼3×10(7) monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+),CD16(low),CD163(+)). Differentiation with M-CSF produces macrophages that are highly phagocytic,HIV-1-infectable,and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate,as they recognise foreign nucleic acids; the lentivector system described here overcomes this,as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells,surmounting issues of transgene silencing. Overall,the method we describe here is an efficient,effective,scalable system for the reproducible production and genetic modification of human macrophages,facilitating the interrogation of human macrophage biology. View Publication

We have shown that epigallocatechin-3-gallate (EGCG),a polyphenolic compound from green tea,inhibits growth and induces apoptosis in human pancreatic cancer cells. However,the preclinical potential of EGCG in a suitable mouse model has not been examined. In this study,we examined the molecular mechanisms by which EGCG inhibited growth,invasion,metastasis and angiogenesis of human pancreatic cancer cells in a xenograft model system. EGCG inhibited viability,capillary tube formation and migration of HUVEC,and these effects were further enhanced in the presence of an ERK inhibitor. In vivo,AsPC-1 xenografted tumors treated with EGCG showed significant reduction in volume,proliferation (Ki-67 and PCNA staining),angiogenesis (vWF,VEGF and CD31) and metastasis (MMP-2,MMP-7,MMP-9 and MMP-12) and induction in apoptosis (TUNEL),caspase-3 activity and growth arrest (p21/WAF1). EGCG also inhibited circulating endothelial growth factor receptor 2 (VEGF-R2) positive endothelial cells derived from xenografted mice. Tumor samples from EGCG treated mice showed significantly reduced ERK activity,and enhanced p38 and JNK activities. Overall,our data suggest that EGCG inhibits pancreatic cancer growth,invasion,metastasis and angiogenesis,and thus could be used for the management of pancreatic cancer prevention and treatment. View Publication

Bone loss in postmenopausal osteoporosis is induced chiefly by an imbalance of bone-forming osteoblasts and bone-resorbing osteoclasts. Salubrinal is a synthetic compound that inhibits de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2$\alpha$). Phosphorylation of eIF2$\alpha$ alleviates endoplasmic reticulum (ER) stress,which may activate autophagy. We hypothesized that eIF2$\alpha$ signaling regulates bone homeostasis by promoting autophagy in osteoblasts and inhibiting osteoclast development. To test the hypothesis,we employed salubrinal to elevate the phosphorylation of eIF2$\alpha$ in an ovariectomized (OVX) mouse model and cell cultures. In the OVX model,salubrinal prevented abnormal expansion of rough ER and decreased the number of acidic vesiculars. It regulated ER stress-associated signaling molecules such as Bip,p-eIF2$\alpha$,ATF4 and CHOP,and promoted autophagy of osteoblasts via regulation of eIF2$\alpha$,Atg7,LC3,and p62. Salubrinal markedly alleviated OVX-induced symptoms such as reduction of bone mineral density and bone volume fraction. In primary bone-marrow-derived cells,salubrinal increased the differentiation of osteoblasts,and decreased the formation of osteoclasts by inhibiting nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Live cell imaging and RNA interference demonstrated that suppression of osteoclastogenesis is in part mediated by Rac1 GTPase. Collectively,this study demonstrates that ER stress-autophagy axis plays an important role in OVX mice. Bone-forming osteoblasts are restored by maintaining phosphorylation of eIF2$\alpha$,and bone-resorbing osteoclasts are regulated by inhibiting NFATc1 and Rac1 GTPase. View Publication

There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL,TEL/PDGFRbeta,FIP1/PDGFRalpha,D816V KIT,NPM/ALK,and FLT3ITD) revealed few common effects on the transcriptome. It is apparent,however,that proteome changes are not directly governed by transcriptome changes. Therefore,we assessed and used a new generation of iTRAQ tagging,enabling eight-channel relative quantification discovery proteomics,to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRalpha. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin,although this did not correspond to changes in motility. However,correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes,illustrating the utility of such a combined approach. View Publication

Leukocyte trafficking to sites of inflammation follows a defined temporal pattern,and evidence suggests that initial neutrophil transendothelial migration modifies endothelial cell phenotype. We tested the hypothesis that preconditioning of human umbilical vein endothelial cells (HUVEC) by neutrophils would also modify the subsequent transendothelial migration of T lymphocytes across cytokine-stimulated HUVEC in an in vitro flow assay. Using fluorescence microscopy,preconditioning of HUVEC by neutrophils was observed to significantly reduce the extent of subsequent stromal cell-derived factor-1alpha (SDF-1alpha [CXCL12])-mediated T lymphocyte transendothelial migration,without reducing accumulation. In contrast,recruitment of a second wave of neutrophils was unaltered. Conditioned medium harvested after transendothelial migration of neutrophils or supernatants from stimulated neutrophils mediated a similar blocking effect,which was negated using a specific neutrophil elastase inhibitor. Furthermore,T lymphocyte transendothelial migration was inhibited by treatment of HUVEC with purified neutrophil elastase,which selectively cleaved the amino terminus of HUVEC-bound SDF-1alpha,which is required for its chemotactic activity. The reduction in T lymphocyte transendothelial migration was not observed using a different chemokine,ELC (CCL19),and was not reversed by replenishment of SDF-1alpha,indicating endothelial retention of the inactivated chemokine. In summary,transmigrating neutrophils secrete localized elastase that is protected from plasma inhibitors,and thereby modulate trafficking of other leukocyte subsets by altering the endothelial-associated chemotactic activities. View Publication

The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications,including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs),a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC),and also the gold standard human embryonic stem cell,we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness,and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly,cells exhibited a noticeable difference in stiffness. From least to most stiff,the order of cell stiffness was as follows: hASC-iPSC,human embryonic stem cell,fibroblast-iPSC,fibroblasts,and,lastly,as the stiffest cell,hASC. In comparing hASC-iPSCs to their origin cell,the hASC,the reprogrammed cell is significantly less stiff,indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence,material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state. View Publication

The delicate “Excitatory/Inhibitory balance” between neurons holds significance in neurodegenerative and neurodevelopmental diseases. With the ultimate goal of creating a faithful in vitro model of the human brain,in this study,we investigated the critical factor of heterogeneity,focusing on the interplay between excitatory glutamatergic (E) and inhibitory GABAergic (I) neurons in neural networks. We used high-density Micro-Electrode Arrays (MEA) with 2304 recording electrodes to investigate two neuronal culture configurations: 100% glutamatergic (100E) and 75% glutamatergic / 25% GABAergic (75E25I) neurons. This allowed us to comprehensively characterize the spontaneous electrophysiological activity exhibited by mature cultures at 56 Days in vitro,a time point in which the GABA shift has already occurred. We explored the impact of heterogeneity also through electrical stimulation,revealing that the 100E configuration responded reliably,while the 75E25I required more parameter tuning for improved responses. Chemical stimulation with BIC showed an increase in terms of firing and bursting activity only in the 75E25I condition,while APV and CNQX induced significant alterations on both dynamics and functional connectivity. Our findings advance understanding of diverse neuron interactions and their role in network activity,offering insights for potential therapeutic interventions in neurological conditions. Overall,this work contributes to the development of a valuable human-based in vitro system for studying physiological and pathological conditions,emphasizing the pivotal role of neuron diversity in neural network dynamics. View Publication

A major road-block in stem cell therapy is the poor homing and integration of transplanted stem cells with the targeted host tissue. Human induced pluripotent stem (hiPS) cells are considered an excellent alternative to embryonic stem (ES) cells and we tested the feasibility of using small,physiological electric fields (EFs) to guide hiPS cells to their target. Applied EFs stimulated and guided migration of cultured hiPS cells toward the anode,with a stimulation threshold of textless30 mV/mm; in three-dimensional (3D) culture hiPS cells remained stationary,whereas in an applied EF they migrated directionally. This is of significance as the therapeutic use of hiPS cells occurs in a 3D environment. EF exposure did not alter expression of the pluripotency markers SSEA-4 and Oct-4 in hiPS cells. We compared EF-directed migration (galvanotaxis) of hiPS cells and hES cells and found that hiPS cells showed greater sensitivity and directedness than those of hES cells in an EF,while hES cells migrated toward cathode. Rho-kinase (ROCK) inhibition,a method to aid expansion and survival of stem cells,significantly increased the motility,but reduced directionality of iPS cells in an EF by 70-80%. Thus,our study has revealed that physiological EF is an effective guidance cue for the migration of hiPS cells in either 2D or 3D environments and that will occur in a ROCK-dependent manner. Our current finding may lead to techniques for applying EFs in vivo to guide migration of transplanted stem cells. View Publication