技术资料

The derivation of astrocytes from human pluripotent stem cells is currently slow and inefficient. We demonstrate that overexpression of the transcription factors SOX9 and NFIB in human pluripotent stem cells rapidly and efficiently yields homogeneous populations of induced astrocytes. In our study these cells exhibited molecular and functional properties resembling those of adult human astrocytes and were deemed suitable for disease modeling. Our method provides new possibilities for the study of human astrocytes in health and disease. View Publication

The vast majority of pedigrees with familial Alzheimer's disease (FAD) are caused by inheritance of mutations in the PSEN1 1 gene. While genetic ablation studies have revealed a role for presenilin 1 (PS1) in embryonic neurogenesis,little information has emerged regarding the potential effects of FAD-linked PS1 variants on proliferation,self-renewal and differentiation,key events that control cell fate commitment of adult brain neural progenitors (NPCs). We used adult brain subventricular zone (SVZ)-derived NPC cultures transduced with recombinant lentivirus as a means to investigate the effects of various PS1 mutants on self-renewal and differentiation properties. We now show that viral expression of several PS1 mutants in NPCs leads to impaired self-renewal and altered differentiation toward neuronal lineage,in vitro. In line with these observations,diminished constitutive proliferation and steady-state SVZ progenitor pool size was observed in vivo in transgenic mice expressing the PS1DeltaE9 variant. Moreover,NPC cultures established from the SVZ of adult mice expressing PS1DeltaE9 exhibit reduced self-renewal capacity and premature exit toward neuronal fates. To these findings,we show that both the levels of endogenous Notch/CBF-1-transcriptional activity and transcripts encoding Notch target genes are diminished in SVZ NPCs expressing PS1DeltaE9. The deficits in self-renewal and multipotency are restored by expression of Notch1-ICD or a downstream target of the Notch pathway,Hes1. Hence,we argue that a partial reduction in PS-dependent gamma-secretase processing of the Notch,at least in part,accounts for the impairments observed in SVZ NPCs expressing the FAD-linked PS1DeltaE9 variant. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease,and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly,as well as scFvs that target the CD133-positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv,and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular nonselective medium. Taken together,these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library,which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. View Publication

p73,a member of the p53 family,is a transcription factor that plays a key role in many biological processes. In the present study,we show that TAp73 is expressed in neural stem cells (NSC) and its expression increases following their differentiation. NSC from p73 null mice have a reduced proliferative potential,together with reduced expression of members of the Sox-2 and Notch gene families known to be important for NSC proliferation. In parallel with this in vitro data,the width of the neurogenic areas was reduced in the brains of embryonic and adult p73-/- mice. These data suggest that p73,and in particular TAp73,is important for maintenance of the NSC pool. View Publication

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently,we elucidated by using round spermatids that,after nuclear transfer,treatment of zygotes with trichostatin A (TSA),an inhibitor of histone deacetylase,can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami,N. Van Thuan,T. Hikichi,H. Ohta,S. Wakayama. E. Mizutani,T. Wakayama,Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids,Dev. Biol. (2005) in press]. Here,we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells,spleen cells,neural stem cells,and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further,we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus,our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques. View Publication

Numerous proteins undergo modification by palmitic acid (S-acylation) for their biological functions including signal transduction,vesicular transport and maintenance of cellular architecture. Although palmitoylation is an essential modification,these proteins must also undergo depalmitoylation for their degradation by lysosomal proteases. Palmitoyl-protein thioesterase-1 (PPT1),a lysosomal enzyme,cleaves thioester linkages in S-acylated proteins and removes palmitate residues facilitating the degradation of these proteins. Thus,inactivating mutations in the PPT1 gene cause infantile neuronal ceroid lipofuscinosis (INCL),a devastating neurodegenerative storage disorder of childhood. Although rapidly progressing brain atrophy is the most dramatic pathological manifestation of INCL,the molecular mechanism(s) remains unclear. Using PPT1-knockout (PPT1-KO) mice that mimic human INCL,we report here that the endoplasmic reticulum (ER) in the brain cells of these mice is structurally abnormal. Further,we demonstrate that the level of growth-associated protein-43 (GAP-43),a palmitoylated neuronal protein,is elevated in the brains of PPT1-KO mice. Moreover,forced expression of GAP-43 in PPT1-deficient cells results in the abnormal accumulation of this protein in the ER. Consistent with these results,we found evidence for the activation of unfolded protein response (UPR) marked by elevated levels of phosphorylated translation initiation factor,eIF2alpha,increased expression of chaperone proteins such as glucose-regulated protein-78 and activation of caspase-12,a cysteine proteinase in the ER,mediating caspase-3 activation and apoptosis. Our results,for the first time,link PPT1 deficiency with the activation of UPR,apoptosis and neurodegeneration in INCL and identify potential targets for therapeutic intervention in this uniformly fatal disease. View Publication

Angiotensin II (Ang II) type 2 (AT2) receptors are abundantly expressed not only in the fetal brain where they probably contribute to brain development,but also in pathological conditions to protect the brain against stroke; however,the detailed mechanisms are unclear. Here,we demonstrated that AT2 receptor signaling induced neural differentiation via an increase in MMS2,one of the ubiquitin-conjugating enzyme variants. The AT2 receptor,MMS2,Src homology 2 domain-containing protein-tyrosine phosphatase 1 (SHP-1),and newly cloned AT2 receptor-interacting protein (ATIP) were highly expressed in fetal rat neurons and declined after birth. Ang II induced MMS2 expression in a dose-dependent manner,reaching a peak after 4 h of stimulation,and this effect was enhanced with AT1 receptor blocker,valsartan,but inhibited by AT2 receptor blocker PD123319. Moreover,we observed that an AT2 receptor agonist,CGP42112A,alone enhanced MMS2 expression. Neurons treated with small interfering RNA of MMS2 failed to exhibit neurite outgrowth and synapse formation. Moreover,the increase in AT2 receptor-induced MMS2 mRNA expression was enhanced by overexpression of ATIP but inhibited by small interfering RNA of SHP-1 and overexpression of catalytically dominant-negative SHP-1 or a tyrosine phosphatase inhibitor,sodium orthovanadate. After AT2 receptor stimulation,ATIP and SHP-1 were translocated into the nucleus after formation of their complex. Furthermore,increased MMS2 expression mediates the inhibitor of DNA binding 1 proteolysis and promotes DNA repair. These results provide a new insight into the contribution of AT2 receptor stimulation to neural differentiation via transactivation of MMS2 expression involving the association of ATIP and SHP-1. View Publication

Doublecortin (DCX) has recently been promulgated as a selective marker of cells committed to the neuronal lineage in both the developing and the adult brain. To explore the potential of DCX-positive (DCX+) cells more stringently,these cells were isolated by flow cytometry from the brains of transgenic mice expressing green fluorescent protein under the control of the DCX promoter in embryonic,early postnatal,and adult animals. It was found that virtually all of the cells (99.9%) expressing high levels of DCX (DCX(high)) in the embryonic brain coexpressed the neuronal marker betaIII-tubulin and that this population contained no stem-like cells as demonstrated by lack of neurosphere formation in vitro. However,the DCX+ population from the early postnatal brain and the adult subventricular zone and hippocampus,which expressed low levels of DCX (DCX(low)),was enriched for neurosphere-forming cells,with only a small subpopulation of these cells coexpressing the neuronal markers betaIII-tubulin or microtubule-associated protein 2. Similarly,the DCX(low) population from embryonic day 14 (E14) brain contained neurosphere-forming cells. Only the postnatal cerebellum and adult olfactory bulb contained some DCX(high) cells,which were shown to be similar to the E14 DCX(high) cells in that they had no stem cell activity. Electrophysiological studies confirmed the heterogeneous nature of DCX+ cells,with some cells displaying characteristics of immature or mature neurons,whereas others showed no neuronal characteristics whatsoever. These results indicate that DCX(high) cells,regardless of location,are restricted to the neuronal lineage or are bone fide neurons,whereas some DCX(low) cells retain their multipotentiality. View Publication

Down's syndrome (DS),caused by trisomy of human chromosome 21,is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons,and fails to promote neuronal ion channel maturation and synapse formation. Transplantation studies show that DS astroglia do not promote neurogenesis of endogenous neural stem cells in vivo. We also observed abnormal gene expression profiles from DS astroglia. Finally,we show that the FDA-approved antibiotic drug,minocycline,partially corrects the pathological phenotypes of DS astroglia by specifically modulating the expression of S100B,GFAP,inducible nitric oxide synthase,and thrombospondins 1 and 2 in DS astroglia. Our studies shed light on the pathogenesis and possible treatment of DS by targeting astrocytes with a clinically available drug. View Publication

In Huntington's disease (HD),whether transneuronal spreading of mutant huntingtin (mHTT) occurs and its contribution to non-cell autonomous damage in brain networks is largely unknown. We found mHTT spreading in three different neural network models: human neurons integrated in the neural network of organotypic brain slices of HD mouse model,an ex vivo corticostriatal slice model and the corticostriatal pathway in vivo. Transneuronal propagation of mHTT was blocked by two different botulinum neurotoxins,each known for specifically inactivating a single critical component of the synaptic vesicle fusion machinery. Moreover,healthy human neurons in HD mouse model brain slices displayed non-cell autonomous changes in morphological integrity that were more pronounced when these neurons bore mHTT aggregates. Altogether,our findings suggest that transneuronal propagation of mHTT might be an important and underestimated contributor to the pathophysiology of HD. View Publication

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However,such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs),which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods,we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene,which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening. View Publication

Despite major advances in the pathophysiological understanding of peripheral nerve damage,the treatment of nerve injuries still remains an unmet medical need. Nerve guidance conduits present a promising treatment option by providing a growth-permissive environment that 1) promotes neuronal cell survival and axon growth and 2) directs axonal extension. To this end,we designed an electrospun nerve guidance conduit using a blend of polyurea and poly-caprolactone with both biochemical and topographical cues. Biochemical cues were integrated into the conduit by functionalizing the polyurea with RGD to improve cell attachment. Topographical cues that resemble natural nerve tissue were incorporated by introducing intraluminal microchannels aligned with nanofibers. We determined that electrospinning the polymer solution across a two electrode system with dissolvable sucrose fibers produced a polymer conduit with the appropriate biomimetic properties. Human neural stem cells were cultured on the conduit to evaluate its ability to promote neuronal growth and axonal extension. The nerve guidance conduit was shown to enhance cell survival,migration,and guide neurite extension. View Publication