技术资料

Neurogenesis decreases during aging and following cranial radiotherapy,causing a progressive cognitive decline that is currently untreatable. However,functional neural stem cells remained present in the subventricular zone of high dose-irradiated and aged mouse brains. We therefore investigated whether alterations in the neurogenic niches are perhaps responsible for the neurogenesis decline. This hypothesis was supported by the absence of proliferation of neural stem cells that were engrafted into the vascular niches of irradiated host brains. Moreover,we observed a marked increase in TGF-β1 production by endothelial cells in the stem cell niche in both middle-aged and irradiated mice. In co-cultures,irradiated brain endothelial cells induced the apoptosis of neural stem/progenitor cells via TGF-β/Smad3 signalling. Strikingly,the blockade of TGF-β signalling in vivo using a neutralizing antibody or the selective inhibitor SB-505124 significantly improved neurogenesis in aged and irradiated mice,prevented apoptosis and increased the proliferation of neural stem/progenitor cells. These findings suggest that anti-TGF-β-based therapy may be used for future interventions to prevent neurogenic collapse following radiotherapy or during aging. View Publication

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically in tumor cells and its efficacy has been tested in pre-clinical models by delivering it systemically as a purified ligand or via engineered stem cells (SC). However,about 50% of tumor lines are resistant to TRAIL and overcoming TRAIL resistance in aggressive tumors,such as glioblastoma-multiforme (GBM),and understanding the molecular dynamics of TRAIL-based combination therapies are critical to broadly use TRAIL as a therapeutic agent. In this study,we developed death receptor (DR)4/5-reporters that offer an imaging-based platform to identify agents that act in concert with a potent,secretable variant of TRAIL (S-TRAIL) by monitoring changes in DR4/5 expression. Utilizing these reporters,we show a differential regulation of DR4/5 when exposed to a panel of clinically relevant agents. A histone deacetylase inhibitor,MS-275,resulted in upregulation of DR4/5 in all GBM cell lines,and these changes could be followed in real time both in vitro and in vivo in mice bearing tumors and they correlated with increased TRAIL sensitivity. To further assess the dynamics of combinatorial strategies that overcome resistance of tumors to SC released S-TRAIL,we also engineered tumor cells to express live-cell caspase-reporters and SCs to express S-TRAIL. Utilizing DR4/5 and caspase reporters in parallel,we show that MS-275 sensitizes TRAIL-resistant GBM cells to stem cell (SC) delivered S-TRAIL by changing the time-to-death in vitro and in vivo. This study demonstrates the effectiveness of a combination of real-time reporters of TRAIL-induced apoptosis pathway in evaluating the efficacy of SC-TRAIL-based therapeutics and may have implications in targeting a broad range of cancers. View Publication

In the search for ways to combat degenerative neurological disorders,neurogenesis-stimulating factors are proving to be a promising area of research. In this study,we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay,we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition,direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely,PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly,no deficit in precursor proliferation was observed in vivo,indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice,as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus,indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory. View Publication

Glioblastoma multiforme is the most malignant type of primary brain tumor with a poor prognosis. These tumors consist of a heterogeneous population of malignant cells,including well-differentiated tumor cells and less differentiated cells with stem cell properties. These cancer stem cells,known as brain tumor initiating cells,likely contribute to glioma recurrence,as they are highly invasive,mobile,resistant to radiation and chemotherapy,and have the capacity to self-renew. Glioblastoma tumor cells release excitotoxic levels of glutamate,which may be a key process in the death of peritumoral neurons,formation of necrosis,local inflammation,and glioma-related seizures. Moreover,elevated glutamate levels in the tumor may act in paracrine and autocrine manner to activate glutamate receptors on glioblastoma tumor cells,resulting in proliferation and invasion. Using a previously described culturing condition that selectively promotes the growth of brain tumor initiating cells,which express the stem cell markers nestin and SOX-2,we characterize the expression of α-amino-3-hydroxy-5-methyl-4-isozolepropionic acid (AMPA)-type glutamate receptor subunits in brain tumor initiating cells derived from glioblastomas. Here we show for the first time that glioblastoma brain tumor initiating cells express high concentrations of functional calcium-permeable AMPA receptors,compared to the differentiated tumor cultures consisting of non-stem cells. Up-regulated calcium-permeable AMPA receptor expression was confirmed by immunoblotting,immunocytochemistry,and intracellular calcium imaging in response to specific agonists. Our findings raise the possibility that glutamate secretion in the GBM tumor microenvironment may stimulate brain tumor derived cancer stem cells. View Publication

The reliable derivation of induced pluripotent stem cells (iPSCs) from a noninvasive autologous source at birth would facilitate the study of patient-specific in vitro modeling of congenital diseases and would enhance ongoing efforts aimed at developing novel cell-based treatments for a wide array of fetal and pediatric disorders. Accordingly,we have successfully generated iPSCs from human fetal chorionic somatic cells extracted from term pregnancies by ectopic expression of OCT4,SOX2,KLF4,and cMYC. The isolated parental somatic cells exhibited an immunophenotypic profile consistent with that of chorionic mesenchymal stromal cells (CMSCs). CMSC-iPSCs maintained pluripotency in feeder-free systems for more than 15 passages based on morphology,immunocytochemistry,and gene expression studies and were capable of embryoid body formation with spontaneous trilineage differentiation. CMSC-iPSCs could be selectively differentiated in vitro into various germ layer derivatives,including neural stem cells,beating cardiomyocytes,and definitive endoderm. This study demonstrates the feasibility of term placental chorion as a novel noninvasive alternative to dermal fibroblasts and cord blood for human perinatal iPSC derivation and may provide additional insights regarding the reprogramming capabilities of extra-embryonic tissues as they relate to developmental ontogeny and perinatal tissue engineering applications. View Publication

Transplantation of human neural progenitor cells (NPCs) into the brain or spinal cord to replace lost cells,modulate the injury environment,or create a permissive milieu to protect and regenerate host neurons is a promising therapeutic strategy for neurological diseases. Deriving NPCs from human fetal tissue is feasible,although problematic issues include limited sources and ethical concerns. Here we describe a new and abundant source of NPCs derived from human induced pluripotent stem cells (iPSCs). A novel chopping technique was used to transform adherent iPSCs into free-floating spheres that were easy to maintain and were expandable (EZ spheres) (Ebert et al. [2013] Stem Cell Res 10:417–427). These EZ spheres could be differentiated towards NPC spheres with a spinal cord phenotype using a combination of all-trans retinoic acid (RA) and epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) mitogens. Suspension cultures of NPCs derived from human iPSCs or fetal tissue have similar characteristics,although they were not similar when grown as adherent cells. In addition,iPSC-derived NPCs (iNPCs) survived grafting into the spinal cord of athymic nude rats with no signs of overgrowth and with a very similar profile to human fetal-derived NPCs (fNPCs). These results suggest that human iNPCs behave like fNPCs and could thus be a valuable alternative for cellular regenerative therapies of neurological diseases. J. Comp. Neurol. 522:2707–2728,2014. textcopyright 2014 Wiley Periodicals,Inc. View Publication

Induced pluripotent stem cells (iPSC) and their differentiated derivatives offer a unique source of human primary cells for toxicity screens. Here,we report on the comparative cytotoxicity of 80 compounds (neurotoxicants,developmental neurotoxicants,and environmental compounds) in iPSC as well as isogenic iPSC-derived neural stem cells (NSC),neurons,and astrocytes. All compounds were tested over a 24-h period at 10 and 100$\$,in duplicate,with cytotoxicity measured using the MTT assay. Of the 80 compounds tested,50 induced significant cytotoxicity in at least one cell type; per cell type,32,38,46,and 41 induced significant cytotoxicity in iPSC,NSC,neurons,and astrocytes,respectively. Four compounds (valinomycin,3,3',5,5'-tetrabromobisphenol,deltamethrin,and triphenyl phosphate) were cytotoxic in all four cell types. Retesting these compounds at 1,10,and 100$\$ using the same exposure protocol yielded consistent results as compared with the primary screen. Using rotenone,we extended the testing to seven additional iPSC lines of both genders; no substantial difference in the extent of cytotoxicity was detected among the cell lines. Finally,the cytotoxicity assay was simplified by measuring luciferase activity using lineage-specific luciferase reporter iPSC lines which were generated from the parental iPSC line. This article is part of a Special Issue entitled SI: PSC and the brain. View Publication

Monge's disease,also known as chronic mountain sickness (CMS),is a disease that potentially threatens more than 140 million highlanders during extended time living at high altitudes (over 2500m). The prevalence of CMS in Andeans is about 15-20%,suggesting that the majority of highlanders (non-CMS) are rather healthy at high altitudes; however,CMS subjects experience severe hypoxemia,erythrocytosis and many neurologic manifestations including migraine,headache,mental fatigue,confusion,and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS,except for phlebotomy. In the current study,we reprogrammed fibroblast cells from both CMS and non-CMS subjects' skin biopsies into the induced pluripotent stem cells (iPSCs),then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties,but instead by a significantly lowered Na+ channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide,for the first time,evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects,hoping that such studies can pave the way to a better understanding of the neuropathology in CMS. View Publication

The role of intermediate methylation states in DNA is unclear. Here,to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity,we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers,exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation,are predominantly allele-independent and are conserved across individuals and between mouse and human,suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons,highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage. View Publication

Physical stimuli can act in either a synergistic or antagonistic manner to regulate cell fate decisions,but it is less clear whether insoluble signals alone can direct human pluripotent stem (hPS) cell differentiation into specialized cell types. We previously reported that stiff materials promote nuclear localization of the Yes-associated protein (YAP) transcriptional coactivator and support long-term self-renewal of hPS cells. Here,we show that even in the presence of soluble pluripotency factors,compliant substrata inhibit the nuclear localization of YAP and promote highly efficient differentiation of hPS cells into postmitotic neurons. In the absence of neurogenic factors,the effective substrata produce neurons rapidly (2 wk) and more efficiently (textgreater75%) than conventional differentiation methods. The neurons derived from substrate induction express mature markers and possess action potentials. The hPS differentiation observed on compliant surfaces could be recapitulated on stiff surfaces by adding small-molecule inhibitors of F-actin polymerization or by depleting YAP. These studies reveal that the matrix alone can mediate differentiation of hPS cells into a mature cell type,independent of soluble inductive factors. That mechanical cues can override soluble signals suggests that their contributions to early tissue development and lineage commitment are profound. View Publication

The enteric nervous system (ENS) is composed of neural crest-derived neurons (also known as ganglion cells) the cell bodies of which are located in the submucosal and myenteric plexuses of the intestinal wall. Intramucosal ganglion cells are known to exist but are rare and often considered ectopic. Also derived from the neural crest are enteric glial cells that populate the ganglia and the associated nerves,as well as the lamina propria of the intestinal mucosa. In Hirschsprung disease (HSCR),ganglion cells are absent from the distal gut because of a failure of neural crest-derived progenitor cells to complete their rostrocaudal migration during embryogenesis. The fate of intramucosal glial cells in human HSCR is essentially unknown. We demonstrate a network of intramucosal cells that exhibit dendritic morphology typical of neurons and glial cells. These dendritic cells are present throughout the human gut and express Tuj1,S100,glial fibrillary acidic protein,CD56,synaptophysin,and calretinin,consistent with mixed or overlapping neuroglial differentiation. The cells are present in aganglionic colon from patients with HSCR,but with an altered immunophenotype. Coexpression of Tuj1 and HNK1 in this cell population supports a neural crest origin. These findings extend and challenge the current understanding of ENS microanatomy and suggest the existence of an intramucosal population of neural crest-derived cells,present in HSCR,with overlapping immunophenotype of neurons and glia. Intramucosal neuroglial cells have not been previously recognized,and their presence in HSCR poses new questions about ENS development and the pathobiology of HSCR that merit further investigation. View Publication

The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently,knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led to genomic instability. Here,we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies,due to downstream suppression of p21,without affecting apoptosis and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns,displayed normal karyotypes,contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion,transient p53 suppression increases reprogramming efficiency without affecting genomic stability,rendering the method suitable for in vitro mechanistic studies with the possibility for future clinical translation. View Publication