技术资料

We have previously demonstrated that lineage negative cells (Lin(neg)) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone,muscle,endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin(neg) UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin,neurofilament,A2B5 and Sox2) are expressed in Lin(neg) cells cultured in FGF4,SCF,Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase,GalC,Olig2 and the late-stage marker MOSP are observed,as is the Schwann cell marker PMP22. In summary,Lin(neg) UCB cells,when appropriately cultured,are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation. View Publication

We report a considerable number of cells in the ventricular and the subventricular zones (SVZ) of newborn mice to stain positive for the PDGF beta-receptor (PDGFRB). Many of them also stained for nestin and/or GFAP but less frequently for the neuroblast marker doublecortin and for the mitotic marker Ki-67. The SVZ of mice with nestin-Cre conditional deletion of PDGFRB expressed the receptor only on blood vessels and was devoid of any morphological abnormality. PDGFRB(-/-) neurospheres showed a higher rate of apoptosis without any significant decrease in proliferation. They demonstrated reduced capacities of migration and neuronal differentiation in response to not only PDGF-BB but also bFGF. Furthermore,the PDGFR kinase inhibitor STI571 blocked the effects of bFGF in control neurosphere cultures. bFGF increased the activity of the PDGFRB promoter as well as the expression and phosphorylation of PDGFRB. These results suggest the presence of the signaling convergence between PDGF and FGF. PDGFRB is needed for survival,and the effects of bFGF in migration and neural differentiation of the cells may be potentiated by induction of PDGFRB. View Publication

Multiple research groups have observed neuropathological phenotypes and molecular symptoms in vitro using induced pluripotent stem cell (iPSC)-derived neural cell cultures (i.e. patient-specific neurons and glia). However,the global differences/similarities that may exist between in vitro neural cells and their tissue-derived counterparts remain largely unknown. In this study,we compared temporal series of iPSC-derived in vitro neural cell cultures to endogenous brain tissue from the same autopsy donor. Specifically,we utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0,35,70,105 and 140 days),and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue,and the following three results support this conclusion: (i) there was a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05),consistent with the transcriptional complexity of the brain; (ii) there was an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue; and (iii) there was a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. Taken together,these results are consistent with in vitro neural development and physiological progression occurring predominantly by transcriptional activation of downregulated genes rather than deactivation of upregulated genes. View Publication

Stress bears a negative impact on adult neurogenesis. High levels of corticoids have been shown to inhibit neural stem cell proliferation,and are considered responsible for the loss of neural precursors. Their effects on the differentiation of the glial and neuronal lineages have been less studied. We examined the effect of dexamethasone (Dex),a synthetic glucocorticoid,on the differentiation of rat neural stem cells in vitro. Dex had no effect on the differentiation of cells cultured under standard conditions. Since we previously determined that NSC,when cultured under classical conditions,were deprived of polyunsaturated fatty acids (PUFA),and displayed phospholipid compositions very different from the in vivo figures [1],we examined the effect of Dex under PUFA supplementation. Dex impaired neuron and oligodendrocyte maturation in PUFA-supplemented cells,demonstrated by the reduction of neurite lengths and oligodendrocyte sizes. This effect was mediated by the glucocorticoid receptor (GR),since it was eliminated by mifepristone,a GR antagonist,and could be relayed by a reduction of ERK phosphorylation. We determined that GR was associated with PPAR β and α under basal conditions,and that this association was disrupted when PUFA were added in combination with Dex. We assumed that this effect on the receptor status enabled the effect of Dex on PUFA supplemented cells,since we determined that the binding to the glucocorticoid response element was higher in cells incubated with PUFA and Dex. In conclusion,corticoids can impair NSC differentiation,and consequently impact the entire process of neurogenesis. View Publication

BACKGROUND Transplantation of enteric neural stem cells (ENSC) holds promise as a potential therapy for enteric neuropathies,including Hirschsprung disease. Delivery of transplantable cells via laparotomy has been described,but we propose a novel,minimally invasive endoscopic method of cell delivery. METHODS Enteric neural stem cells for transplantation were cultured from dissociated gut of postnatal donor mice. Twelve recipient mice,including Ednrb(-/-) mice with distal colonic aganglionosis,underwent colonoscopic injection of ENSC under direct vision using a 30-gauge Hamilton needle passed through a rigid cystoureteroscope. Cell engraftment,survival,and neuroglial differentiation were studied 1-4 weeks after the procedure. KEY RESULTS All recipient mice tolerated the procedure without complications and survived to sacrifice. Transplanted cells were found within the colonic wall in 9 of 12 recipient mice with differentiation into enteric neurons and glia. CONCLUSIONS & INFERENCES Endoscopic injection of ENSC is a safe and reliable method for cell delivery,and can be used to deliver a large number of cells to a specific area of disease. This minimally invasive endoscopic approach may prove beneficial to future human applications of cell therapy for neurointestinal disease. View Publication

MicroRNA-10b (miR-10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes,while absent in normal neuroglial cells of the brain. miR-10b inhibition strongly impairs proliferation and survival of cultured glioma cells,including glioma-initiating stem-like cells (GSC). Although several miR-10b targets have been identified previously,the common mechanism conferring the miR-10b-sustained viability of GSC is unknown. Here,we demonstrate that in heterogeneous GSC,miR-10b regulates cell cycle and alternative splicing,often through the non-canonical targeting via 5'UTRs of its target genes,including MBNL1-3,SART3,and RSRC1. We have further assessed the inhibition of miR-10b in intracranial human GSC-derived xenograft and murine GL261 allograft models in athymic and immunocompetent mice. Three delivery routes for the miR-10b antisense oligonucleotide inhibitors (ASO),direct intratumoral injections,continuous osmotic delivery,and systemic intravenous injections,have been explored. In all cases,the treatment with miR-10b ASO led to targets' derepression,and attenuated growth and progression of established intracranial GBM. No significant systemic toxicity was observed upon ASO administration by local or systemic routes. Our results indicate that miR-10b is a promising candidate for the development of targeted therapies against all GBM subtypes. View Publication

Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro,and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously,we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this NIA Mouse ESC Bank we generated and characterized 48 additional mouse ESC lines,in which single TFs in each line could be induced in a doxycycline-controllable manner. Together,with the previous ESC lines,the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g.,neural lineages by Myt1 Isl1,and St18; mesodermal lineages by Pitx1,Pitx2,Barhl2,and Lmx1a; white blood cells by Myb,Etv2,and Tbx6,and ovary by Pitx1,Pitx2,and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. View Publication

A recent study has suggested that fibroblasts can be converted into mouse-induced neural stem cells (miNSCs) through the expression of defined factors. However,successful generation of human iNSCs (hiNSCs) has proven challenging to achieve. Here,using microRNA (miRNA) expression profile analyses,we showed that let-7 microRNA has critical roles for the formation of PAX6/NESTIN-positive colonies from human adult fibroblasts and the proliferation and self-renewal of hiNSCs. HMGA2,a let-7-targeting gene,enables induction of hiNSCs that displayed morphological/molecular features and in vitro/in vivo differentiation potential similar to H9-derived NSCs. Interestingly,HMGA2 facilitated the efficient conversion of senescent somatic cells or blood CD34+ cells into hiNSCs through an interaction with SOX2,whereas other combinations or SOX2 alone showed a limited conversion ability. Taken together,these findings suggest that HMGA2/let-7 facilitates direct reprogramming toward hiNSCs in minimal conditions and maintains hiNSC self-renewal,providing a strategy for the clinical treatment of neurological diseases. View Publication

Abstract

Ideal tissue-engineered scaffold materials regulate proliferation,apoptosis and differentiation of cells seeded on them by regulating gene expression. In this study,aligned and randomly oriented collagen nanofiber scaffolds were prepared using electronic spinning technology. Their diameters and appearance reached the standards of tissue-engineered nanometer scaffolds. The nanofiber scaffolds were characterized by a high swelling ratio,high porosity and good mechanical properties. The proliferation of spinal cord-derived neural stem cells on novel nanofiber scaffolds was obviously enhanced. The proportions of cells in the S and G2/M phases noticeably increased. Moreover,the proliferation rate of neural stem cells on the aligned collagen nanofiber scaffolds was high. The expression levels of cyclin D1 and cyclin-dependent kinase 2 were increased. Bcl-2 expression was significantly increased,but Bax and caspase-3 gene expressions were obviously decreased. There was no significant difference in the differentiation of neural stem cells into neurons on aligned and randomly oriented collagen nanofiber scaffolds. These results indicate that novel nanofiber scaffolds could promote the proliferation of spinal cord-derived neural stem cells and inhibit apoptosis without inducing differentiation. Nanofiber scaffolds regulate apoptosis and proliferation in neural stem cells by altering gene expression.

Research Highlights

(1) Electronic spinning technology was used to obtain randomly oriented nanofiber membranes and aligned nanofiber membranes. The aligned and randomly oriented collagen nanometer scaffolds were shown to alter the biological behaviors of neural stem cells and induce changes in gene expression.

(2) The effects of the aligned nanofiber membranes on promoting neural stem cell proliferation and on inhibiting apoptosis of neural stem cells were better than those of the randomly oriented nanofiber membranes. Aligned and randomly oriented collagen nanometer scaffolds did not significantly induce apoptosis or differentiation in stem cells.

(3) Aligned and randomly oriented collagen nanometer scaffolds regulated the expression of apoptosis and cell cycle genes in neural stem cells.

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The role of epigenetic regulators in the control of adult neurogenesis is largely undefined. We show that the histone demethylase enzyme Kdm5b (Jarid1b) negatively regulates neurogenesis from adult subventricular zone (SVZ) neural stem cells (NSCs) in culture. shRNA-mediated depletion of Kdm5b in proliferating adult NSCs decreased proliferation rates and reduced neurosphere formation in culture. When transferred to differentiation culture conditions,Kdm5b-depleted adult NSCs migrated from neurospheres with increased velocity. Whole-genome expression screening revealed widespread transcriptional changes with Kdm5b depletion,notably the up-regulation of reelin (Reln),the inhibition of steroid biosynthetic pathway component genes and the activation of genes with intracellular transport functions in cultured adult NSCs. Kdm5b depletion increased extracellular reelin concentration in the culture medium and increased phosphorylation of the downstream reelin signaling target Disabled-1 (Dab1). Sequestration of extracellular reelin with CR-50 reelin-blocking antibodies suppressed the increase in migratory velocity of Kdm5b-depleted adult NSCs. Chromatin immunoprecipitation revealed that Kdm5b is present at the proximal promoter of Reln,and H3K4me3 methylation was increased at this locus with Kdm5b depletion in differentiating adult NSCs. Combined the data suggest Kdm5b negatively regulates neurogenesis and represses Reln in neural stem cells from the adult SVZ. View Publication

We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells,and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse,we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality. View Publication

Amyloid-ß (Aß) precursor protein (APP) metabolism engages neuronal endolysosomal pathways for Aß processing and secretion. In Alzheimer's disease (AD),dysregulation of APP leads to excess Aß and neuronal dysfunction; suggesting that neuronal APP/Aß trafficking can be targeted for therapeutic gain. Cathepsin B (CatB) is a lysosomal cysteine protease that can lower Aß levels. However,whether CatB-modulation of Aß improves learning and memory function deficits in AD is not known. To this end,progenitor neurons were infected with recombinant adenovirus expressing CatB and recovered cell lysates subjected to proteomic analyses. The results demonstrated Lamp1 deregulation and linkages between CatB and the neuronal phagosome network. Hippocampal injections of adeno-associated virus expressing CatB reduced Aß levels,increased Lamp1 and improved learning and memory. The findings were associated with the emergence of c-fos + cells. The results support the idea that CatB can speed Aß metabolism through lysosomal pathways and as such reduce AD-associated memory deficits. View Publication