Roof plate secretion of bone morphogenetic proteins(BMPs)directs the cellular fate of sensory neurons during spinal cord development,including the formation of the ascending sensory columns,though their biology is not...Roof plate secretion of bone morphogenetic proteins(BMPs)directs the cellular fate of sensory neurons during spinal cord development,including the formation of the ascending sensory columns,though their biology is not well understood.Type-ⅡBMP receptor(BMPRⅡ),the cognate receptor,is expressed by neural precursor cells during embryogenesis;however,an in vitro method of enriching BMPRⅡ^(+)human neural precursor cells(hNPCs)from the fetal spinal cord is absent.Immunofluorescence was undertaken on intact second-trimester human fetal spinal cord using antibodies to BMPRⅡand leukemia inhibitory factor(LIF).Regions of highest BMPRⅡ^(+)immunofluorescence localized to sensory columns.Parenchymal and meningeal-associated BMPRⅡ^(+)vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers,CD34/CD39.LIF immunostaining identified a population of somas concentrated in dorsal and ventral horn interneurons,mirroring the expression of LIF receptor/CD118.A combination of LIF supplementation and high-density culture maintained culture growth beyond 10 passages,while synergistically increasing the proportion of neurospheres with a stratified,cytoarchitecture.These neurospheres were characterized by BMPRⅡ^(+)/MAP2ab^(+/–)/βⅢ-tubulin^(+)/nestin^(–)/vimentin^(–)/GFAP^(–)/NeuN^(–)surface hNPCs surrounding a heterogeneous core ofβⅢ-tubulin^(+)/nestin^(+)/vimentin^(+)/GFAP^(+)/MAP2ab^(–)/NeuN^(–)multipotent precursors.Dissociated cultures from tripotential neurospheres contained neuronal(βⅢ-tubulin^(+)),astrocytic(GFAP+),and oligodendrocytic(O4+)lineage cells.Fluorescence-activated cell sorting-sorted BMPRⅡ^(+)hNPCs were MAP2ab^(+/–)/βⅢ-tubulin^(+)/GFAP^(–)/O4^(–)in culture.This is the first isolation of BMPRⅡ^(+)hNPCs identified and characterized in human fetal spinal cords.Our data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres,characterized by surface BMPRⅡ^(+)hNPCs.Our study has provided a new methodology for an in vitro model capable of amplifying human fetal spinal cord cell numbers for>10 passages.Investigations of the role BMPRⅡplays in spinal cord development have primarily relied upon mouse and rat models,with interpolations to human development being derived through inference.Because of significant species differences between murine biology and human,including anatomical dissimilarities in central nervous system(CNS)structure,the findings made in murine models cannot be presumed to apply to human spinal cord development.For these reasons,our human in vitro model offers a novel tool to better understand neurodevelopmental pathways,including BMP signaling,as well as spinal cord injury research and testing drug therapies.展开更多
This study aimed to investigate the effect and mechanism of valproic acid(VPA)on the neurosphere formation in rat embryonic cortical cells.We used free-floating neurosphere formation as a model system to evaluate the ...This study aimed to investigate the effect and mechanism of valproic acid(VPA)on the neurosphere formation in rat embryonic cortical cells.We used free-floating neurosphere formation as a model system to evaluate the VPA on the proliferation of neural stem cells(NSCs).We found a time-and dose-dependent increase in neurosphere formation and NSC proliferation after VPA treatment.Further RNA-seq analysis demonstrated that the upregulated TGFβ1 signaling might attribute to the effect of VPA on the neurosphere formation and NSC proliferation.Consistently,the neurosphere formation and NSC proliferation were blocked by the treatment with SB431542,an inhibitor of TGFβ1 receptor.Moreover,in a coculture system,NSCs treated with VPA significantly reduced the oxygen-glucose deprivation-induced neuronal apoptosis.Taken together,our results showed that VPA could enhance neurosphere formation and NSC proliferation by activating TGFβ1,which might be a novel therapeutic strategy for neurological disorders.展开更多
The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the...The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells(NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3 r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor(IP3 R) type-1 and ryanodine receptor(RyR) type-2 are differentially expressed in cells with neuron-or radial glial-like properties. Furthermore, ATP but not caffeine(IP3 R and RyR agonists, respectively), induced the generation of Ca^(2+) transients in cells exhibiting neuron-or glial-like morphology. These results indicate the differential expression of components of the Ca^(2+) -signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca^(2+) signaling as a read-out.展开更多
BACKGROUND Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss.Cells extracted from peripheral corneas can form stem cell-enriched s...BACKGROUND Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss.Cells extracted from peripheral corneas can form stem cell-enriched spheres,which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic tissue.AIM To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic tissue.METHODS Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal cells.These spheres were implanted into incisions created in full thickness and onto the surface of 10μm thin sections of keratoconic and normal stromal tissues in vitro.Tissue sections were used to maximise use of limited keratoconic tissue available for research.Living cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d(D14)from implantation.Sphere cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus treatment.RESULTS Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10μm thin en face tissue sections.No observable differences were seen in sphere migration,proliferation or differentiation in keratoconic tissue compared to normal between day 0 and D14.Spheres stained positively with Calcein-AM up to D14.Cell migration increased from day 0 to D14,occurring radially in three dimensions from the sphere and in alignment with tissue edges.Cell proliferation marker,EdU,was detected at day 10.Implanted spheres stained positively for putative stem cell markersΔNp63αand ABCB5,while ABCG2,ABCB5,ΔNp63 and p63αwere detectable by droplet digital PCR up to D14.Double immunolabelling revealed absence of ABCB5 staining in migrated cells but positive staining of alpha smooth muscle actin(myofibroblast marker)in some migrated cells.Droplet digital PCR showed similar expression patterns of differentiation markers but a reduction in stem cell markers between normal and keratoconic tissue with an increase in stromal cell markers and a reduction in epithelial cell markers,indicating an appropriate response to repopulating diseased tissue.CONCLUSION Cells from implanted stem cell-enriched spheres can repopulate a keratoconic corneal stromal surface in a directed manner and exhibit migratory stromal cell phenotypes.展开更多
AIM: To determine the role for the intermediate filament protein nestin in glioma invasion. METHODS: We examined the expression and function of nestin in gliomas(Grades Ⅱ-Ⅳ as defined by the World Health Organizatio...AIM: To determine the role for the intermediate filament protein nestin in glioma invasion. METHODS: We examined the expression and function of nestin in gliomas(Grades Ⅱ-Ⅳ as defined by the World Health Organization). We determined nestin expression using Immunohistochemical methods. To elucidate nestin's biological function(s), we reduced m RNA levels by 61% and 87% in two glioblastomaderived neurosphere lines using short hairpin RNAs and determined the effect of reduced nestin expression on glioma cell proliferation and invasion using MTS and matrigel migration assays, respectively. We also utilized quantitative real time polymerase chain reaction assaysto determine the effect of reduced nestin expression on the expression of other markers associated with glioma stem cells and their differentiated progenies. RESULTS: We found a significant correlation between nestin immunoreactivity and astrocytoma tumor grade, with 36% of grade Ⅱ, 75% of grade Ⅲ, and 100% of grade Ⅳtumors expressing significant levels of the protein when assessed using immunohistochemistry. Reduction in nestin expression had no effect on cell growth in culture, but did retard the capacity of one line to migrate in-vitro on matrigel. Interestingly, in the line whose migration was not affected, m RNA levels of a second intermediate filament, synemin(also knowns as desmuslin), were elevated following introduction of sh RNA targeting nestin. As synemin was not induced in the line which required nestin for migration, it is a possibility that synemin may compensate for the loss of nestin in this process. CONCLUSION: Nestin expression is prominent in high-grade astrocytomas. Nestin is not required for cell growth but it may, however, be required for cell motility.展开更多
基金supported by grants from the National Health and Medical Research Council(NHMRC)of Australia(Nos.571100 and 1048082)the Baxter Charitable Foundation(to TCL)+1 种基金Medical Research grants from the Rebecca L.Cooper Medical Research Foundation(to MWW,TCL,and MDL)supported by a Charles D.Kelman,M.D.Postdoctoral Award(2010)from the International Retinal Research Foundation(USA)。
文摘Roof plate secretion of bone morphogenetic proteins(BMPs)directs the cellular fate of sensory neurons during spinal cord development,including the formation of the ascending sensory columns,though their biology is not well understood.Type-ⅡBMP receptor(BMPRⅡ),the cognate receptor,is expressed by neural precursor cells during embryogenesis;however,an in vitro method of enriching BMPRⅡ^(+)human neural precursor cells(hNPCs)from the fetal spinal cord is absent.Immunofluorescence was undertaken on intact second-trimester human fetal spinal cord using antibodies to BMPRⅡand leukemia inhibitory factor(LIF).Regions of highest BMPRⅡ^(+)immunofluorescence localized to sensory columns.Parenchymal and meningeal-associated BMPRⅡ^(+)vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers,CD34/CD39.LIF immunostaining identified a population of somas concentrated in dorsal and ventral horn interneurons,mirroring the expression of LIF receptor/CD118.A combination of LIF supplementation and high-density culture maintained culture growth beyond 10 passages,while synergistically increasing the proportion of neurospheres with a stratified,cytoarchitecture.These neurospheres were characterized by BMPRⅡ^(+)/MAP2ab^(+/–)/βⅢ-tubulin^(+)/nestin^(–)/vimentin^(–)/GFAP^(–)/NeuN^(–)surface hNPCs surrounding a heterogeneous core ofβⅢ-tubulin^(+)/nestin^(+)/vimentin^(+)/GFAP^(+)/MAP2ab^(–)/NeuN^(–)multipotent precursors.Dissociated cultures from tripotential neurospheres contained neuronal(βⅢ-tubulin^(+)),astrocytic(GFAP+),and oligodendrocytic(O4+)lineage cells.Fluorescence-activated cell sorting-sorted BMPRⅡ^(+)hNPCs were MAP2ab^(+/–)/βⅢ-tubulin^(+)/GFAP^(–)/O4^(–)in culture.This is the first isolation of BMPRⅡ^(+)hNPCs identified and characterized in human fetal spinal cords.Our data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres,characterized by surface BMPRⅡ^(+)hNPCs.Our study has provided a new methodology for an in vitro model capable of amplifying human fetal spinal cord cell numbers for>10 passages.Investigations of the role BMPRⅡplays in spinal cord development have primarily relied upon mouse and rat models,with interpolations to human development being derived through inference.Because of significant species differences between murine biology and human,including anatomical dissimilarities in central nervous system(CNS)structure,the findings made in murine models cannot be presumed to apply to human spinal cord development.For these reasons,our human in vitro model offers a novel tool to better understand neurodevelopmental pathways,including BMP signaling,as well as spinal cord injury research and testing drug therapies.
基金This work was supported by grants from the Key R&D Program of Jiangsu Province(Grant No.2017CX010)the National Natural Science Foundation of China(Grant No.81973308)to J.G.the Nanjing Science and Technology Development Program(Grant No.201402021)to H.L.
文摘This study aimed to investigate the effect and mechanism of valproic acid(VPA)on the neurosphere formation in rat embryonic cortical cells.We used free-floating neurosphere formation as a model system to evaluate the VPA on the proliferation of neural stem cells(NSCs).We found a time-and dose-dependent increase in neurosphere formation and NSC proliferation after VPA treatment.Further RNA-seq analysis demonstrated that the upregulated TGFβ1 signaling might attribute to the effect of VPA on the neurosphere formation and NSC proliferation.Consistently,the neurosphere formation and NSC proliferation were blocked by the treatment with SB431542,an inhibitor of TGFβ1 receptor.Moreover,in a coculture system,NSCs treated with VPA significantly reduced the oxygen-glucose deprivation-induced neuronal apoptosis.Taken together,our results showed that VPA could enhance neurosphere formation and NSC proliferation by activating TGFβ1,which might be a novel therapeutic strategy for neurological disorders.
基金supported by the ANR/RGC Joint Research Scheme Award (A-HKUST601/ 13)the HK RGC General Research Fund awards (662113, 16101714, 16100115)Funding from the HKITC (ITCPD/17-9)
文摘The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells(NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3 r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor(IP3 R) type-1 and ryanodine receptor(RyR) type-2 are differentially expressed in cells with neuron-or radial glial-like properties. Furthermore, ATP but not caffeine(IP3 R and RyR agonists, respectively), induced the generation of Ca^(2+) transients in cells exhibiting neuron-or glial-like morphology. These results indicate the differential expression of components of the Ca^(2+) -signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca^(2+) signaling as a read-out.
基金Supported by Save Sight Society of New Zealand,No.37116543New Zealand Wound Care Society,No.3713325John Hamel MacGregor Trust
文摘BACKGROUND Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss.Cells extracted from peripheral corneas can form stem cell-enriched spheres,which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic tissue.AIM To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic tissue.METHODS Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal cells.These spheres were implanted into incisions created in full thickness and onto the surface of 10μm thin sections of keratoconic and normal stromal tissues in vitro.Tissue sections were used to maximise use of limited keratoconic tissue available for research.Living cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d(D14)from implantation.Sphere cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus treatment.RESULTS Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10μm thin en face tissue sections.No observable differences were seen in sphere migration,proliferation or differentiation in keratoconic tissue compared to normal between day 0 and D14.Spheres stained positively with Calcein-AM up to D14.Cell migration increased from day 0 to D14,occurring radially in three dimensions from the sphere and in alignment with tissue edges.Cell proliferation marker,EdU,was detected at day 10.Implanted spheres stained positively for putative stem cell markersΔNp63αand ABCB5,while ABCG2,ABCB5,ΔNp63 and p63αwere detectable by droplet digital PCR up to D14.Double immunolabelling revealed absence of ABCB5 staining in migrated cells but positive staining of alpha smooth muscle actin(myofibroblast marker)in some migrated cells.Droplet digital PCR showed similar expression patterns of differentiation markers but a reduction in stem cell markers between normal and keratoconic tissue with an increase in stromal cell markers and a reduction in epithelial cell markers,indicating an appropriate response to repopulating diseased tissue.CONCLUSION Cells from implanted stem cell-enriched spheres can repopulate a keratoconic corneal stromal surface in a directed manner and exhibit migratory stromal cell phenotypes.
文摘AIM: To determine the role for the intermediate filament protein nestin in glioma invasion. METHODS: We examined the expression and function of nestin in gliomas(Grades Ⅱ-Ⅳ as defined by the World Health Organization). We determined nestin expression using Immunohistochemical methods. To elucidate nestin's biological function(s), we reduced m RNA levels by 61% and 87% in two glioblastomaderived neurosphere lines using short hairpin RNAs and determined the effect of reduced nestin expression on glioma cell proliferation and invasion using MTS and matrigel migration assays, respectively. We also utilized quantitative real time polymerase chain reaction assaysto determine the effect of reduced nestin expression on the expression of other markers associated with glioma stem cells and their differentiated progenies. RESULTS: We found a significant correlation between nestin immunoreactivity and astrocytoma tumor grade, with 36% of grade Ⅱ, 75% of grade Ⅲ, and 100% of grade Ⅳtumors expressing significant levels of the protein when assessed using immunohistochemistry. Reduction in nestin expression had no effect on cell growth in culture, but did retard the capacity of one line to migrate in-vitro on matrigel. Interestingly, in the line whose migration was not affected, m RNA levels of a second intermediate filament, synemin(also knowns as desmuslin), were elevated following introduction of sh RNA targeting nestin. As synemin was not induced in the line which required nestin for migration, it is a possibility that synemin may compensate for the loss of nestin in this process. CONCLUSION: Nestin expression is prominent in high-grade astrocytomas. Nestin is not required for cell growth but it may, however, be required for cell motility.
