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
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.
