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In the developing cortex, postmigratory neurons accumulate in the cortical plate (CP) to properly differentiate consolidating subtype identities. Microglia, despite their extensive surveying activity, temporarily disappear from the midembryonic CP. However, the mechanism and significance of this absence are unknown. Here, we show that microglia bidirectionally migrate via attraction by CXCL12 released from the meninges and subventricular zone and thereby exit the midembryonic CP. Upon nonphysiological excessive exposure to microglia in vivo or in vitro, young postmigratory and in vitro-grown CP neurons showed abnormal differentiation with disturbed expression of the subtype-associated transcription factors and genes implicated in functional neuronal maturation. Notably, this effect is primarily attributed to interleukin 6 and type I interferon secreted by microglia. These results suggest that "sanctuarization" from microglia in the midembryonic CP is required for neurons to appropriately fine-tune the expression of molecules needed for proper differentiation, thus securing the establishment of functional cortical circuit.

DOI： 10.1038/s41467-020-15409-3

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

In the developing central nervous system, cell departure from the apical surface is the initial and fundamental step to form the 3D, organized architecture. Both delamination of differentiating cells and repositioning of progenitors to generate outer radial glial cells (oRGs) contribute to mammalian neocortical expansion; however, a comprehensive understanding of their mechanisms is lacking. Here, we demonstrate that Lzts1, a molecule associated with microtubule components, promotes both cell departure events. In neuronally committed cells, Lzts1 functions in apical delamination by altering apical junctional organization. In apical RGs (aRGs), Lzts1 expression is variable, depending on Hes1 expression levels. According to its differential levels, Lzts1 induces diverse RG behaviors: planar division, oblique divisions of aRGs that generate oRGs, and their mitotic somal translocation. Loss-of-function of lzts1 impairs all these cell departure processes. Thus, Lzts1 functions as a master modulator of cellular dynamics, contributing to increasing complexity of the cerebral architecture during evolution.

DOI： 10.1038/s41467-019-10730-y

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Authorship：Corresponding author  

DOI： 10.1016/j.neures.2018.09.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer New York LLC  

Spatiotemporally ordered production of cells is essential for brain development. Normally, most undifferentiated neural progenitor cells (NPCs) face the apical (ventricular) surface of embryonic brain walls. Pathological detachment of NPCs from the apical surface and their invasion of outer neuronal territories, i.e., formation of NPC heterotopias, can disrupt the overall structure of the brain. Although NPC heterotopias have previously been observed in a variety of experimental contexts, the underlying mechanisms remain largely unknown. Yes-associated protein 1 (Yap1) and the TEA domain (Tead) proteins, which act downstream of Hippo signaling, enhance the stem-like characteristics of NPCs. Elevated expression of Yap1 or Tead in the neural tube (future spinal cord) induces massive NPC heterotopias, but Yap/Tead-induced expansion of NPCs in the developing brain has not been previously reported to produce NPC heterotopias. To determine whether NPC heterotopias occur in a regionally characteristic manner, we introduced the Yap1-S112A or Tead-VP16 into NPCs of the telencephalon and diencephalon, two neighboring but distinct forebrain regions, of embryonic day 10 mice by in utero electroporation, and compared NPC heterotopia formation. Although NPCs in both regions exhibited enhanced stem-like behaviors, heterotopias were larger and more frequent in the diencephalon than in the telencephalon. This result, the first example of Yap/Tead-induced NPC heterotopia in the forebrain, reveals that Yap/Tead-induced NPC heterotopia is not specific to the neural tube, and also suggests that this phenomenon depends on regional factors such as the three-dimensional geometry and assembly of these cells.

DOI： 10.1007/s11064-017-2390-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CURRENT BIOLOGY LTD  

Neural stem cells go through a sequence of timely regulated gene expression and pattern of division mode to generate diverse neurons during brain development. During vertebrate cerebral cortex development, neural stem cells begin with proliferative symmetric divisions, subsequently undergo neurogenic asymmetric divisions, and finally gliogenic divisions. In this review, we explore the relationship between stem cell versus neural fate specification and the division mode. Specifically, we discuss recent findings on the mechanisms of asymmetric divisions, division mode, and developmental progression of neural progenitor identity.

DOI： 10.1016/j.conb.2016.11.009

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Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

DOI： 10.3389/fcell.2016.00139

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and -extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode.

DOI： 10.1038/ncomms11349

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Authorship：Corresponding author   Language：English   Publisher：TAYLOR & FRANCIS INC  

DOI： 10.1080/15384101.2016.1204857

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

The neuroepithelium (NE) or ventricular zone (VZ), from which multiple types of brain cells arise, is pseudostratified. In the NE/VZ, neural progenitor cells are elongated along the apicobasal axis, and their nuclei assume different apicobasal positions. These nuclei move in a cell cycledependent manner, i.e., apicalward during G2 phase and basalward during G1 phase, a process called interkinetic nuclear migration (INM). This review will summarize and discuss several topics: the nature of the INM exhibited by neural progenitor cells, the mechanical difficulties associated with INM in the developing cerebral cortex, the community-level mechanisms underlying collective and efficient INM, the impact on overall brain formation when NE/VZ is overcrowded due to loss of INM, and whether and how neural progenitor INM varies among mammalian species. These discussions will be based on recent findings obtained in live, three-dimensional specimens using quantitative and mechanical approaches. Experiments in which overcrowding was induced in mouse neocortical NE/VZ, as well as comparisons of neocortical INM between mice and ferrets, have revealed that the behavior of NE/VZ cells can be affected by cellular densification. A consideration of the physical aspects in the NE/VZ and the mechanical difficulties associated with high-degree pseudostratification (PS) is important for achieving a better understanding of neocortical development and evolution.

DOI： 10.3389/fncel.2014.00473

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

To achieve highly sensitive and comprehensive assessment of the morphology and dynamics of cells committed to the neuronal lineage in mammalian brain primordia, we generated two transgenic mouse lines expressing a destabilized (d4) Venus controlled by regulatory elements of the Neurogenin2 (Neurog2) or Gadd45g gene. In mid-embryonic neocortical walls, expression of Neurog2-d4Venus mostly overlapped with that of Neurog2 protein, with a slightly (1h) delayed onset. Although Neurog2-d4Venus and Gadd45g-d4Venus mice exhibited very similar labeling patterns in the ventricular zone (VZ), in Gadd45g-d4Venus mice cells could be visualized in more basal areas containing fully differentiated neurons, where Neurog2-d4Venus fluorescence was absent. Time-lapse monitoring revealed that most d4Venus(+) cells in the VZ had processes extending to the apical surface; many of these cells eventually retracted their apical process and migrated basally to the subventricular zone, where neurons, as well as the intermediate neurogenic progenitors that undergo terminal neuron-producing division, could be live-monitored by d4Venus fluorescence. Some d4Venus(+) VZ cells instead underwent nuclear migration to the apical surface, where they divided to generate two d4Venus(+) daughter cells, suggesting that the symmetric terminal division that gives rise to neuron pairs at the apical surface can be reliably live-monitored. Similar lineage-committed cells were observed in other developing neural regions including retina, spinal cord, and cerebellum, as well as in regions of the peripheral nervous system such as dorsal root ganglia. These mouse lines will be useful for elucidating the cellular and molecular mechanisms underlying development of the mammalian nervous system.

DOI： 10.1111/dgd.12131

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Neural progenitors exhibit cell cycle-dependent interkinetic nuclear migration (INM) along the apicobasal axis. Despite recent advances in understanding its underlying molecular mechanisms, the processes to which INM contributes mechanically and the regulation of INM by the apicobasally elongated morphology of progenitors remain unclear. We found that knockdown of the cell-surface molecule TAG-1 resulted in retraction of neocortical progenitors' basal processes. Highly shortened stem-like progenitors failed to undergo basalward INM and became overcrowded in the periventricular (subapical) space. Surprisingly, the overcrowded progenitors left the apical surface and migrated into basal neuronal territories. These observations, together with the results of in toto imaging and physical tests, suggest that progenitors may sense and respond to excessive mechanical stress. Although, unexpectedly, the heterotopic progenitors remained stem-like and continued to sequentially produce neurons until the late embryonic period, histogenesis was severely disrupted. Thus, INM is essential for preventing overcrowding of nuclei and their somata, thereby ensuring normal brain histogenesis.

DOI： 10.1038/nn.3525

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Cerebellar corticogenesis begins with the assembly of Purkinje cells into the Purkinje plate (PP) by embryonic day 14.5 (E14.5) in mice. Although the dependence of PP formation on the secreted protein Reelin is well known and a prevailing model suggests that Purkinje cells migrate along the 'radial glial' fibers connecting the ventricular and pial surfaces, it is not clear how Purkinje cells behave in response to Reelin to initiate the PP. Furthermore, it is not known what nascent Purkinje cells look like in vivo. When and how Purkinje cells start axonogenesis must also be elucidated.
Results: We show that Purkinje cells generated on E10.5 in the posterior periventricular region of the lateral cerebellum migrate tangentially, after only transiently migrating radially, towards the anterior, exhibiting an elongated morphology consistent with axonogenesis at E12.5. After their somata reach the outer/dorsal region by E13.5, they change 'posture' by E14.5 through remodeling of non-axon (dendrite-like) processes and a switchback-like mode of somal movement towards a superficial Reelin-rich zone, while their axon-like fibers remain relatively deep, which demarcates the somata-packed portion as a plate. In reeler cerebella, the early born posterior lateral Purkinje cells are initially normal during migration with anteriorly extended axon-like fibers until E13.5, but then fail to form the PP due to lack of the posture-change step.
Conclusions: Previously unknown behaviors are revealed for a subset of Purkinje cells born early in the posteior lateral cerebellum: tangential migration; early axonogenesis; and Reelin-dependent reorientation initiating PP formation. This study provides a solid basis for further elucidation of Reelin's function and the mechanisms underlying the cerebellar corticogenesis, and will contribute to the understanding of how polarization of individual cells drives overall brain morphogenesis.

DOI： 10.1186/1749-8104-5-23

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Notch signaling is essential for the self-renewal of mammalian neural progenitor cells. A variety of mechanisms modulate Notch signaling to balance the self-renewal and differentiation of progenitor cells. Fringe is a major Notch regulator and promotes or suppresses Notch signaling, depending on the Notch ligands. In the developing brain. Lunatic fringe (Lfng) is expressed in self-renewing progenitors, but its roles are unknown. In this study, in vivo mosaic analyses using in utero electroporation were developed to investigate the roles of Lfng in neural progenitor cells. We found that Lfng potentiates Notch signaling cell-autonomously. Its depletion did not affect the balance between neuronally committed cells and self-renewing progenitors, however, irrespective of the cell density of Lfng-depleted cells, and caused no obvious defects in brain development. In vivo overexpression experiments with Notch ligands suggest that Lfng strongly augments Notch signaling mediated by Delta-like 1 but not Jagged 1. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.mcn.2010.05.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CURRENT BIOLOGY LTD  

Cortical development progresses through an early phase of progenitor expansion, a middle phase of neurogenesis, and a final phase of gliogenesis. During the middle phase, the neurogenic phase, the neocortical primordium balances the production of neurons against the maintenance of neural precursor cells (NPCs). The final number of neurons is determined by the duration of the neurogenic phase, the rate of NPC division, and the mode of NPC division, that is, whether a division gives rise to two NPCs, one NPC and one cell committed to the neuronal lineage, or two committed cells. We discuss here recent advances in understanding these key aspects that are fundamental for normal brain development.

DOI： 10.1016/j.conb.2010.01.001

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Cellular diversity of the brain is largely attributed to the spatial and temporal heterogeneity of progenitor cells. In mammalian cerebral development, it has been difficult to determine how heterogeneous the neural progenitor cells are, owing to dynamic changes in their nuclear position and gene expression. To address this issue, we systematically analyzed the cDNA profiles of a large number of single progenitor cells at the mid-embryonic stage in mouse. By cluster analysis and in situ hybridization, we have identified a set of genes that distinguishes between the apical and basal progenitors. Despite their relatively homogeneous global gene expression profiles, the apical progenitors exhibit highly variable expression patterns of Notch signaling components, raising the possibility that this causes the heterogeneous division patterns of these cells. Furthermore, we successfully captured the nascent state of basal progenitor cells. These cells are generated shortly after birth from the division of the apical progenitors, and show strong expression of the major Notch ligand delta-like 1, which soon fades away as the cells migrate in the ventricular zone. We also demonstrated that attenuation of Notch signals immediately induces differentiation of apical progenitors into nascent basal progenitors. Thus, a Notch-dependent feedback loop is likely to be in operation to maintain both progenitor populations.

DOI： 10.1242/dev.022616

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Progenitor cells that generate neuron pairs ("pair progenitor cells") are implicated in mammalian cortical development, and their division has been thought to be "symmetric." However, asymmetric growth of two sister neurons generated by the division of a pair progenitor cell would lead to more efficient generation of neuronal diversity in the cortex. To explore mechanisms by which pair progenitor cells provide neuronal diversity, we examined molecular differences between a pair of neurons generated in clonal-density culture. Time-course analysis for the acquisition of neuronal markers and the disappearance of Pax6 and Neurogenin2 (Ngn2) demonstrated that 1) these transcription factors are expressed transiently in some but not all young neurons and 2) some neuron pairs showed uneven/asymmetric expression of Pax6 (19.5%) or Ngn2 (23.8%), whereas other pairs were either symmetrically positive or negative. Asymmetric Pax6 distribution in neuron pairs was not associated with asymmetric distribution of Numb, which raises an intriguing possibility, that Pax6 asymmetry in neuron pairs is produced by an alternative mode of the cell autonomous mechanisms. Stage-dependent changes were noted in the pattern of Ngn2 retention in daughter neurons, reflecting qualitative changes in the pair progenitor population. We suggest that pair progenitor cells contribute to the generation of neuronal diversity through cell-intrinsic heterogeneity and asymmetric division. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/jnr.20347

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Mature neocortical layers all derive from the cortical plate (CP), a transient zone in the dorsal telencephalon into which young neurons are continuously delivered. To understand cytogenetic and histogenetic events that trigger the emergence of the CP, we have used a slice culture technique. Most divisions at the ventricular surface generated paired cycling daughters (P/P divisions) and the majority of the P/P divisions were asymmetric in daughter cell behavior; they frequently sent one daughter cell to a non-surface (NS) position, the subventricular zone (SVZ), within a single cell-cycle length while keeping the other mitotic daughter for division at the surface. The NS-dividing cells were mostly Hu(+) and their daughters were also Hu+, suggesting their commitment to the neuronal lineage and supply of early neurons at a position much closer to their destiny than from the ventricular surface. The release of a cycling daughter cell to SVZ was achieved by collapse of the ventricular process of the cell, followed by its NS division. Neurogenin2 (Ngn2) was immunohistochemically detected in a certain cycling population during G1 phase and was further restricted during G2-M phases to the SVZ-directed population. Its retroviral introduction converted surface divisions to NS divisions. The asymmetric P/P division may therefore contribute to efficient neuron/progenitor segregation required for CP initiation through cell cycle-dependent and lineage-restricted expression of Ngn2.

DOI： 10.1242/dev.01173

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

For the understanding of histogenetic events in the 3-D retinal neuroepithelium, direct observation of the progenitor cells and their morphological changes is required. A slice culture method has been developed by which the behavior of single progenitor cells can be monitored. Although it has been believed that each retinal progenitor cell loses its basal process while it is in M phase, it is reported here that the process is retained throughout M phase and is inherited by one daughter cell, which can be a neuron or a progenitor cell. Daughter neurons used an inherited process for neuronal translocation and positioning. In divisions that produced two mitotic daughters, both of which subsequently divided to form four granddaughter cells, only one daughter cell inherited the original basal process while the other extended a new process. Interestingly, behavioral differences were often noted between such mitotic sisters in the trajectory of interkinetic nuclear movement, cell cycle length, and the composition of the granddaughter pair. Therefore, 'symmetric' (progenitor --> progenitor + progenitor) divisions are in fact morphologically asymmetric, and the behavior of the mitotic daughters can often be asymmetric, indicating the necessity for studying possible associations between the process inheritance and the cell fate choice.

DOI： 10.1046/j.1524-4725.2003.690.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Despite recent progress in the neural stem cell biology, their cellular characteristics have not been described well. We investigated various characteristics of neural stem cells (NSCs) in vivo during CNS development, using FACS to identify the NSCs. We first examined stage-dependent changes in the physical parameters, using forward scatter (FSC) and side scatter (SSC) profiles, of NSCs from the developing striatum, where they appear to be active throughout the life of mammals. NSCs were divided into several fractions according to their FSC/SSC profile. With development, their number decreased in the FSChigh fractions but increased in the FSClow/SSChigh fraction, whereas NSCs were significantly concentrated in the fraction containing the largest cells (about 20 mum in diameter) at any stage, which were mostly the cells with the highest nestin-enhancer activity. Furthermore, we demonstrated that, at all stages examined, the "side population" (SP), defined as the Hoechst 33342 low/negative fraction, which is known to be a stem cell-enriched population in bone marrow, was also enriched for Notch1-positive immature neural cells (about 60%) from the developing striatum. However, these immature SP cells were not detected in the large-cell fraction, however, but were concentrated instead in the FSClow/mid fractions. FACS analysis showed that SP cells from adults were included to some extent in the CD24(low)/ PNA(low) fraction, where NSCs were greatly concentrated. Collectively, the characteristics of NSCs were not uniform and changed developmentally. (C) 2002 Wiley-Liss, Inc.

DOI： 10.1002/jnr.10339

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Slice culture combined with the use of fluorescent dyes and/or the introduction of fluorescent protein genes provides live and three-dimensional information on cytogenetic and histogenetic events at the level of the individual cell. Using slices prepared from midembryonic mouse cerebral wall tissue upon which fine Dil crystals were placed on the pial or ventricular surface, we recently found that dividing progenitor cells do not lose their pia-connected (basal) processes and that the processes are inherited by daughter cells, including neurons (Miyata et al. [2001] Neuron 31:727-741). To understand more fully the biological significance of this inheritance process, the fate of each daughter cell should be monitored over a culture period extended long enough to allow a neuron to migrate up to the cortex or for a progenitor to proceed to the next round of division. Exposure of slices to 40%, instead of 20%, O-2 significantly improved their overall thickening, cell production, and layer formation and also provided better spatial resolution by preventing the loss of transparency that accompanies cell death. (C) 2002 Wiley-Liss, Inc.

DOI： 10.1002/jnr.10335

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Recent studies demonstrated the neuronogenic role of radial glial cells (RGCs) in the rodent. To reveal the fate of radial glial processes, we intensively monitored divisions of RGCs in Dil-labeled slices from the embryonic day 14 mouse cortex. During RGC division, each pia-connected fiber becomes thin but is neither lost nor divided; it is inherited asymmetrically by one daughter cell. In divisions that produce a neuron and a progenitor, the neuron inherits the pial fiber, also grows a thick ventricular process for several hours, and is therefore indistinguishable from the progenitor RGC. The ventricular process in the radial glial-like neuron ("radial neuron") then collapses, leading to ascent of the neuron by using the "recycled" radial fiber.

DOI： 10.1016/S0896-6273(01)00420-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Many tissues arise from pluripotent stem cells through cell-type specification and maturation. In the bone marrow, primitive stem cells generate all the different types of blood cells via the sequential differentiation of increasingly committed progenitor cells. Cell-surface markers that clearly distinguish stem cells, restricted progenitors, and differentiated progeny have enabled researchers to isolate these cells and to study the regulatory mechanisms of hematopoiesis. Neuronal differentiation appears to involve similar mechanisms. However, neural progenitor cells that are restricted to a neuronal fate have not been characterized in vivo, because specific cell-surface markers are not available. We have developed an alternative strategy to identify and isolate neuronal progenitor cells based on dual-color fluorescent proteins. To identify and isolate directly progenitor cells from brain tissue without the need for either transfection or intervening cell culture, we established lines of transgenic mice bearing fluorescent transgenes regulated by neural promoters. One set of transgenic lines expressed enhanced yellow fluorescent protein (EYFP) in neuronal progenitor cells and neurons under the control of the T alpha1 alpha -tubulin promoter. Another line expressed enhanced green fluorescent protein (EGFP) in immature neural cells under the control of the enhancer/promoter elements of the nestin gene. By crossing these lines we obtained mice expressing both transgenes. To isolate neuronal progenitor cells directly from the developing brain, we used flow cytometry, selecting cells that expressed EGFP and EYFP simultaneously. We expect this strategy to provide valuable material with which to study the mechanisms of neurogenesis and to develop cell-based therapies for neurological disorders. J. Neurosci. Res. 65:220-227, 2001. (C) 2001 Wiley-Liss, Inc.

DOI： 10.1002/jnr.1145

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：IGAKU-SHOIN LTD  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We generated transgenic mice carrying enhanced green fluorescent protein (EGFP) under the control of the nestin second-intronic enhancer (E/nestin:EGFP). Flow cytometry followed by in vitro assays revealed that in situ EGFP expression in the embryonic brain correlated with the mitotic index, the cogeneration of both neurons and glia, and the frequency of neurosphere formation in vitro. High-level EGFP expressors derived from embryos included a distinct subpopulation of cells that were self-renewable and multipotent, criteria that define neural stem cells (NSCs). Such cells were largely absent among lower-level or non-EGFP expressors, thereby permitting us to enrich for NSCs using EGFP expression level. In adults, although E/nestin:EGFP-positive cells included the NSC population, the frequency of neurosphere formation did not correlate directly with the level of EGFP expression. However, moderately EGFP-expressing cells in adults gained EGFP intensity when they formed neurospheres, suggesting embryonic and adult NSCs exist in different microenvironments in vivo.

DOI： 10.1006/mcne.2000.0925

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE AMERICA INC  

Neurogenesis persists in the adult mammalian hippocampus. To identify and isolate neuronal progenitor cells of the adult human hippocampus, we transfected ventricular zone-free dissociates of surgically-excised dentate gyrus with DNA encoding humanized green fluorescent protein (hGFP), placed under the control of either the nestin enhancer (E/nestin) or the T alpha 1 tubulin promoter (P/T alpha 1), two regulatory regions that direct transcription in neural progenitor cells. The resultant P/T alpha 1:hGFP(+) and E/nestin:enhanced (E)GFP(+) cells expressed beta III-tubulin or microtubule-associated protein-2; many incorporated bromodeoxyuridine, indicating their genesis in vitro. Using fluorescence-activated cell sorting, the E/nestin:EGFP(+) and P/T alpha 1:hGFP(+) cells were isolated to near purity, and matured antigenically and physiologically as neurons. Thus, the adult human hippocampus contains mitotically competent neuronal progenitors that can be selectively extracted. The isolation of these cells may provide a cellular substrate for re-populating the damaged or degenerated adult hippocampus.

DOI： 10.1038/73119

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Adult humans, like their nonhuman mammalian counterparts, harbor persistent neural progenitor cells in the forebrain ventricular lining. In the absence of adequate surface markers, however, these cells have proven difficult to isolate for study. We have previously identified and selected neural progenitor cells from both the fetal and adult rodent ventricular zone (VZ), by sorting forebrain cells transfected with plasmid DNA encoding the gene for green fluorescent protein driven by the early neuronal promoter for T alpha 1 tubulin (P/T alpha 1:hGFP). We have now extended this approach by purifying both P/T alpha 1:hGFP tubulin-defined neuronal progenitors, as well as potentially less committed E/nestin:hGFP-defined neural progenitor cells, from the adult human VZ. The ventricular wall of the temporal horn of the lateral ventricle was dissected from temporal lobes obtained from four adult patients undergoing therapeutic lobectomy. These samples were dissociated, and the cultured cells transduced with either PT alpha 1:hGFP or E/nestin:EGFP plasmid DNA. A week later, the cells were redissociated, selected via fluorescence-activated cell sorting (FACS) on the basis of neural promoter-driven GFP expression, and replated. The majority of these cells expressed the early neuronal protein beta III-tubulin upon FAGS; within the week thereafter, most matured as morphologically evident neurons that coexpressed beta III-tubulin and microtubule-associated protein (MAP)-2. Many of these neurons had incorporated bromodeoxyuridine in vitro in the days before FAGS, indicating their mitogenesis in vitro. Thus, the use of fluorescent transgenes under the control of early neural promoters permits the enrichment of neuronal progenitor cells from the adult human ventricular zone. The specific acquisition, in both purity and number, of residual neural progenitor cells from the adult human brain may now permit hitherto unfeasible studies of both their biology and practical application. (C) 2000 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-4547(20000201)59:3<321::AID-JNR5>3.0.CO;2-9

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Language：Japanese   Publisher：金原一郎記念医学医療振興財団  

DOI： 10.11477/mf.2425902273

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