Enlarged lateral ventricles and aberrant behavior in mice overexpressing PDGF-B in embryonic neural stem cells

Platelet-derived growth factor (PDGF) is important in central nervous system (CNS) development, and aberrant expression of PDGF and its receptors has been linked to developmental defects and brain tumorigenesis. We previously found that neural stem and progenitor cells in culture produce PDGF and respond to it by autocrine and/or paracrine signaling. We therefore aimed to examine CNS development after PDGF overexpression in neural stem cells in vivo.Transgenic mice were generated with PDGF-B under control of a minimal nestin enhancer element, which is specific for embryonic expression and will not drive adult expression in mice.The resulting mouse showed increased apoptosis in the developing striatum, which suggests a disturbed regulation of progenitor cells. Later in neurodevelopment, in early postnatal life, mice displayed enlarged lateral ventricles. This enlargement remained into adulthood and it was more pronounced in male mice than in transgenic female mice. Nevertheless, there was an overall normal composition of cell types and numbers in the brain and the transgenic mice were viable and fertile. Adult transgenic males, however, showed behavioral aberrations and locomotor dysfunction. Thus, a tightly regulated expression of PDGF during embryogenesis is required for normal brain development and function in mice.     

Id4 is required for the correct timing of neural differentiation

Complex intrinsic and extrinsic mechanisms determine neural cell fate during development of the nervous system. Using Id4 deficient mice, we show that Id4 is required for normal development of the central nervous system (CNS), timing neural differentiation in the developing forebrain. In the absence of Id4, the ventricular zone of the neocortex, future hippocampus as well as lateral and medial ganglionic eminences exhibited a 20-30% reduction in mitotic neural precursor cells (NPCs). Although the number of apoptotic cells was significantly increased, the neocortex of Id4(-/-) embryos was consistently thicker due to premature neuronal differentiation, which resulted in an increase in early-born neurons in the adult Id4(-/-) cortex. Late-born cortical neurons and astrocytes in the cortex, septum, hippocampus and caudate putamen of Id4(-/-) adult brains were decreased, however, likely due to the depletion of the NPC pool. Consequently, adult Id4(-/-) brains were smaller and exhibited enlarged ventricles. In vitro analysis of neurosphere cultures revealed that proliferation of Id4-deficient NPCs was impaired and that BMP2-mediated astrocyte differentiation was accelerated in the absence of Id4. Together, these in vivo and in vitro data suggest a crucial role for Id4 in regulating NPC proliferation and differentiation.     

Fbxw7-dependent degradation of Notch is required for control of "stemness" and neuronal-glial differentiation in neural stem cells

Control of the growth and differentiation of neural stem cells is fundamental to brain development and is largely dependent on the Notch signaling pathway. The mechanism by which the activity of Notch is regulated during brain development has remained unclear, however. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members. We now show that mice with brain-specific deletion of Fbxw7 (Nestin-Cre/Fbxw7(F/F) mice) die shortly after birth with morphological abnormalities of the brain and the absence of suckling behavior. The maintenance of neural stem cells was sustained in association with the accumulation of Notch1 and Notch3, as well as up-regulation of Notch target genes in the mutant mice. Astrogenesis was also enhanced in the mutant mice in vivo, and the differentiation of neural progenitor cells was skewed toward astrocytes rather than neurons in vitro, with the latter effect being reversed by treatment of the cells with a pharmacological inhibitor of the Notch signaling pathway. Our results thus implicate Fbxw7 as a key regulator of the maintenance and differentiation of neural stem cells in the brain.     

Structurally distinct LewisX glycans distinguish subpopulations of neural stem/progenitor cells

There is increasing evidence that the stem and progenitor cell population that builds the central nervous system is very heterogeneous. Stem cell markers with the potential to divide this cell pool into subpopulations with distinct characteristics are sparse. We were looking for new cell type-specific antigens to further subdivide the progenitor pool. Here, we introduce the novel monoclonal antibody clone 5750. We show that it specifically labels cell surfaces of neural stem and progenitor cells. When 5750-expressing cells were isolated by fluorescence-activated cell sorting from embryonic mouse brains, the sorted population showed increased neurosphere forming capacity and multipotency. Neurospheres generated from 5750-positive cells could self-renew and remained multipotent even after prolonged passaging. Carbohydrate binding assays revealed that the 5750 antibody specifically binds to LewisX-related carbohydrates. Interestingly, we found that the LewisX epitope recognized by clone 5750 differs from those detected by other anti-LewisX antibody clones like 487(LeX), SSEA-1(LeX), and MMA(LeX). Our data further reveal that individual anti-LewisX clones can be successfully used to label and deplete different subpopulations of neural cells in vivo and in vitro. In conclusion, we present a new tool for the isolation and characterization of neural subpopulations and provide insights into the complexity of cell surface glycosylation.     

Neural progenitors from human embryonic stem cells.

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.     

Nanog-induced dedifferentiation of p53-deficient mouse astrocytes into brain cancer stem-like cells

Self-renewal, differentiation, and tumorigenicity characterize cancer stem cells (CSCs), which are rare and maintained by specific cell fate regulators. CSCs are isolated from glioblastoma multiforme (GBM) and may be responsible for the lethality of incurable brain tumors. Brain CSCs may arise from the transformation of undifferentiated, nestin-positive neural stem or progenitor cells and GFAP-expressing astrocytes. Here, we report a role of Nanog in the genesis of cancer stem-like cells. Using primary murine p53-knockout astrocytes (p53^−/− astrocytes), we provide evidence that enforced Nanog expression can increase the cellular growth rate and transform phenotypes in vitro and in vivo. In addition, Nanog drives p53^−/− astrocytes toward a dedifferentiated, CSC-like phenotype with characteristic neural stem cell/progenitor marker expression, neurosphere formation, self-renewal activity, and tumor development. These findings suggest that Nanog promotes dedifferentiation of p53-deficient mouse astrocytes into cancer stem-like cells by changing the cell fate and transforming cell properties.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.     

Botch promotes neurogenesis by antagonizing Notch

Regulation of self-renewal and differentiation of neural stem cells is still poorly understood. Here we investigate the role of a developmentally expressed protein, Botch, which blocks Notch, in neocortical development. Downregulation of Botch in vivo leads to cellular retention in the ventricular and subventricular zones, whereas overexpression of Botch drives neural stem cells into the intermediate zone and cortical plate. In vitro neurosphere and differentiation assays indicate that Botch regulates neurogenesis by promoting neuronal differentiation. Botch prevents cell surface presentation of Notch by inhibiting the S1 furin-like cleavage of Notch, maintaining Notch in the immature full-length form. Understanding the function of Botch expands our knowledge regarding both the regulation of Notch signaling and the complex signaling mediating neuronal development.     

Bmi-1 over-expression in neural stem/progenitor cells increases proliferation and neurogenesis in culture but has little effect on these functions in vivo

The polycomb gene Bmi-1 is required for the self-renewal of stem cells from diverse tissues, including the central nervous system (CNS). Bmi-1 expression is elevated in most human gliomas, irrespective of grade, raising the question of whether Bmi-1 over-expression is sufficient to promote self-renewal or tumorigenesis by CNS stem/progenitor cells. To test this we generated Nestin-Bmi-1-GFP transgenic mice. Analysis of two independent lines with expression in the fetal and adult CNS demonstrated that transgenic neural stem cells formed larger colonies, more self-renewing divisions, and more neurons in culture. However, in vivo, Bmi-1 over-expression had little effect on CNS stem cell frequency, subventricular zone proliferation, olfactory bulb neurogenesis, or neurogenesis/gliogenesis during development. Bmi-1 transgenic mice were born with enlarged lateral ventricles and a minority developed idiopathic hydrocephalus as adults, but none of the transgenic mice formed detectable CNS tumors, even when aged. The more pronounced effects of Bmi-1 over-expression in culture were largely attributable to the attenuated induction of p16^Ink4a and p19^Arf in culture, proteins that are generally not expressed by neural stem/progenitor cells in young mice in vivo. Bmi-1 over-expression therefore has more pronounced effects in culture and does not appear to be sufficient to induce tumorigenesis in vivo.     

Characterization of Neural Stem/Progenitor Cells Expressing VEGF and its Receptors in the Subventricular Zone of Newborn Piglet Brain

Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multipotent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.     

Neural precursor cells differentiating in the absence of Rb exhibit delayed terminal mitosis and deregulated E2F 1 and 3 activity

The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis we examined the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural stem cells derived from Rb-deficient mice. Although Rb -/- neural precursor cells are able to differentiate in vitro we show that these cells exhibit a significant delay in terminal mitosis relative to wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts with the putative Rb target E2F3 in neural precursor cells, our results indicate that it clearly cannot restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3 activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In these studies we show that Rb is essential for the regulation of E2F 1 and 3 activity as well as the onset of terminal mitosis in neural precursor cells.     

Age-dependent changes in the subcallosal zone neurogenesis of mice

There are several known neurogenic areas including subventricular zone and subgranular layer in the dentate gyrus of the hippocampus. Both germinal centers exhibit an age-dependent decline in cell proliferation and neurogenesis, which may be associated with age-related decline in brain function. We recently identified the subcallosal zone (SCZ) as a novel neural stem cell niche with a potential to spontaneously produce new neuroblasts. We examined whether SCZ neurogenesis is also regulated by the age of mice. The number of newly generated neuroblasts was reduced in the SCZ with age, and only marginal number of DCX-labeled neuroblasts was found in 6-month-old SCZ, which is most likely due to reduced proliferation of progenitor cells and loss of neural stem cells (NSCs). This age-dependent changes in the SCZ occurred earlier than that of other neurogenic brain regions. The neurosphere assay in vitro confirmed the depletion of NSCs within the SCZ of young adults. However, marked induction of neuroblast production in the SCZ was seen in 6-month-old mice after traumatic brain injury. Taken together, these results indicate that a rapid decline in SCZ neurogenesis in mice is due to depletion of NSCs and reduced capacity to produce neuroblasts.     

Neural stem cells transplanted into intact brains as neurospheres form solid grafts composed of neurons, astrocytes and oligodendrocyte precursors

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential that can give rise to neurons and glia in vivo and in vitro. The aim of this study was to transplant NSCs as whole neurospheres into intact brain and assess the fate and phenotype of their progeny generated in vivo. We isolated NSCs from E14 foetal rat forebrains and cultured them in basic fibroblast and epidermal growth factor-supplemented serum-free medium in the form of neurospheres in vitro. Neurospheres were transplanted into the intact brains of 2 Wistar rats and after a period of 3 weeks, grafted brains were examined immunohistochemically. Neurospheres formed solid grafts that were found in the lateral ventricle and in the velum interpositum under the hippocampus. The majority of cells in the transplanted tissue were identified as beta-III-tubulin(+), NeuN(+), PanNF(+) and synaptophysin(+) neurons and were accumulated throughout the graft centre. GFAP(+) astrocytes were scattered throughout the entire graft and astrocyte processes delimited the outer and perivascular surfaces. A great number of NG2(+) oligodendrocyte precursors was detected. Nestin(+) endothelial cells were found to line capillaries growing in the transplant. These data indicate that nestin(+) NSCs prevailing in neurospheres differentiate following transplantation into nestin(-) neuronal and glial cells which confirms the multipotency of NSCs. Three weeks posttransplantation neuronal and astrocyte cells reached terminal differentiation (formation of synaptic vesicles and superficial and perivascular limiting membranes) while elements of oligodendroglial cell lineage remained immature. Grafting stem cells as non-dissociated neurospheres provide cells with favourable conditions which facilitate cell survival, proliferation and differentiation. However, in the intact brain, grafted neurosphere cells were not found to integrate with the brain parenchyma and formed a compact structure demarcated from its surroundings.     

Characterization of neurosphere cell phenotypes by flow cytometry

BACKGROUND: Neural stem cell research regularly utilizes neurosphere cultures as a continuous source of primitive neural cells. Results from current progenitor cell assays show that these cultures contain a low number of neural progenitors. Our goal is to characterize neurosphere cultures and define subpopulations in order to purify neural progenitor cells. METHODS: Cells from embryonic mouse neurosphere cultures were stained with Hoechst 33342 and analyzed by flow cytometry. Subpopulations were sorted based on their relative fluorescence intensity in the blue and red regions of the spectrum. Individual sorted subpopulations were reanalyzed after 7 days in culture. RESULTS: Neurosphere cultures contain a relatively high number of cells that stain weakly with Hoechst 33342. This subpopulation is present when cultured as an entire batch in the presence of epidermal growth factor (EGF). When cultured separately, this subpopulation gives rise to a neurosphere population with essentially the same characteristics as freshly isolated embryonic mouse brain cells but contains substantially fewer weakly Hoechst-stained cells. CONCLUSIONS: Similar to hemopoietic systems, neurosphere cultures contain a subpopulation that can be characterized by a low emission of Hoechst fluorescence. When cultured separately, this subpopulation gives rise to a phenotype similar to freshly isolated, uncultured neural cells.     

Midkine, a heparin-binding growth factor, is expressed in neural precursor cells and promotes their growth

Midkine, a heparin-binding growth factor, was found to be expressed in neural precursor cells, which consist of neural stem cells and the progenitor cells. When embryonic brain cells were allowed to form neurospheres enriched in neural precursor cells, numbers were significantly smaller from the midkine-deficient brain than from the wild-type brain. Dissociated neurosphere cells yielded nestin-positive neural precursor cells and differentiated neuronal cells upon culture on a substratum. Neural precursor cells from the midkine-deficient brain spread poorly and grew less effectively on a substratum coated with poly-l-lysine than the cells on midkine-coated substratum. Neural precursor cells from the wild-type brain spread and grew well on both the substrata. Differentiation to neurons and glia cells was not affected by the absence of midkine. Heparitinase digestion of dissociated neurosphere cells resulted in poor growth of neural precursor cells, while chondroitinase digestion had no effect. These results indicate that midkine is involved in the growth of neural precursor cells and suggest that the interaction with heparan sulfate proteoglycans is important in midkine action to these cells.     

Cell cycle kinetics of expanding populations of neural stem and progenitor cells in vitro

Neural stem cells (NSCs) are undifferentiated, primitive cells with important potential applications including the replacement of neural tissue lost due to neurodegenerative diseases, including Parkinson's disease, as well as brain and spinal cord injuries, including stroke. We have developed methods to rapidly expand populations of mammalian stem and progenitor cells in neurosphere cultures. In the present study, flow cytometry was used in order to understand cell cycle activation and proliferation of neural stem and progenitor cells in suspension bioreactors. First, a protocol was developed to analyze the cell cycle kinetics of NSCs. As expected, neurosphere cells were found to cycle slowly, with a very small proportion of the cell population undergoing mitosis at any time. Large fractions (65-70%) of the cells were detected in G1, even in rapidly proliferating cultures, and significant fractions (20%) of the cells were in G0. Second, it was observed that different culturing methods influence both the proportion of neurosphere cells in each phase of the cell cycle and the fraction of actively proliferating cells. The results show that suspension culture does not significantly alter the cell cycle progression of neurosphere cells, while long-term culture (>60 days) results in significant changes in cell cycle kinetics. This suggests that when developing a process to produce neural stem cells for clinical applications, it is imperative to track the cell cycle kinetics, and that a short-term suspension bioreactor process can be used to successfully expand neurosphere cells.