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Identification of neural stem cells in the adult vertebrate brain.

Neurogenesis continues into adult life in restricted germinal layers. The identification of the neural stem cells that give rise to these new neurons has important clinical implications and provides fundamental information to understand the origins of the new neurons. Work in adult birds and rodents yielded a surprising result: the neural stem cells appear to have characteristics of glia. In adult birds, the primary neuronal precursors are radial glia. In adult mammals, the primary neuronal precursors have properties of astrocytes. Radial glial cells have previously been shown to transform into astrocytes; both cell types are classically considered part of a committed astroglial lineage. Instead, we propose that neural stem cells are contained within this astroglial lineage. These findings in adult vertebrate brain, together with recent work in the developing mammalian cerebral cortex, force us to reexamine traditional concepts about the origin of neurons and glia in the central nervous system. In particular, neural stem cells possess a surprisingly elaborate structure, suggesting that in addition to their progenitor role, they have important structural and metabolic support functions. The very same cells that give birth to new neurons also seem to nurture their maturation and support their function.

Animals↗

Local environmental factors determine hematopoietic differentiation of neural stem cells.

Stem cells exhibit unique properties and hold high therapeutic promise, but factors influencing their differentiation after transplantation need to be recognized and defined for this promise to be fully met. Here, we demonstrate that endogenous colony-forming unit spleen (CFU-S) colonies are not generated in lethally irradiated mice transplanted with neural stem cells obtained from brain tissue of syngeneic donors. We investigated the proportion of transplanted neural stem cells that contributed to hematopoietic reconstitution and compared the distribution of transplanted cells in nonsplenectomized to that of splenectomized mice following sublethal whole-body irradiation. We also used clonogenic assays, colony assays, and histochemical analyses to explore conditions under which transplanted, beta-galactosidase-tagged neural stem cells underwent hematopoietic differentiation. Our results suggest that neural stem cells do undergo extramedullary hematopoiesis, even while no endogenous hematopoietic colonies develop in the spleen. Furthermore, we found that neural stem cells effectively colonized the bone marrow of splectomized recipients. We conclude that the hematopoietic differentiation of neural stem cells is highly dependent on the extramedullary environment. We also conclude that the bone marrow does not provide an environment supportive of hematopoietic differentiation by neural stem cells.

Animals↗

Identification and functions of chondroitin sulfate in the milieu of neural stem cells.

The behavior of cells is generally considered to be regulated by environmental factors, but the molecules in the milieu of neural stem cells have been little studied. We found by immunohistochemistry that chondroitin sulfate (CS) existed in the surroundings of nestin-positive cells or neural stem/progenitor cells in the rat ventricular zone of the telencephalon at embryonic day 14. Brain-specific chondroitin sulfate proteoglycans (CSPGs), including neurocan, phosphacan/receptor-type protein-tyrosine phosphatase beta, and neuroglycan C, were detected in the ventricular zone. Neurospheres formed by cells from the fetal telencephalon also expressed these CSPGs and NG2 proteoglycan. To examine the structural features and functions of CS polysaccharides in the milieu of neural stem cells, we isolated and purified CS from embryonic day 14 telencephalons. The CS preparation consisted of two fractions differing in size and extent of sulfation: small CS polysaccharides with low sulfation and large CS polysaccharides with high sulfation. Interestingly, both CS polysaccharides and commercial preparations of dermatan sulfate CS-B and an E-type of highly sulfated CS promoted the fibroblast growth factor-2-mediated proliferation of neural stem/progenitor cells. None of these CS preparations promoted the epidermal growth factor-mediated neural stem cell proliferation. These results suggest that these CSPGs are involved in the proliferation of neural stem cells as a group of cell microenvironmental factors.

Animals↗

Culture and identification of monoclonal neural stem cells derived from cerebral cortex.

To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to detect the specific marker of neuroepithelial stem cells (Nestin) of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.

Animals↗

Temporal profile of neural stem cell proliferation in the subventricular zone after ischemia/hypoxia in the neonatal rat brain.

OBJECTIVES: Ischemia/hypoxia (I/H) causes severe neonatal brain injury, such as periventricular leukomaracia and hypoxic/ischemic encephalopathy. Neural stem cell research could lead to a treatment for such disorders. In order to elucidate the dynamic changes in neural stem cells in the neonatal brain after I/H, we investigated the proliferation of new cells in the subventricular zone (SVZ). METHODS: Seven-day-old Wister rats were subjected to ligation of the left carotid artery followed by 2 hours of hypoxic stress (8% O(2) and 92% N(2), at 33 degrees C). In order to elucidate the dynamic change of neural stem cells in the SVZ, single bromodeoxyuridine (BrdU; 50 mg/kg) was administered 2 hours before death 1, 7, 14 and 21 days after I/H. Immunohistochemical and immunofluorescent studies for BrdU and doublecortin (DCX) were carried out. As a control, a group of rats was subjected to sham surgery (incision of skin, but no ligation of the carotid artery) and no I/H. RESULTS: The numbers of BrdU-labeled cells in the SVZ, for both the ipsilateral side and the contralateral side of the I/H brain, were twice the level of the control at 7 days after I/H, but the numbers for both sides returned to the control level at 21 days. In the ipsilateral side of the I/H brain, the number of BrdU-labeled cells in the SVZb (lining the upper wall of lateral ventricle) was 4-fold at 7 days and 15-fold at 21 days after I/H compared with the control level. This chronological pattern is very similar to the pattern for I/H results of the posterior periventricle (pPV). DCX appeared in most BrdU-labeled cells in the SVZb and pPV. DISCUSSION: These findings indicate that I/H enhances neural stem cell proliferation in the SVZ, and some newborn cells migrate as neural precursors to the SVZb and pPV after I/H in the neonatal rat brain.

Animals↗

Nestin-expressing neural stem cells identified in the scar following myocardial infarction.

Nerve fiber innervation of the scar following myocardial damage may have occurred either via the growth of pre-existing fibers and/or the mobilization of neural stem cells. The present study examined whether neural stem cells were recruited to the infarct region of the rat heart following coronary artery ligation. The neural stem cell marker nestin was detected in the infarct region of 1-week post-myocardial infarct (MI) male rats and cultured scar-derived neural-like cells. By contrast, nestin staining was undetected in either scar myofibroblasts or cardiac myocytes residing in the non-infarcted left ventricle. Reactive astrocytes were isolated from the infarct region and characterized by the co-expression of nestin, glial fibrillary acidic protein, and vimentin. Specific staining of oligodendrocytes and neurons was also detected in the infarct region and cultured scar-derived neural-like cells. Furthermore, neurofilament-M positive fibers were identified in the scar and tyrosine hydroxylase immunoreactivity was observed in peripherin-positive neurons. Neurite formation was induced in PC12 cells treated with the conditioned-media of primary passage scar-derived cells, highlighting the synthesis and secretion of neurotrophic factors. Nerve growth factor (NGF) and brain-derived neurotrophic factor were detected in myofibroblasts and neural cells, and both cell types expressed the NGF receptors trkA and p75. These data highlight the novel observation that neural stem cells were recruited to the infarct region of the damaged rat heart and may contribute in part to nerve fiber growth and subsequent innervation of the scar.

Animals↗

Microarray analysis of neural stem cell differentiation in the striatum of the fetal rat.

1. Gene expression profiles in neural stem cell differentiation in vitro were determined by cDNA microarray analysis. 2. Total RNA was extracted and reverse transcripted into cDNA from differentiated and undifferentiated neural stem cells. The 33P labeled cDNA was hybridized with a cDNA microarray consisting of 14,000 human genes. 3. The results showed that a total of 1406 genes were differentially expressed, of which 148 genes exhibited more than twofold differences. Some genes were obviously activated while others were strongly repressed. These changes in gene expression suggest that differentiation is regulated by different genes at different expressional levels. By biological classification, the differentially expressed genes were divided into four functional categories: molecular function, biological process, cellular component, and new functional genes or ESTs. 4. These findings will be a valuable contribution for gene expression profiling and elucidation of neural stem cell differentiation mechanisms.

Animals↗

Expression of steroid receptor coactivator-1 is elevated during neuronal differentiation of murine neural stem cells.

The cell-lineage-specific fate of neural stem cells (NSCs) is defined by spatial and temporal gene expression regulated by various transcription factors and their coregulators, including coactivators and corepressors. To clarify the cellular distribution of the steroid receptor coactivator-1 (SRC-1), a ligand-dependent nuclear receptor coactivator, during neurogenesis, we examined the expression profiles of SRC-1 during the proliferation and differentiation in culture of NSCs derived from the ganglionic eminence of mouse embryos. We found that SRC-1 was rarely expressed in proliferating cells and multipotent precursors with the typical characters of NSCs. Under conditions that promote the differentiation of NSCs, both the SRC-1 transcript and protein levels were elevated and the population of SRC-1-positive cells was found to be higher in the mature neurons than in the immature neurons. In contrast, SRC-1 expression was rarely localized in the glial lineage cells, including astrocytes and oligodendrocytes. These results indicate that SRC-1 expression accompanies the appearance of neuronal-fate-committed cells derived from multipotent NSCs and is preferentially expressed in neuronal lineage cells during the differentiation of NSCs.

Analysis of Variance↗

[Blocking with double- stranded RNA of the expression of Hes5 in rat bone marrow-derived neural stem cells].

OBJECTIVE: To examine the efficiency of exogenous small double-stranded RNA (dsRNA) in knocking down the gene expression at the post-transcription level, and investigate the factors that may influence the transfection. METHOD: The bone marrow stromal cells of SD rat were separated and cultured in vitro, followed by induction of the cells to evolve into neural stem cells using special culture medium prepared by our laboratory. Synthetic dsRNA was then transferred into the cells at varied concentrations, and the results were analyzed by Western blotting. RESULTS: The concentrations ranging from 200 to 300 nmol/L were optimal for specifically blocking the expression of Hes5, whereas the suitable concentrations for the cell survival were between 50 and 200 nmol/L. CONCLUSION: dsRNA is capable of triggering RNA interference in neural stem cells, and at appropriate concentration, it may specifically and effectively knock down endogenous gene expression without sacrificing the viability of the cells.

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Engineering of the extracellular matrix: working toward neural stem cell programming and neurorestoration--concept and progress report.

In the concept of neurorestoration, cellular and structural elements that have been lost are replaced, and their function is restored. Central to this therapeutic strategy is the transplantation of neural progenitor cells such as clonogenically expanded stem cells. Stem cells make decisions regarding fate and patterning in response to external environmental signals. The therapeutic effectiveness of neural stem cells may be facilitated by the ability to manipulate these signals in a temporal and spatially appropriate fashion. Artificial deoxyribonucleic acid and artificial protein technology combines elements of protein engineering, molecular biology, and recombinant deoxyribonucleic acid technology to produce proteins with functional domains derived from naturally occurring proteins and represents a potentially powerful tool to modulate stem cell behavior. To this end, we have developed three artificial extracellular matrix proteins that incorporate the active domain of hJagged1 and hDelta1 into an elastin backbone. hJagged1 and hDelta1 are members of the DSL family of ligands to the Notch receptor, a signaling system that is very important in development and is the strongest known signal to instruct neural progenitor cells to choose glial fates over neuronal fates. The successful cloning of the artificial genes was confirmed by test digestions with appropriate restriction enzymes as well as direct deoxyribonucleic acid sequencing. In addition, we have demonstrated that all three artificial extracellular matrix proteins express at a high level in a prokaryotic host. This report describes the concept and progress in an entirely novel and previously unreported approach to modulate neural stem cell behavior. Its future application could include in vitro processing of stem cells before transplantation, supporting and programming the cells after transplantation, as well as the development of bioactive biomaterials.

Cell Physiological Phenomena↗

Generation and transplantation of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice.

EGF-responsive neural stem cells isolated from murine striatum have the capacity to differentiate into both neurons and glia in vitro. Genetic modification of these cells is hindered by a number of problems such as gene stability and transfection efficiency. To circumvent these problems we generated transgenic mice in which the human GFAP promoter directs the expression of human NGF. Neural stem cells isolated from the forebrain of these transgenic animals proliferate and form clusters, which appear identical to stem cells generated from control animals. Upon differentiation in vitro, the transgenic stem cell-derived astrocytes express and secrete bioactive hNGF. Undifferentiated GFAP-hNGF or control stem cells were transplanted into the striatum of adult rats. One and 3 weeks after transplantation, hNGF was detected immunocytochemically in an halo around the transplant sites. In GFAP-hNGF-grafted animals, intrinsic striatal neurons proximal to the graft appear to have taken up hNGF secreted by the grafted cells. Ipsilateral to implants of GFAP-hNGF-secreting cells, choline acetyltransferase-immunoreactive neurons within the striatum were hypertrophied relative to the contralateral side or control-grafted animals. Further, GFAP-hNGF-grafted rats displayed a robust sprouting of p75 neurotrophin receptor-positive fibers emanating from the underlying basal forebrain. These studies indicate that EGF-responsive stem cells which secrete hNGF under the direction of the GFAP promoter display in vitro and in vivo properties similar to that seen following other methods of NGF delivery and this source of cells may provide an excellent avenue for delivery of neurotrophins such as NGF to the central nervous system.

Animals↗

Lavendustin A enhances axon elongation in VHL gene-transfected neural stem cells.

Axonal elongation is necessary for neuronal regeneration of the spinal cord after spinal injury. Recently neural stem cells have been proposed as hopeful graft donors for regeneration of the central nervous system. However, most grafted stem cells are not able to differentiate into neurons, and grafted stem cells cannot usually grow axons. Here, we show the effect of the protein tyrosine kinase inhibitor lavendustin A on axonal growth of neurons differentiated from neural stem cells obtained from adult rat hippocampal cells transfected with the von Hippel-Lindau (VHL) gene. Significantly greater axonal outgrowth was observed for the transfected cells treated with the inhibitor than for those not so treated. Thus, protein-tyrosine kinase inhibition is effective for axonal outgrowth of neurons differentiated from neural stem cells and may prove to be useful for neuronal regeneration via transplanted stem cells, particularly in the case of spinal cord injuries.

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Green fluorescent protein-labeled mapping of neural stem cells migrating towards damaged areas in the adult central nervous system.

Neural stem cells, which are clonogenic cells with multilineage differentiation properties from regions of the fetal brain, cortex and hippocampus, are currently considered as powerful candidates for cell replacement therapy in neurodegenerative disorders, such as Parkinson's disease. A key issue is whether stem cells can survive, migrate and differentiate following transplantation into the adult CNS. Here, enhanced green fluorescent protein plasmid electroporation-transfected neural stem cells from the fetal cortex were grafted into the striatum of a rat model of Parkinson's disease. We found most of the grafted cells could survive in the adult parkinsonian rat brain and migrated towards damaged areas, while they moved randomly in the normal brain. Several grafted cells differentiated into neurons.

Animals↗

Developmental potential of hematopoietic and neural stem cells: unique or all the same?

Like many other animals, mammals develop from fertilized oocytes - the ultimate stem cells. As embryogenesis proceeds, most cells lose developmental potential and eventually become restricted to a specific cell lineage. The result is the formation of a complete and structured mature organism with complex organs composed of a great variety of mature, mostly mitotically quiescent effector cells. However, along the way, some exceptional cells, known as somatic stem cells (SSCs) are set aside and maintain a high proliferation and tissue-specific differentiation potential. SSCs, in contrast to embryonic stem (ES) cells, which are able to give rise to all cell types of the body, have been regarded as being more limited in their differentiation potential in the sense that they were thought to be committed exclusively to their tissue of origin. However, recent studies have demonstrated that somatic stem cells from a given tissue can also contribute to heterologous tissues and thus show a broad nontissue restricted differentiation potential. The question arises: how plastic are somatic stem cells? To provide a tentative answer, we describe and review here recent investigations into the developmental potentials of two somatic stem cell types, namely hematopoietic and neural stem cells.

Animals↗

Trophic support of mouse inner ear by neural stem cell transplantation.

In the auditory system, efforts to reduce degeneration of spiral ganglion neurons have the immediate objective of improving clinical benefits of cochlear implants, which are small devices designed to stimulate spiral ganglion neurons electronically. Recent studies have indicated several neurotrophins can enhance survival of spiral ganglion neurons. However, the strategy for application of neurotrophins in inner ear is still a matter of debate. In this study, we examined the potential of cell therapy as a strategy for application of neurotrophins in the inner ear. Neural stem cells obtained from green fluorescent protein-transgenic mice were used as donor cells. Medium containing neural stem cells was injected into mouse inner ear. Histological analysis 4 weeks later revealed that transplant-derived cells survived in inner ear and that most transplant-derived cells in the cochlea had differentiated into glial cells. Moreover, expression of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor was observed in transplant-derived cells. These findings indicate that transplantation of neural stem cells can be a useful strategy for application of neurotrophins in inner ear.

Animals↗

Differential neurogenesis and gliogenesis by local and migrating neural stem cells in the olfactory bulb.

The rostral migratory stream (RMS) is a unique forebrain structure that provides a long-distance migratory route for the neural stem cells of the periventricular region towards the olfactory bulb (OB). The purpose of the study presented here is to examine the extent of neurogenesis and gliogenesis by the neural stem cells of different origins (periventricular vs. intrabulbar) in the OB. After the RMS had been subjected to injury, the rats received intraperitoneal injections of 5-bromodeoxyuridine (BrdU) and were further reared for 2 weeks. Neuronal and glial differentiations of the BrdU(+) cells in the olfactory bulbar granule cell (OB-GCL) and the olfactory glomerular (OB-GL) layers were examined immunohistochemically using antibodies against neuronal (NeuN, neuronal nuclei) and glial (GFAP, glial fibrillary acidic protein) markers in the OBs with injured and uninjured (control) RMS. In the completely RMS-lesioned OB, where migration of the periventricular neural stem cells was inhibited, a small number of BrdU(+) NeuN(+) cells were found in both the OB-GCL and OB-GL. The BrdU(+) NeuN(+) cells accounted for a much higher percentage of the BrdU(+) cells on the control side (OB-GCL, 36.7%; OB-GL, 8.8%) than on the completely RMS-lesioned side (OB-GCL, 3.7%; OB-GL, 0.6%). The percentage of the BrdU(+) GFAP(+) cells relative to the BrdU(+) cells did not show any major difference between the control and completely RMS-lesioned sides. This study revealed differences in neurogenesis and gliogenesis between the local and migrating neural stem cells in the OB of the adult rodent.

Animals↗

Manipulation of proliferation and differentiation of human bone marrow-derived neural stem cells in vitro and in vivo.

Recent evidence has demonstrated that neural stem cells (NSC) can be expanded from a variety of sources, including embryos, fetuses, and adult bone marrow and brain tissue. We have previously reported the generation of adult rat bone marrow-derived cellular spheres that are morphologically and phenotypically similar to neurospheres derived from brain NSC. Here we show that adult human bone marrow-derived neural stem cells (HBM-NSC) are capable of generating spheres that are similar to brain neural-derived neurospheres. Additionally, we sought to promote proliferation and differentiation of HBM-NSC through transduction with nonreplicative recombinant adenovirus encoding the cDNA sequence for Gli, rADV-Gli-1; sonic hedgehog, rADV-Shh; or Nurr1, rADV-Nurr1. Immunocytochemistry and RT-PCR analysis showed that HBM-NSC could be efficiently expanded and differentiated in vitro and that HBM-NSC transduced with rADV-Gli-1 or rADV-Shh dramatically increased NSC time-related proliferation; however, Nurr1 had no effect on proliferation. We also transplanted HBM-NSC into chicken embryos to examine their potential function in vivo. We found that transduction of HBM-NSC with rADV-Gli-1 or rADV-Shh and subsequent transplantation into chicken embryos increased HBM-NSC proliferation, whereas rADV-Nurr1 promoted migration and differentiation in vivo. Our findings suggest that HBM-NSC can be efficiently expanded and differentiated in vitro and in vivo by overexpressing Gli-1, Shh or Nurr1.

Animals↗