Stem Cells and Tissue Regeneration

Bone Marrow Transplantation (2003) 32, S13–S17. doi:10.1038/sj.bmt.1703937

Neural stem cells

D L Clarke1

1ES Stem Cell International, Cambridge, MA, USA

Correspondence: Dr DL Clarke, ES Cell International, 61 Moulton Street, Cambridge, MA 02138, USA



The adult vertebrate central nervous system (CNS) consists of four major differentiated cell types: neurons, astrocytes, oligodendrocytes and ependymal cells. Historically, there has been a disagreement on how these differentiated cell types are generated in the CNS. Progress remains hindered by the complexity of cell structure in this system, the lack of specific cell surface markers to identify distinct cell types and the presence of numerous transit amplifying cell populations that rapidly generate early progenitors. At present, it is clear that some cells, termed neural stem cells, can generate neurons as well as astrocytes and oligodendrocytes of the glial lineage both in vitro and in vivo. Additionally, specific neural stem cell populations have also exhibited tissue lineage plasticity.


neural, stem cell, progenitor, differentiation, somatic contribution


Origin of neural and glial progenitors in the central nervous system

All cells present in the central nervous system (CNS) are originally derived from the early neuroepithelium that forms as the neural plate along the midline of the developing embryo. As development proceeds, this single layer of pseudostratified epithelium folds to form the neural tube. The differentiation of the neuroepithelial stem cells into neurons and glia then proceeds in a temporal specific manner that is specific for each region of the developing neural tube.1,2 Generally neurogenesis occurs first, followed by gliogenesis. This patterning of the neural tube is thought to begin at the neural plate stage of development through inductive cellular interactions that create organizing centers at the dorsal and ventral poles.3,4,5 These specialized neuroepithelial cells generate signals that induce, often in a concentration-dependent manner, the expression of patterning genes in adjacent neuroepithelial cells. Patterning genes generally encode homeodomain transcription factors, and their expression patterns divide the cells in the neuroepithelium into different domains along the rostral–caudal and dorsoventral axes of the neural tube.6,7 These patterning genes are thought to specify neuronal subtype identity and control the duration of specific types of neurogenesis occurring in the brain and spinal cord during each developmental stage.

By midgestation the early neuroepithelial cells present in the developing cortex of the brain have given rise to young neurons. These neurons have migrated beyond the germinal ventricular zone (VZ) of the neuroepithelium with the aid of newly formed glial cells in this region (Figure 1). The radial glia contact the inner ventricular surface and the outer pial surface of the neural tube, guiding neuronal migration away from the VZ and forming the second germinal zone, the subventricular zone (SVZ). When early neuroblast formation has ceased, the remaining neuro-epithelial cells in the VZ begin to differentiate into glioblasts. Clonal studies suggest that most glia originate from stem cells in the neuroepithelium.8,9 These cells migrate out into the adjacent SVZ where they proliferate and become astrocytes and oligodendrocytes. Lineage tracing studies using stereotactically injected retrovirus support the view that the majority of progenitors within this germinal matrix are glial precursors that generate either astrocytes or oligodendrocytes.10,11 Some SVZ cells give rise to both oligodendrocytes and astrocytes, and a rare cell will develop into both neurons and glia,12 although this remains a controversial issue.13

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Early specification of the neuroepithelium in the neocortex of the brain. Early in development, neuroepithelial stem cells reside in the luminal cellular layer of the neural tube termed the ventricular zone (VZ). These cells begin to divide rapidly, initially generating radial glia and early-restricted neuroblasts. The neuroblasts delaminate from the neuroepithelium, accumulate within the VZ and activate a number of genes involved in neuronal differentiation. Activation of these genes is thought to put into motion the expression of specific cascades of factors that specify both neuronal determination and differentiation in diffrent regions of the cortex. As the neuroblasts mature, the radial glia provide a substrate for the migration of these postmitotic neurons from the germinal VZ to the emerging layers of the neocortex. Soon after neuroblast formation subsides, neuroepithelial stem cells in the VZ generate glioblasts. These glioblasts migrate out into the adjacent subventricular zone (SVZ) where they proliferate and become primary astrocytes and oligodendrocytes. These cells then migrate away from this region to populate the generating layers of the cortex. Illustration by Cheng-Jung Lai.

Full figure and legend (26K)

When glioblast formation ceases shortly after birth, the germinal VZ disappears throughout the neuroaxis and many of the remaining neuroepithelial cells become ependymal cells. The ependymal cells persist throughout adulthood lining the luminal surface of the ventricular system of the brain and the central canal of the spinal cord. These cells possess multiple cilia on their apical surface that effectively move the cerebral spinal fluid throughout these regions. Similarly, the SVZ decreases in size and persists immediately adjacent to the ependymal cell layer, throughout most of the ventricular regions of the brain. However, a SVZ region is not present in the developing or mature regions of the spinal cord.

As development proceeds and compartmentalization of the CNS becomes apparent, neural stem and early progenitor cells in the mammalian fetal CNS are considered to be concentrated in seven major areas: the olfactory bulb, VZ and SVZ of the forebrain; the hippocampus, cerebellum, cerebral cortex and the spinal cord. Their number and pattern of development vary in different species.1,14,15 However, once the patterning of the different CNS compartments is in place, it is believed that stem cells located in these different regions of the developing CNS are developmentally distinct and are not a single population of stem cells that are dispersed over multiple sites.16 Stem cells isolated from the spinal cord generate spinal cord progeny.17 Stem cells isolated from the basal forebrain generate more GABA containing neurons than stem cells derived from dorsal regions.18 Whether this distinction persists in the adult remains controversial.


Adult neural stem cells

Proliferative stem cell compartments are not exclusive to the developing or fetal CNS. Numerous pioneering experiments have demonstrated that specific regions of the mammalian CNS undergo a moderate, yet continuous level of neurogenesis postnatally and throughout adult life19,20,21,22 (Figure 2). To date, neurogenesis in the adult mammalian CNS is known to utilize at least one dividing progenitor cell population23 and two different multipotential stem cell populations.24,25,26 The putative progenitor cell population resides in the subgranular zone of the dentate gyrus located in the hippocampus, the region of the brain involved in learning and memory. The two remaining stem cell populations have been reported to exist in and near the anterior lateral ventricular wall of the cerebral cortex, both of which exist in the adult as highly differentiated glial cell types – SVZ astrocytes and ventricular ependymal cells. While there is only a limited amount of neurogenesis that occurs in the adult hippocampus, both ependymal cells and subventricular zone astrocytes are thought to continuously replace interneurons in the olfactory bulb. It remains controversial whether the rapidly dividing multipotential stem cells in the SVZ are a distinct stem cell population that contributes to the generation of olfactory interneurons. It has been suggested that the adjacent ependymal cell layer, which has been shown to divide at a comparatively slower rate in vivo, may give rise to the SVZ cells.24,27

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact or the author

Neurogenic regions of the adult mammalian CNS. Neural stem cells have been identified in three different regions of the adult CNS: the ependymal cell layer lining the lateral vertical regions of the brain, the adjacent subventricular zone and the hippocampus. The ventricular system of the brain is continuous with the central canal of the spinal cord (a). Coronal sections through different regions of the brain show the location of the hippocampus near the posterior region of the cerebral hemispheres (b). The single ependymal cell layer lining the luminal surface of the lateral ventricles and the adjacent multilayered subventricular zone are shown in (c). Neural stem cells in these two regions of the brain migrate rostrally to give rise to interneurons in the olfactory bulb (OB). Illustration by Cheng-Jung Lai.

Full figure and legend (17K)

The identification and existence of adult neural stem cells in the ventricular and SVZ was a surprising finding. Even more surprising is the suggestion that they are highly differentiated glial cell types and not remnants of nascent undifferentiated stem cells that are specified early during embryonic development. Numerous trophic factors have been shown to influence the actual developmental fate of a progenitor or multipotential stem cell from these regions, which may differ from its developmental potential.28,29 In culture, cells from these regions can differentiate into neurons in response to instructive extracellular signals that promote the production or activity of proneural transcription factors. Similarly, they can differentiate into glia in response to a variety of extracellular signals such as ciliary neurotrophic factor (CNTF), bone morphogenic proteins (BMPs), transforming growth factor-alpha (TGFalpha) and neuregulin-1 (Nrg-1)/glial growth factor-2 (GGF2). However, it is not known whether these same factors directly induce neural and glial differentiation in vivo. Since environmental conditions can be provided in vitro by adding specific trophic factors to the culture medium, neural stem cells differentiating in cultures have been shown to exhibit considerable plasticity not normally observed in vivo. Thus, understanding the environmental conditions necessary to promote specific types of differentiation from a stem cell may show that the limitations that these cells perceive in vivo are controlled by an environmental cues and not necessarily by an intrinsic commitment of the stem cell itself.


Oligodendroglial progenitors

The multipotential neural stem cells or early neural progenitor cells present in the central nervous system of embryonic, neonatal and adult animals can also differentiate into intriguing lineage-restricted progenitors, termed oligodendroglial progenitors. Most oligodendrocyte precursor cells in the developing central nervous system terminally differentiate into oligodendrocytes that myelinate axons. Terminally differentiated oligodendrocytes do not divide, dedifferentiate, or re-enter the cell cycle. However, precursors to oligodendrocytes do exist and their division persists, albeit at a slow rate, throughout life, with a certain potentiality bias for myelin repair.

Recently, oligodendrocyte progenitors have been isolated from the adult brain. These cells have been propagated extensively in vitro as clonal spheroid cell aggregates that detach from the tissue culture dish and grow in suspension to generate a large number of multipotential stem cell progeny that maintain their myelinating potential.30 Characterization of these cells has indicated that these oligodendrocyte progenitors resemble neonatal rather than adult progenitors. Generation of such cells from the adult brain opens new possibilities to explore the potential of these cells for repairing myelin disorders.


Are neural stem cells derived from the adult CNS irreversibly determined?

Adult spinal-cord-derived cells, which normally generate only glia,1,24,31 can differentiate into granular neurons when injected into the adult hippocampus.31 Similarly, adult hippocampus-derived stem cells can make olfactory interneurons after transplantation into the SVZ.32 However, the ability of adult-derived cells to produce complex projection neurons that span long distances in the mature CNS, has not been demonstrated.

Only recently have people investigating the differentiation potential of adult neural stem cells discovered that their differentiation repertoire extends well beyond the boundaries of the cell types found in the CNS. It is possible that adult neural stem cells, when cultured in vitro, exhibit a renewed potentiality that has not been tested or observed in direct transplantation assays. This renewed potential may involve complex inter- and intracellular mechanisms that may have been unwittingly imparted to these cells in culture. Alternatively, the need to restrict the potential of a stem cell may decrease as an organism matures.33 Thus, during embryonic development, cells may be exposed to overlapping sets of extracellular signals. This may initially necessitate their use of cell-autonomous mechanisms to restrict their differentiation. Therefore, transplanting restricted cells from one location to another does not affect their differentiation potential. However, once an organism matures, stem cells in different tissues may be spatially segregated into specific niches where they no longer actively encounter signals that restrict their differentiation potential. These cells may be released from their cell autonomous programs making them more responsive to environmental cues that can influence their differentiation.


Differentiation of neural stem cells to hematopoietic cells

Intriguingly, in the past few years several reports have indicated that both hematopoietic and neural stem cells demonstrate surprising plasticity.34 Bone-marrow-derived cells have been shown to generate cells expressing neuronal markers in the brain.35,36 Similarly, embryonic or adult-derived neural stem cells from the brain, when injected intravenously into sublethally irradiated mice, have been shown to generate hematopoietic derivatives.37 In this particular study, in vitro clonogenic assays, immunocytochemistry and flow cytometric analysis were used to test whether these cells had adopted a hematopoietic identity. In the clonogenic assays, cells from the bone marrow of transplanted animals were plated in the presence of defined cytokines. Colonies founded by single hematopoietic precursors of neural stem cell origin were 13% pure granulocyte, 30% granulocyte–macrophage, 22% pure macrophage and 19% mixed colonies. A few colonies did not originate from the donor stem cell population, confirming that some endogenous hematopoietic progenitors had survived the irradiation. The neural stem cells used in this study also generated neurons, astrocytes and oligodendrocytes in vitro, arguing against the possibility that the blood-forming cells from the brain were stray hematopoietic stem cells that contributed to the repopulation of the hematopoietic lineages.


Differentiation of neural stem cells to other somatic cell lineages

The myogenic potential of adult neural stem cells has also been recently demonstrated. Coculture of primary mouse or human neural stem cells with myoblasts or injection of the neural stem cells into skeletal muscle of adult mice resulted in the differentiation of these cells to myocytes.38 A conceptually different approach has also demonstrated the myogenic differentiation potential of neural stem cells.39 This approach took advantage of the large number of inductive signals present in embryonic stem cells undergoing differentiation in vitro, a process that ultimately results in the differentiation of the ES cells to various cell lineages. In order to expose neural stem cells to such a milieu, the cells were cultured in close proximity to differentiating embryoid bodies. In these experiments, differentiated neural stem cells gave rise primarily to myocytes, identified by the expression of the markers desmin and myosin heavy chain, and exhibited morphological features such as syncytium formation.

Two separate methods have been used to simultaneously demonstrate the broad differentiation potential of neural stem cells.39 These experiments have relied heavily on the abundance of factors present in the early embryonic environment to demonstrate such potential. In a separate set of experiments, adult neural stem cells were injected into early developing chick or mouse embryos. In the developing chick, neurospheres or clusters of clonally derived neural stem cells were placed directly on or near the primitive streak of a gastrulating embryo. This allowed the neural stem cells to integrate into the epiblast, move with the primitive ectoderm cells through the primitive streak during gastrulation and be distributed throughout the three definitive germ layers. In the mouse, dissociated neural stem cells, or small neurospheres, were aggregated with morulae or injected into blastocysts. In both of these early embryo models, neural stem cell progeny were found in numerous tissues and were morphologically indistinguishable from neighboring host cells. Contributions to tissues were large and occasionally comprised as much as 30% of the entire organ. Cell type specific antibodies were used to verify whether the donor neural stem cells had remained neural or if they had taken on a phenotype appropriate for the tissue into which they had integrated. In both the chick and mouse assays, neural stem cell progeny had indeed differentiated to many embryonic cell types including hepatocytes, cardiomyocytes and epidermal cells, thus representing cells that originate from all three germ layers. Importantly, in experiments where multipotential secondary clonal cultures were established, it was shown that a single neural stem cell had the potential to generate progeny for all three germ layers.

While many of the transplantation and embryonic differentiation studies have demonstrated the ability of adult neural stem cell populations to populate and differentiate into cell types specific for a particular tissue in vivo, their longevity and functional contribution to a living organism have not been demonstrated. Many of the obstacles hampering the definitive detection of donor cells in the adult can now be addressed as new genetically marked strains of mice, and methods increasing the efficiency of cellular contribution have been identified. It will be of particular interest to determine whether the contribution of a donor neural stem cell to a particular adult stem cell niche will allow the original donor stem cell to retain its ability to self-renew, continuously contributing to the neogenesis of cell types particular to that tissue.



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