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“One must show the greatest respect towards any thing that increases exponentially, no matter how small.”
Garrett Hardin (1968)

Stem cells, despite their microscopic size, have the remarkable capacity to generate a whole tissue or even a whole living organism. From the zygote to adult tissues, stem cells play powerful roles in tissue generation and maintenance. In the nervous system, the discovery that neural stem cells are present in embryos (1) and adults (2) brings the promise of repair to a tissue that is normally nonregenerative yet highly vulnerable to degenerative diseases and damage.

Neural stem cells exist in many regions of the embryonic central nervous system (CNS) and peripheral nervous system (PNS) (3). They are multipotent and can generate all three types of neural progeny--neurons, oligodendrocytes, and astrocytes--as well as more stem cells, maintaining themselves by self-renewal, which is a hallmark of the stem cell state (4-6).

At early stages of nervous system formation, neural stem cells undergo symmetric cell divisions that expand the stem cell pool (7-9). When neurogenesis begins, they switch to asymmetric cell divisions that generate neurons as well as perpetuating the stem cell population. By watching the growth of individual stem cells over several days, using time-lapse recording, we have seen that cortical stem cells in tissue culture undergo repeated asymmetric cell divisions, generating neural lineage trees akin to those of Caenorhabditis elegans and Drosophila (10-12). As in invertebrates (13), asymmetric localization of Numb during mitosis is important for cortical stem cells to generate two different progeny (14, 15). Mutations in numb lead to reduced incidence of asymmetric cell divisions, overproliferation of neural progenitor cells, and defects in cortical morphogenesis (15, 16).

As neural stem cells divide asymmetrically, their capacity to make different cell types changes: Their ability to generate neurons diminishes (11, 17-19) and they lose the capacity to produce early neuron types (20). Hence the ability of a neural stem cell to make a particular neuron type is transitory during development and is lost as the stem cell ages. This presents a problem for using neural stem cells in CNS replacement therapies. After expansion in vitro to create enough cells for transplantation, they become biased toward making glia and can no longer form early cells such as principal projection neurons.

How can we amplify stem cells of a particular stage to allow the production of select neuron types? Are there factors that cause stem cells to self-renew without losing their neurogenic potential? In vivo, stem cells reside in specialized niches consisting of diverse cells that organize the extracellular matrix and secrete growth factors to maintain stem cell properties and protect them from harm (21). Niches have been identified in a variety of tissues, including the bone marrow, ovary germarium, hair follicle bulge, intestinal crypts, and subventricular zone (SVZ) of the adult brain. Recently, vascular involvement in neural stem cell niches has been suggested. In the hippocampus, stem cells lie close to actively dividing blood vessels, suggesting a link between angiogenesis and neurogenesis (22). In the adult SVZ, stem cells localized with the marker LeX often occur close to blood vessels (23).

To examine their relationship, we cocultured single, isolated, neural stem cells with purified endothelial cells, separated by a membrane. We found that endothelial cells release soluble factors that promote neural stem cell self-renewal, leading to an expanded stem cell pool. In contrast to the predominantly asymmetric cell divisions of stem cells observed in control culture conditions, stem cells cocultured with endothelial cells divide symmetrically, similar to their preneurogenic behavior in vivo. Strikingly, endothelial-expanded stem cells generate thousands of neurons as well as glia when transwells are removed, triggering differentiation (24) (see the figure).

Promoting neural stem cell self-renewal: Endothelial cocultured stem cells (Endo) have massive neuron production compared to production under control conditions (CTX), shown by β-tubulin-III expression (in green). From (24).

This effect was seen with embryonic and adult neural stem cells, indicating that endothelial coculture is a powerful approach for enhancing neuron production from a variety of stages. Enhanced neuron production could result simply from increased stem cell number. Alternatively, endothelial factors could increase the neurogenic potential of individual stem cells. To analyze these possibilities, we subcloned cultures to examine secondary clone formation. Endothelial-expanded cells generate more neurospheres, more secondary clones, and a higher percentage of neurons in secondary clones than do control cells, suggesting that endothelial factors both enlarge the stem cell pool and enhance its neurogenic capacity. Moreover, the repertoire of neuron production is preserved: Endothelial factors stimulate massive expansion of early embryonic cortical stem cells and are still able to generate pyramidal neurons that are normally made in the early embryo. Thus, by promoting symmetric rather than asymmetric cell division, the aging of neural stem cells can be halted. Interestingly, stem cells from older embryonic stages or adults have limited ability to generate projection neurons even after exposure to endothelial factors, indicating that (unfortunately) they cannot reverse the aging process.

Our results show that endothelial cells are critical components of the neural stem cell niche. We speculate that endothelial factors promote neural stem cell-cell interactions, which maintain Notch activation and stimulate self-renewal (25-27). Indeed, we found that self-renewal was disrupted when Notch activity was inhibited by γ-secretase inhibitor II. In PNS stem cells, stimulation of Notch leads to enhanced gliogenesis (28) rather than neurogenesis; perhaps downstream effectors could explain this difference. Neural stem cells expanded in the presence of endothelial factors show increased expression of the Notch effector Hes1, which has been linked to self-renewal (29, 30), but not of Hes-5, which has been linked to gliogenesis (31). It will be worthwhile to explore whether Notch signaling interacts with other key factors in stem cell maintenance and differentiation such as BMP and Wnts to preserve self-renewal and neurogenesis of neural stem cells.

Understanding the interaction between neural stem cells and their niche microenvironment is an important step in the process of developing effective therapies using endogenous or transplanted stem cells. By discovering that endothelial cell factors promote exponential expansion of neural stem cells, we hope to open new avenues of exploration into the neural stem cell niche and the mechanisms of self-renewal, and to provide a better means of harnessing the great potential for which these small cells deserve so much respect.

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