Stem Cell Biology in the Central Nervous System

The Subventricular Zone

In the early 1990s Reynolds and Weiss discovered cells in the adult mouse brain that proliferated and differentiated into neurons and astrocytes. These cells were also immunopositive for nestin, an intermediate filament of neuroepithelial stem cells prevalent prior to differentiation. These results led to the identification of the subventricular zone (SVZ) as one of the neurogenic niches present in the adult brain. Within the SVZ is a layer of proliferative cells along the lateral aspect of the lateral ventricle ( Fig. 64.2 ).

These cells express glial fibrillary acidic protein (GFAP), a classic marker for astrocytes, and polysialylated neural cell adhesion molecule (PSA-NCAM). Separating the GFAP ⁺ /PSA-NCAM ⁺ cells from the ventricular lumen is a layer of ependymal cells, with most of the astrocytes extending minute apical processes directly contacting the ventricle. Astrocytes from the SVZ differentiate into immature neurons called neuroblasts that travel via a migratory pathway to the olfactory bulb, where they differentiate into neurons. This pathway is known as the rostral migratory stream (RMS). The astroglial cells of the SVZ also generate astrocytes, oligodendrocyte precursor cells (OPCs, discussed later) and myelinating oligodendrocytes in response to chemical demyelinating lesions. This multipotential nature of SVZ astrocytes earns them the classification of NSCs. FLOAT NOT FOUND

Models of the cytoarchitecture of the SVZ have been in a state of flux, but the basic format has been determined. The primary precursors in vivo are the slowly dividing astrocytes mentioned earlier, called type B cells. B cells divide asymmetrically, meaning that mitosis results in two different daughter cells. One is an SVZ astrocyte, just like the parent cell. The other is a short-lived transit-amplifying cell called a type C cell. C cells are antigenically distinct from B cells in that C cells express neither GFAP nor PSA-NCAM. After a brief period of increased mitotic activity, C cells ultimately give rise to migrating neuroblasts (type A cells). The neuroblasts travel from the SVZ via the RMS to the olfactory bulb, where they generate two types of inhibitory interneurons: granule cells and periglomerular neurons. C and A cells in the SVZ and subgranular zone (SGZ) can be identified by their expression of the microtubule-associated protein doublecortin and can be used as a marker to reflect levels of neurogenesis. The transcription factor Tbr2 is also expressed in early postmitotic neurons and their intermediate progenitors.

In addition to their lineage relations (i.e., B cells giving rise to C cells giving rise to A cells), all three types are closely associated spatially. As neuroblasts migrate toward the olfactory bulb, they coalesce to form a network of chains moving rostrally. ^, They do so through cellular tunnels consisting mainly of type B cells with occasional C cells interspersed among them.

Newly born neurons in the adult contend with markedly different circumstances than those of the embryonic brain. Adult neuroblasts migrate through more intricate terrain, frequently over longer distances. They do so first in a tangential fashion toward the olfactory bulb, then radially away from the RMS. In order to execute this switch, the neuroblasts must detach from the migrating chain, a process regulated by the extracellular matrix protein tenascin-R. Tangential and radial migration combined takes place in less than a week, after which the cells must integrate into fully functional circuits. For this reason, it is likely that the differentiation patterns do not simply recapitulate development. Soon after migration, the neurons display spontaneous activity, and over the course of the next 5 to 10 days, spiking activity. Overall it takes weeks for cells to be born in the SVZ, migrate rostrally in the RMS, migrate radially after reaching the olfactory bulb, differentiate, and fully integrate into the existing neuronal circuitry. Even then, a large fraction of the cells (approximately 50%) will not survive without a sufficient level of sensory input.

In early studies of adult neurogenesis, some researchers suggested that the ependymal cells in the periventricular region comprise the NSC population. Indeed, NSCs have been shown to contain a prominent cilium, albeit smaller and more specialized than that of the motile cilia characteristic of brain ependyma. However, studies involving antimitotic drugs and viral labeling, among others, strongly suggest that it is, in fact, the type B cells that comprise the NSC population, and this is the generally accepted view. Some work suggests that one of the roles of the ependymal cells in adult neurogenesis is to produce noggin, an antagonist of bone morphogenetic proteins (BMPs). Because BMPs are known to inhibit neurogenesis as well as induce astrogliogenesis, noggin production by ependymal cells may be a means to maintain the neurogenic niche. Although the presence of the SVZ in humans is clear (reviewed later), the existence of the RMS is controversial. ^,

The Subgranular Zone

The second principal site of adult neurogenesis exists in the hippocampal formation, in a region known as the subgranular zone (SGZ) ( Fig. 64.3 ).

The SGZ lies between the dentate gyrus and the hilus of the hippocampus. In this region of the brain, the primary precursors of new neurons are, as in the SVZ, astrocytes. These astrocytes are also referred to as B cells, but much of the rest of the terminology for the SGZ cytoarchitecture differs from that of the SVZ. In the hippocampus, B cells give rise to a set of immature D cells that divide less frequently and are smaller and more differentiated than the C cells of the SVZ. Because D cells are less mitotic than transit-amplifying cells, the increase in production of these intermediates is probably restricted. The D cells then generate the excitatory granule neurons that migrate to the granule cell layer (GCL) of the hippocampus. These newly formed neurons integrate into a circuitry that is relatively close to their astrocytic forebears as compared with the distances that cells born in the SVZ must migrate to reach their ultimate destination in the olfactory bulb. These fully differentiated neurons then extend axonal projections called mossy fibers to hippocampal region CA3, a subfield implicated in learning and memory processes. ^, The newly formed neurons are integrated into the hippocampal circuitry within a week, forming excitatory synaptic connections with both interneurons and CA3 pyramidal cells. As in the SVZ, the various cell types can be distinguished on the basis of expression of certain molecular markers. Whereas SGZ astrocytes express GFAP, D cells and their progeny (i.e., neurons) do not, expressing PSA-NCAM instead. FLOAT NOT FOUND

The rate at which the hippocampus produces new neurons varies considerably, depending on age, internal factors such as neurotransmitter levels, and external factors such as exercise, stress, and sleep. Thousands of new cells may be produced each day, but it is only a very small percentage that ultimately survives, differentiate fully, and integrate into neural networks.

Neurogenesis in Other Areas of the Brain

Several studies have suggested that neurogenesis also occurs to varying degrees in areas outside of the SGZ and SVZ niches, particularly in the context of injury. Striatal astrocytes, for example, have been shown to give rise to neuroblasts in the post-stroke rodent brain, and cerebral cortical astrocytes have been shown to form self-renewing and multipotent neurospheres through an SHH-mediated pathway in vitro in response to invasive (i.e., stab) injury. Similarly, ependymal cells within the rodent hypothalamus can be seen to exhibit neurogenic potential in response to a high-fat diet. ^,

Regulation of Neurogenesis

Several factors have been shown to regulate the birth and integration of new neurons in rodents, including environmental cues, learning-related stimuli and neuronal activity. For example, postnatal unilateral olfactory deprivation results in significant reduction in bulb volume, an effect that is reversed with sufficient restoration of stimulation. Furthermore, the exposure of a mouse to a complex odor environment results in both an increase in incorporation of newborn neurons and enhancement of short-term olfactory memory. ^, Other physiologic states such as pheromone stimulation and pregnancy also modulate olfactory bulb neurogenesis. ^, Other stimuli regulating neurogenesis include exercise, learning, memory, environment, and stress. While most of these are positive regulators, stress and the associated changes in corticosteroid levels result in a decline in neurogenesis and memory. For instance, stress in adult marmoset monkeys results in a decrease in the number of proliferating granule cell precursors in the dentate gyrus.

Neurogenesis in the hippocampus is also subject to activity-dependent regulation. Because of the diversity of inputs to and complexity of connections in the dentate gyrus, ^, this control may be more elaborate in the SGZ than the SVZ. One of the key functions of the dentate gyrus is the formation of distinct representations of contexts, places, and episodes, a role that may render the region sensitive to the environment and/or cortical activity. The dentate gyrus, as part of the limbic system, also modulates emotional processes such as stress and depression. Consistent with these roles, hippocampal neurogenesis is regulated by the environment, cognitive and emotional processes, and exercise.

On the cellular and molecular levels, γ-aminobutyric acid (GABA) and glutamate exert numerous effects on neurogenesis, including in the realms of proliferation, survival, and integration. In addition, a plethora of growth factors and extracellular matrix components influence the birth and maturation of new neurons in an activity-dependent manner.

Astrocytes as Stem Cells

At first it may seem surprising that an astrocyte, a purported terminally differentiated cell, would serve as the de facto stem cell of the CNS. Stem cells are by conventional logic specialized to be unspecialized—that is, undifferentiated. That astrocytes can function both as mature glial cells and as precursors to other CNS cell types indicates a more complex function set for these cells than previously appreciated. For a cell that was once thought to simply be “glue” or to be a relatively uninteresting default cell fate, astrocytes are turning out to play an increasingly wide range of roles in the brain, an attribute appropriate for the most numerous cell type in the CNS.

Current evidence suggests that B cells are derived from radial glial cells, the principal NSCs in the embryonic mammalian forebrain. Both are NSCs, occupying the same periventricular region of the brain, and both contact the ventricle proper via an apical process. Radial glia also possess a basal process that extends to the pial surface, and some adult SVZ GFAP ⁺ cells also have such a process.

On closer inspection, it turns out that astrocytes are in some ways ideally suited to fulfill the role of primary progenitor. Their numerous processes contact many cell types and the basal lamina of blood vessels, and interastrocyte connections exist in the form of gap junctions. These structural features poise astrocytes to integrate signals from a variety of sources to effectively regulate the stem cell niche. Astrocytes are also able to produce signals that drive neurogenesis in vitro. ^,

Parenchymal astrocytes in regions other than the SVZ and SGZ do not appear to be neurogenic in vivo, although astrocytes derived from the cortex, cerebellum, and spinal cord in the first 2 postnatal weeks can all give rise to neurospheres in vitro.

Functional Significance of Neurogenesis

A growing body of evidence points to neurogenesis as a critical element in an array of adult brain functions. NCAM-deficient mice show a 40% reduction in bulb size that is restricted to the GCL and results in impaired odor discrimination. Antidepressants increase neurogenesis in the hippocampus, but only after chronic, not acute, treatment. An abundance of evidence suggests that adult neurogenesis is directly involved in learning and memory processes, demonstrating plasticity distinct from that occurring at the level of the synapse.