Role of astrocytes as neural stem cells in the adult brain

Abstract

In the adult mammalian brain, bona fide neural stem cells were discovered in the subventricular zone (SVZ), the largest neurogenic niche lining the striatal wall of the lateral ventricles of the brain. In this region resides a subpopulation of astrocytes that express the glial fibrillary acidic protein (GFAP), nestin and LeX. Astonishingly, these GFAP-expressing progenitors display stem-cell-like features both in vivo and in vitro. Throughout life SVZ astrocytes give rise to interneurons and oligodendrocyte precursors, which populate the olfactory bulb and the white matter, respectively. The role of the progenies of SVZ astrocytes has not been fully elucidated, but some evidence indicates that the new neurons play a role in olfactory discrimination, whereas oligodendrocytes contribute to myelinate white matter tracts. In this chapter, we describe the astrocytic nature of adult neural stem cells, their organization into the SVZ and some of their molecular and genetic characteristics.

INTRODUCTION

In 1931, Santiago Ramon y Cajal stated “The Nature has granted us with a limited endowment of cerebral cells. This is a resource, large or little, which nobody can increase because the neuron is unable to reproduce itself” (Cajal’s speech for the “Archivo de la Palabra”, Spain). This statement soon became a dogma that was passionately supported by many other prominent neuroscientists. However, at the 1960’s, Joseph Altman (Altman, 1962) challenged this assumption by showing that the postnatal brain was not a quiescent organ incapable of neurogenesis. In a series of experiments Altman and colleagues, as well as other groups, described the presence of neurons labeled with tritiated thymidine (3H-TdR) in the subependymal zone lining the lateral wall of the lateral ventricles, which suggested the presence of neurogenesis in the adult brain (Altman and Gopal, 1965; Altman and Das, 1967; Kaplan and Hinds, 1977). Later studies from other groups also described ongoing neurogenesis in female canaries (Goldman and Nottebohm, 1983) and lizards (Pérez-Cañellas and García-Verdugo, 1996). Since 3H-TdR may also induce cell cycle arrest and apoptosis, these initial results were somehow criticized and could not unequivocally determine the presence of neurogenesis in the adult brain. This controversy was finally finished by Arturo Alvarez-Buylla and coworkers, whom in serial experiments not only demonstrated the presence of new born neurons (neuroblasts) in vivo, but also proved that the primary progenitor of these neuroblasts is a subpopulation of astrocytes resident in the lateral walls of the lateral ventricles of the adult mammalian brain. These newly generated cells migrate anteriorly and form functional circuits with the resident neurons in the olfactory bulb (Lois and Alvarez-Buylla, 1993, 1994b; Lois et al., 1996; Doetsch et al., 1997; Wichterle et al., 1997; Doetsch et al., 1999b; Sanai et al., 2004; Spassky et al., 2005; Jackson et al., 2006; Merkle et al., 2007).

Since the discovery of neurogenesis in the adult mammalian brain, a lot of research has been done in several brain regions to determine the presence new born neurons but, so far, it is well-accepted that this process is mainly confined to the subventricular zone (SVZ) at the forebrain and the subgranular zone (SGZ) in the dentate gyrus at the hippocampus (Reznikov, 1991; Luskin, 1993; Lois and Alvarez-Buylla, 1994a). The SVZ is the largest neurogenic niche in the adult mammalian brain (Luskin, 1993; Alvarez-Buylla and Garcia-Verdugo, 2002). In this region resides a subpopulation of glial cells with stem-cell-like features, both in vivo and in vitro, which can give rise to neurons, oligodendrocytes, NG2-glia and astrocytes (Doetsch et al., 1999c; Laywell et al., 2000; Imura et al., 2003; Morshead et al., 2003; Garcia et al., 2004; Menn et al., 2006; Gonzalez-Perez et al., 2009; Gonzalez-Perez and Quinones-Hinojosa, 2010; Gonzalez-Perez and Alvarez-Buylla, 2011). The role of the new born neurons and oligodendrocytes derived from the adult SVZ has not been fully elucidated, but some evidence indicates that the new neurons play a role in odor discrimination tasks (Gheusi et al., 2000), whereas SVZ oligodendrocyte precursors contribute to maintain the population of oligodendrocytes in the white matter (Menn et al., 2006; Gonzalez-Perez et al., 2009; Gonzalez-Perez and Alvarez-Buylla, 2011).

In contrast to the SVZ progenitors, the precursor cells found in the SGZ of dentate gyrus in the hippocampus appear not to be bona fide neural stem cells, because they only give rise to neurons in vivo. In fact, some studies showed that adult SGZ progenitors were not multipotent self-renewing progenitors (Seaberg and van der Kooy, 2002; Bull and Bartlett, 2005). Therefore, SGZ neurogenic progenitors are not considered neural stem cells, but neuronal precursor cells (Song et al., 2002; Jagasia et al., 2006). In this chapter, we discuss the characteristics and functions of adult neural stem cells per se, as well as, we describe the evidence that demonstrated the astrocytic identity of multipotent cells resident within the adult SVZ.

Neural stem cells in the adult brain

Early experiments in vitro found that tissue harvested from the SVZ contained a subpopulation of multipotent precursor cells, which under growth-factor-enriched conditions were able to produce self-renewing clones (Reynolds and Weiss, 1992; Morshead et al., 1994; Weiss et al., 1996). These clones proliferated and gave rise to spherical cell aggregates known as ‘neurospheres’, which if subsequently subjected to growth factor removal and serum-free conditions, formed neurons, oligodendrocytes, and astrocytes, suggesting that these self-renewing cells are also multipotent. In consequence, the neurosphere assay has been extensively utilized as a method to indicate the presence of stem cells in tissue samples. However, more recent evidence indicates that this assay might not accurately identify stem cell capacity in vivo. Therefore in vivo experiments are always required to fully demonstrate the ‘stemness’ potential in the mammalian brain. The capacity of SVZ progenitor cells to behave as putative stem cells in vivo was subsequently demonstrated in rodents and humans (Doetsch et al., 1999b; Laywell et al., 2000; Sanai et al., 2004).

The presence of bona fide neural stem cells in the adult brain has been demonstrated through multiple experimental approaches. Primary precursors were identified in vivo by using deoxythymidine or bromodeoxyuridine (BrdU) as cell proliferation markers. Thus, it was found that a subpopulation of astrocytes remain labeled into the SVZ and SGZ after long survival times, which suggested that these glial cells corresponded to stem cells (Doetsch et al., 1999b; Alvarez-Buylla et al., 2002). These multipotent astrocytes are denominated as type-B cells, which typically express the glial fibrillary acidic protein (GFAP) and have very well-defined ultrastructural characteristics (see Chapter 5). The first evidence indicating that SVZ astrocytes are neural stem cells was provided by Fiona Doetsch et al., she discovered that a subpopulation of astrocytes that survived to an intracerebroventricular administration of a cytotoxic drug (cytosine-β-D-arabinofuranoside, Ara-C) was able to fully regenerate all germinal progenitors within the SVZ over a period of 14 days (Doetsch et al., 1999b; Doetsch et al., 1999a). After that, also using Ara-C to identify the primary progenitor in the SGZ, Betina Seri et al. found that SGZ radial astrocytes (named type-D cells) were capable of reconstituting the germinal layers in the dentate gyrus (Seri et al., 2001). Later reports confirmed these observations indicating that the GFAP-expressing astrocytes are candidate stem cells in the SVZ (Chiasson et al., 1999; Capela and Temple, 2002; Garcia et al., 2004; Spassky et al., 2005). Interestingly, astrocytes collected from multiple brain regions before postnatal day 10 may behave as neural stem cells in vitro. After that time, only the SVZ astrocytes retain this ‘stemness’ capacity (Lim and Alvarez-Buylla, 1999; Laywell et al., 2000).

Organization of the subventricular zone

The subventricular zone, the largest niche of adult neural stem cells, is located along the lateral walls of the lateral ventricles in the forebrain (Figure 1) (Doetsch et al., 1997; Doetsch, 2003). To have a complete view of the SVZ as a neurogenic niche, we have to study not only the cell organization and ultrastructure of the SVZ progenitors, but also their interrelationships with surrounding microenvironment. Thus, some authors have denominated the SVZ niche as the ventricular-subventicular (VZ-SVZ) compartment. For the purposes of this chapter we will refer to this region simply as the SVZ. The adult SVZ has a center-surround cell arrangement, denominated as ‘pinwheel’ organization, for ependymal cells (type-E cells) and astrocytes (type-B cells) within ventricular walls (Mirzadeh et al., 2008). These pinwheel-like compartments contain the apical endings of type-B cells and of multiciliated (type-E1) and bi-ciliated (type-E2) ependymal cells (Figure 1). Remarkably, SVZ astrocytes extend two cell processes: a petite cilium that contact the ventricular lumen and a long basal process attached to blood vessels (Figure 1)(Mirzadeh et al., 2008; Shen et al., 2008). Some authors have proposed that these contacts are important to regulate adult neurogenesis through signals derived from extracellular matrix proteins, soluble factors or blood-vessel secretion (Leventhal et al., 1999; Mercier et al., 2002; Shen et al., 2008; Tavazoie et al., 2008). Some of these chemical mediators include the CXC chemokine, the stromal-derived factor 1 (SDF-1), the epidermal (EGF), the platelet-derived (PDGF) and the fibroblast (FGF) growth factors (Reynolds and Weiss, 1992; Vescovi et al., 1993; Gritti et al., 1995; Craig et al., 1996; Gritti et al., 1999; Jackson et al., 2006; Kokovay et al., 2010).

Figure 1.

The adult subventricular zone (SVZ) is lining the striatal wall of the lateral ventricles in the brain (coronal section left). A 3-D model of the SVZ is shown to the right. This region contains astrocytic neural stem cells (type-B cells depicted in blue). Type-B cells give rise to rapidly dividing intermediate progenitor cells (type-C cells depicted in green), which in turn produce migrating neuroblasts (type-A cells depicted in red). Type-B astrocytes have a long basal process contacting blood vessels (BV) and an apical ending at the ventricle surface. Note the pinwheel-like organization composed of multiciliated (Type-E1) and bi-ciliated (Type-E2) ependymal cells encircling apical surfaces of astrocytes. Reprinted from Brain Research Reviews, Vol 67, Gonzalez-Perez & Alvarez-Buylla, Oligodendrogenesis in the subventricular zone and the role of epidermal growth factor, page 148, Copyright 2011, with permission from Elsevier.

Interestingly, the cell organization of the adult human SVZ is quite different to that found in rodents and most of the mammalians. In the human SVZ, astrocytes are not adjacent to the ependymal, instead, they accumulate into the parenchyma and form a ‘ribbon’ of cells separated from the ependymal layer by a gap that is largely devoid of cell bodies (Sanai et al., 2004; Quinones-Hinojosa et al., 2006).

SVZ astrocytes as neural stem cells

The adult SVZ contains slowly dividing type-B cells, the putative neural stem cells, which are a subpopulation of astrocytes that expresses nestin, GFAP and LeX (Doetsch et al., 1999b; Rietze et al., 2001; Capela and Temple, 2002; Garcia et al., 2004). Type-B astrocytes divide to self-renew and generate highly proliferating transit-amplifying progenitors (type-C cells), which in turn produce neuroblasts (type-A cells) (Figure 1 and 2) (Doetsch et al., 1997; Doetsch et al., 1999b). The cytoplasmic processes of type B cells sheath type-A cells and contribute to form tangential neuroblast chains. The confluence of neuroblast chains forms a brain structure, called the rostral migratory stream (RMS), which goes through the olfactory tract and connects the anterior SVZ with the olfactory bulb (Figure 2) (Lois and Alvarez-Buylla, 1994b; Lois et al., 1996). Interestingly, type-A cells follow a gradient of signaling proteins dissolved in the cerebrospinal fluid (Sawamoto et al., 2006; Gonzalez-Perez, 2012). Once in the olfactory bulb, type-A cells differentiate into mature neuronal cells that continually replace interneurons in the glomerular and the mitral cell layers (Lois and Alvarez-Buylla, 1993, 1994b; Merkle et al., 2007).

Figure 2.

Cellular organization of the adult SVZ, rostral migratory stream (RMS) and the olfactory bulb. Neuroblast (type-A cells) in the SVZ migrate to the olfactory bulb via the RMS. Migrating neuroblasts differentiate into granular and periglomerular interneurons in the olfactory bulb. B1: Type-B1 cell; C: Type-C cell; A: Type-A cell; V: Ventricle; CC: Corpus callosum; RMS: Rostral migratory stream; SVZ: Subventricular zone. Reprinted from Current Immunology Reviews, Vol 6, Gonzalez-Perez et al., Immune System Modulates the Function of Adult Neural Stem Cells, Page 169, Copyright 2010, with permission from Bentham Science Publishers Ltd..

Remarkably, type-B stem cells are not a homogeneous population; instead, it seems they comprise restricted subpopulations with diverse neurogenic potential (Merkle et al., 2007). In addition to interneurons, type-B astrocytes of the SVZ produce oligodendrocyte precursor cells that help maintain the oligodendrocyte population in the corpus callosum, fimbria fornix and striatum (Menn et al., 2006; Gonzalez-Perez et al., 2009; Gonzalez-Perez and Quinones-Hinojosa, 2010; Gonzalez-Perez and Alvarez-Buylla, 2011).

The identification of type-B cells in the SVZ as primary neural progenitors of new neurons and oligodendrocytes was demonstrated by labeling SVZ astrocytes in transgenic mice denominated as GFAP-tva mice (Figure 3) (Doetsch et al., 1999b; Doetsch et al., 2002; Menn et al., 2006). These animals express the receptor for an avian leukosis retrovirus (RCAS) controlled by the GFAP gene promoter (Holland and Varmus, 1998). Thus, the progeny of dividing astrocytes can be permanently traced by injecting engineered avian RCAS retroviruses carrying an inheritable reporter gene, which drives the expression of green fluorescent protein. With this method, it has been demonstrated that type-B astrocytes in vivo can generate both new neurons and new oligodendrocytes, and both of them become permanent and functional populations of cells in the adult brain (Doetsch et al., 1999b; Menn et al., 2006; Gonzalez-Perez et al., 2009). Interestingly, parenchymal astrocytes from cortex, striatum, septum or white matter appear not to have multipotential properties.

Figure 3.

Schematic representation of the RCAS/GFAP-tva system. After injection of the RCAS-GFP virus, proliferating astrocytes incorporate into their genome the gene of green fluorescent protein, which is inherited to their progeny. With this system only dividing GFAP-expressing cells are permanently label and can be efficiently traced throughout life. Reprinted from Brain Research Reviews, Vol 67, Gonzalez-Perez & Alvarez-Buylla, Oligodendrogenesis in the subventricular zone and the role of epidermal growth factor, page 150, Copyright 2011, with permission from Elsevier.

After these initial reports, other studies using viral transfection or mutant mice have fully demonstrated that GFAP-expressing astrocytes are neural stem cells in the adult mammalian brain. Conditional ablation of GFAP-expressing cells produces a complete loss of neurogenesis (Imura et al., 2003; Morshead et al., 2003). Interestingly, a recent study using a Cre-lox-based strategy demonstrated that radial glial cells (multipotent neural precursors in the developing brain) and SVZ zone astrocytes (multipotent neural precursors in the adult brain) belong to the same lineage (Merkle et al., 2004). This study showed that radial glial cells generate SVZ astrocytes, which act as adult neural stem cells both in vivo and in vitro. Taken together, these findings confirm that SVZ astrocytes function as neural stem cells in the adult brain

CONCLUSION

A subpopulation of astrocytes derived from the SVZ acts as neural stem cells both in vivo and in vitro. These findings have changed our perception about glia and though we have greatly advanced our understanding of neural stem cells, there are still many unanswered questions. For instance, what are the fundamental characteristics that distinguish neurogenic astrocytes from the vast population of non-neurogenic astrocytes elsewhere in the brain? What genetic profiles induce SVZ astrocytes to act as a stem cell? Can SVZ astrocytes be induced to generate a specific repertoire of neural cell types? Addressing these questions may shed light on the genetic and molecular basis of stem-cell behavior in SVZ astrocytes, which in turn will allow designing novel therapies against neurodegenerative diseases.