2.2.1 Astrocytes – underappreciated cells of the brain
There are four major cell types in the brain: neurons, microglia, oligodendrocytes and astrocytes. The latter two are also often referred to as (macro-)glial cells. The primary function of neurons is the intercellular signal transmission by electric and chemical signalling and between synapses. Oligodendrocytes myelinate axons of neurons to enable faster and better electric signal conduction. Therefore, they are crucial for signal transmission of neurons and play a role in neurodegenerative and inflammatory diseases like multiple sclerosis (Compston and Coles 2002).
In contrast to neurons, oligodendrocytes and astrocytes, microglia do not originate from the neuroectoderm but are of mesodermal origin and are closely related to blood cells (Prinz and Mildner 2011). They correspond to macrophages in peripheral tissues and act as primary defense line against pathogen invasion.
Astrocytes are generated later in development than neurons (Miller and Gauthier 2007). The first glial precursor cells (A2B5+) have been observed in rodents at around embryonic day 13.5 (E13.5), and are known to generate various phenotypically-different cell populations (Bignami et al. 1972; Lendahl et al. 1990;
Miller and Szigeti 1991). The first astrocytes positive for glial fibrillary acidic protein (GFAP; a marker for astrocytes) are found on E16, develop further beyond birth, and do not reach maturity until several weeks later.
While neurons have been extensively studied, and much is known about neuronal subtypes and their specific functions, only little is known about the diversity
levels, astrocytes differ in function and morphology and regional specific subtypes of astrocytes are known (Allen and Barres 2009; Kimelberg 2004; Kimelberg and Nedergaard 2010; Matyash and Kettenmann 2010; Walz 2000; Wang and Bordey 2008). Astrocytes of the white matter are recognised by their typical elongated and fibrillary structures, while protoplasmic astrocytes of the grey matter usually adopt a flat morphology with multiple branches (Kimelberg 2004).
As far as we know today, astrocyte heterogeneity is dependent on brain region, local cellular environment, the activation state and age. The exact definition of astrocyte subtypes is difficult due to a lack of markers. Therefore, only regional differences or differences in marker expression patterns can be observed. The same population can express a different set of markers after partial activation. This is further complicated by the lack of a pan-astrocytic marker that is present in all astrocyte subpopulations. This impedes cell type identification in mixed cultures.
2.2.2 Astrocyte function
The crucial role of astrocytes in brain function has long been underestimated. After their discovery, they were named “astrocyte” (greek: astro = star) due to their star-shaped morphology. Astroglial cells (glia = “glue” in greek) were historically thought to merely provide structural guidance in the developing brain (Kettenmann and Ransom 2005).
In recent years however, knowledge about astrocyte functions has greatly improved. Today it is known that astrocytes play vital roles in almost all processes in the brain. In the developing brain they provide guidance cues for migratory neuronal progenitor cells and are involved in the physical structuring of the brain. Using their endfeet, they interconnect with neurons and are also tightly associated with the blood brain barrier (BBB) (Bauer et al. 2005). Together with endothelial cells of the BBB, astrocytes regulate the pore size and fenestration of the BBB. Furthermore, they are responsible for recruiting lymphocytes through the BBB into the inflamed brain by secretion of chemotactic proteins, such as IL-8 (Aloisi et al. 1992). They actively support neuronal signalling by regulating ion homeostasis through ion transport channels (mainly potassium) (Coles and Deitmer 2005). They also directly contribute
instance, astrocytes remove excess glutamate or γ-aminobutyric acid (GABA) from the synaptic cleft, thus preventing toxic accumulations of these neurotransmitters (Swanson 2005). By secreting neurotrophic factors such as IL-6, NGF and nutrients (e.g. lactate) they support neuronal survival. Together with microglia, astrocytes are responsible for the detection of pathogen associated molecular patterns (PAMP), tissue damage and toxic events (Falsig et al. 2008). Upon detection of such noxious events, they switch from a “resting” to an activated state and mount an inflammatory response (reactive gliosis) by secreting soluble factors (e.g. NO, IL-6) (Falsig et al. 2004). An overshooting inflammatory reaction by astrocytes, called reactive gliosis, can lead to severe damage in the brain. Chronic brain inflammation is a common symptom in most neurodegenerative diseases and is usually associated with reactive gliosis (Eng et al.
2000).
Activated astrocytes rapidly proliferate into damaged tissue and build up a
“glial scar”. This can be a problem in traumatic nerve injuries (e.g. paraplegia) as the astrocytic tissue prevents regeneration of neuronal circuits.
In vitro cultures of astrocytes are usually done with astrocytes isolated from the brain of newborn rodents. Astrocytes have been isolated from the brain and cultured in vitro from all developmental stages of rats, mice and humans (Garcia-Abreu et al. 1995;
Lovatt et al. 2007; Zhang and Barres 2010). Most data has however been obtained from neonatal astrocytes, and experiments with adult astrocytes are rarely done and have only become possible in recent years. For mostly historical reasons, most isolation procedures for astrocytes have been optimised for GFAP-positive astrocytes.
Functional astrocytes can be obtained in reasonable quantities from postnatal mice by manual dissection of the cortex, removal of the meninges, and subsequent purification by gradient centrifugation (Weinstein 2001). Thereby, pure cultures of astrocytes virtually devoid of microglial contamination can be obtained (Henn et al. 2011).
However, this procedure requires intensive expert training and is very operator dependent. Furthermore, the quality and purity of the culture has to be determined for each preparation. Normally, mature astrocytes do not divide. However, in the dentate gyrus and the ventricular zone, rapidly dividing populations of astrocyte-like cells can be found that behave like neural stem cells (Buffo et al. 2008; Doetsch 2003; Doetsch et al. 1999; Kriegstein and Alvarez-Buylla 2009; Seri et al. 2001; Seri et al. 2004).
These astrocytic cells are most likely radial glia cells, a special type of neural progenitor cell that can generate subtypes of astrocytes and neurons (Goldman 2003).
Mature astrocytes do not divide however when taken into culture, astrocytes start to proliferate. One possible explanation is that in vitro cultures are most likely generated from a pool of isolated astrocyte progenitor cells which mature in culture.
Furthermore, the origin of these cells is restricted to only a few regions of the brain.
This is supported by the finding that in vitro cultures of astrocytes have an immature phenotype (Zhang and Barres 2010). Progress has been made on the isolation of (more) mature astrocytes (Cahoy et al. 2008; Pihlaja et al. 2011) though maintaining these cells in culture is still difficult. Primary astrocyte cultures are isolated regionally-specific from brain, and thus only reflect a limited population of the diverse set of astrocyte subpopulations. A lack of subtype-markers makes the identification of astrocyte-subtype difficult. The definition of astrocytic subtypes is unclear in terms of marker expression, and is usually based on morphology and the location of the brain
Because of these restrictions, in vitro cultures generated from primary astrocytes most likely do reflect the astrocyte populations in the brain. But, due to a lack of alternatives, they have served as gold standards for in vitro astrocytes and have nevertheless helped to bring light into the mystery of astrocyte function. Nowadays, in vitro cultures of primary astrocyte cultures are routinely used to elucidate the role of astrocytes in brain inflammation (Falsig et al. 2004; Falsig et al. 2006b; Henn et al.
2011) or the contribution to onset and progression of degenerative brain diseases (Di Giorgio et al. 2007; Nagai et al. 2007).
Embryonic stem cells (ESC) can differentiate into all cell types of the body and thus may provide alternative sources of astrocytes (Figure 2.2-1). Apart from ESC, more specialised neural stem also offer access to differentiated astrocytes in vitro.
Neural stem cells are already committed to the neural lineage, and can only give rise to oligodendrocytes, neurons and astrocytes. Astrocytes have been obtained by differentiation from various neural stem cell populations isolated from brain (Gritti et al. 1994; Reynolds and Weiss 1992; Vanhoutte et al. 2004). In a similar way, the generation of astrocytic cultures from human and murine embryonic stem cell (ESC) derived neural stem cells has been described (Conti et al. 2005; Glaser et al. 2007;
Pollard et al. 2006). Attempts to differentiate astrocytes directly from ESC have also been made. For instance, Rao and colleagues report the generation of astrocyte progenitor cells from murine ESC (Mujtaba and Rao 2002) and Kamnasaran and colleagues isolated GFAP-positive astrocytes from differentiated ESC. However, stem cell derived astrocyte cultures are characterised usually only by expression of GFAP and little is known about functional competence of these cultures.