Comparison of in vitro cultures of astrocytes from different sources

Many subtypes of astrocytes are known. Unfortunately for in vitro astrocyte research, most of them are distinguished only by brain regions (Reichenbach and Wolburg 2005). Little is known about the differential expression of marker sets or about different functionalities of the diverse astrocytes subtypes in vivo. Further research into the phenotypic and functional differences of astrocytes in different regions of the brain is needed to allow subtype specification in in vitro derived astrocytes. Recently, Krencik et al. succeeded in differentiating human ESC into neural epithelial progenitor cells. These neural epithelial progenitors were then differentiated to progenitor cells in the presence of the patterning factors FGF8 (anterior) or retinoic acid (posterior). After

in stem cell-derived astrocytes. The exact subtype identity of our MEDA cultures remains to be elucidated.

Figure 4-4: Methods to derive astrocytes from embryonic stem cells developed in this thesis.

Two methods to derive functional astrocytes from murine ESC were developed in this thesis. (A) Mouse embryonic stem cell derived astrocytes (MEDA), generating GFAP-positive and GFAP-negative astrocytes, were differentiated according to a two-step protocol. In the first step, neural induction was triggered in suspension cultures for 21 days. Afterwards, the neural cell-containing aggregates were further differentiated adherently to astrocytes. The protocol generated also GFAP-negative astrocytes which, for the first time, allowed the study of functional properties of GFAP-negative astrocytes in vitro.

(B) The second protocol relied on a different strategy: First, neural progenitor cells (NPC) were derived from embryonic stem cells. These NPC can be expanded in vitro and cryopreserved or induced to differentiate into astrocytes within 3-5 days, generating pure populations of non-dividing, GFAP-positive astrocytes. gel: gelatine; PorL: poly-L-ornithin/laminin; A: no cytokines; B: EGF/bFGF; C:

CNTF/BMP4.

It is important to compare stem cell-derived astrocytes with primary isolated astrocytes, being the “gold standard” in the field. MEDA shared many of the tested functional aspects such as inflammatory activation and metabolic competence with primary astrocytes, however their staining pattern was different. Similar to primary astrocytes, the ability of the MEDA cultures to respond to CCM, and to remain quiescent without stimulation, was retained upon replating or thawing after cryopreservation (Crocker et al. 2008; Falsig et al. 2004; Henn et al. 2011).

disease, however, as mentioned before, they most likely represent a subpopulation of all astrocytes capable to grow in culture conditions they are isolated with. Primary astrocytes proliferate in vitro. MEDA obtained by the first protocol proliferate and also express nestin. This is indicative of an either activated or immature phenotype (or both). Astrocytes derived from ESC via NPC neither proliferated nor expressed nestin, which indicates a more mature phenotype. Nevertheless, the high levels of GFAP expression in these cells indicates an either immature or an activated phenotype. In vivo, high expression of GFAP is associated with astrogliosis. However as of now, this is only speculation and requires further analysis. We initiated a project that compares the mRNA expression levels in astrocytes from various sources to gain some insight into the degree of maturity of in vitro astrocytes and also to identify new suitable astrocyte markers (Götz et al., personal communication).

4.5.4 Inflammation in neurotoxicity and brain disease

Astrocytes are implicated in degenerative processes in the brain. Activation of astrocytes in a diseased brain or by exogenous substances can lead to (chronic) inflammation. An overshooting activation can result in a strong immune response including cytokine secretion ROS formation (Figure 4-5) (Dong and Benveniste 2001).

This process called astrogliosis is usually observed in patients with chronic brain diseases like AD or PD (Carpentier et al. 2008; Falsig et al. 2008).

Apart from inflammation in disease, astrocytes can also become activated upon exposure to chemicals. For example, trimethyltin derivatives induce astrogliosis in astrocytes which leads to neurotoxicity in vitro (Cristòfol et al. 2004; Holden and Coleman 2007).

Insights on a molecular detail of the contribution of astrocytes to the development of degenerative diseases have been gained in studies of the fragile X

generation of amyotrophic laterals sclerosis (ALS). This disease is characterised by a progressive loss of peripheral motor neurons. Astrocytes carrying a mutated form of superoxide dismutase (SOD1) that has been linked to ALS before (Rosen et al. 1991), were shown to secrete soluble factors that selectively kill motor neurons in co-cultures, thereby causing the disease (Di Giorgio et al. 2008; Nagai et al. 2007).

In cases where astrocytes contribute to neurotoxicity or to complex degenerative diseases such as ALS, AD or PD, standard in vitro neuronal cultures will fail as a model, because the complex interaction of the two (or more) cell types can not be studied in detail. Assays that detect neurotoxicity using co-cultures of neurons and astrocytes have already been developed (Anderl et al. 2009; Woehrling et al. 2007) and report a generally higher resistance to toxicants of neuron, when co-cultured with astrocytes. Also, as stated before, the presence of contaminating microglia, as often described for primary astrocyte preparations (Saura 2007), would hamper functional studies as microglia can produce similar inflammatory mediators as astrocytes .

Our microglia free MEDA were developed in order to use them in co-culture models to study aspects of brain inflammation and the development of neurotoxicity (Figure 4-5). Our DNT-test system based on neurons differentiated from mESC (Chapter 3.2) also contained “contaminating” astrocytes, and therefore astrocyte-mediated influences on toxicity or disease generation might also be picked up in these cultures. However, the few astrocytes were restricted to small astrocytic “islands” in the culture well and were not evenly distributed. The generation of defined co-cultures of neurons and astrocytes is therefore preferable and would yield more consistent results when studying the interplay of astrocytes and neurons in co-cultures.

4.5.5 Astrocyte metabolism: the double edged sword

Everything comes at a price. In the healthy brain, the metabolic activity of astrocytes for maintaining physiological conditions is crucial for a functioning brain.

Occasionally however, the metabolic activity of astrocytes can result in toxic metabolites. We used a substance commonly used for the induction of a Parkinson disease phenotype in mice as a model substance to demonstrate the metabolic functions of our astrocytes. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a

enzyme monoamineoxidase B (MaoB) into 1-methyl-4-phenyl-1,2-dihydropyridinium (MPDP⁺) which spontaneously forms the potent neurotoxin 1-methyl-4-phenyl-pyridinium (MPP⁺). Using ESI-MS we showed that astrocytes differentiated from ESC converted MPTP and released MPDP⁺ and MPP⁺ into the supernatant, underlining their suitability for toxicological studies (Chapter 3.3 and 3.4). Similar data has also previously been reported for primary astrocytes (Schildknecht et al. 2009).

Figure 4-5: Astrocytes in neurotoxicity and developmental neurotoxicity.

The involvement of glial cells (astrocytes and microglia) in the generation of indirect neurotoxicity is indicated. Astrocyte activation can lead to an overshooting immune response that can lead to neuronal damage or cell death. Soluble factors and reactive oxygen species secreted by activated glial cells are

neurotoxicity testing. If omitted, substances that are converted into toxins by astrocytes will be overlooked in neuronal cultures devoid of astrocytes. Besides, their metabolising activity is important for detoxification of neurotoxins (e.g. removal of excess extracellular glutamate), and testing in cultures devoid of astrocytes could lead to hazard overestimation, because the substance might not be eliminated by metabolic activity or their concentration would not be reduced to non-cytotoxic levels in absence

of astrocytes, as it would be the case in vivo. In vitro neurotoxicity assays based on astrocyte-neuronal co-cultures should complement conventional testing with neuron mono-cultures, to guarantee a better safety assessment of potential toxicants.

I have demonstrated that astrocytes derived from stem cells can serve as a substitute for primary isolated astrocytes. They behaved similar in all functional characteristics tested and, using the protocols I developed, can be obtained in large amounts under chemically defined conditions. Furthermore, they can be cryopreserved without loss of functionality, which makes the preparation of large batches possible, which makes them ideal candidates for next generation assay development.

The future challenge is now to transfer the methods I developed to human ESC (hESC) and to delineate functional astrocytes. In human development, astrocytes do not appear before the 3^rd months of development. In vitro generation of astrocytes from hESC can therefore be expected to take longer than from the mouse (Liu and Zhang 2011). In a very promising approach, Krencik et al. recently differentiated hESC to neuroepithelium and then into astroglial progenitor cells. These progenitor cells were expanded in suspension cultures as aggregates and were differentiated to astrocytes using CNTF and LIF or serum. Apart from phenotypic marker expression, these hESC-derived astrocytes also showed functional characteristics of astrocytes (Krencik et al.

2011; Liu and Zhang 2011). However, the differentiation phase required over 180 days, which raises doubts as to their practical applicability in the near future.

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Im Dokument Generation of astrocytes from embryonic stem cells (Seite 169-197)