The Generation of Astrocytes from Stem Cells and their Potency to Regain Stem Cell Potential

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Stem Cells and their Potency to Regain Stem Cell Potential

Dissertation submitted for the degree of Doctor of Natural Sciences (Dr. rer. Nat.)

Presented by

Susanne Maria Kleiderman, née Kirner

at the

Faculty of Sciences Department of Biology

Date of the oral examination: 19.04.2016 First referee: Prof. Marcel Leist Second referee: Prof. Thomas Brunner

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-363060

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Abstract ... v

Zusammenfassung ... vii

1 Introduction ... 1

1.1 Astrocytes in general – functions and heterogeneity ... 1

1.1.1 Astrocyte functions ... 1

1.1.2 Heterogeneity of astrocytes ... 6

1.1.3 How to define a cell as an astrocyte? ... 8

1.2 Research with primary astrocytes in vitro ... 9

1.3 The generation of astrocytes from stem cells ... 11

1.3.1 Neural stem cells and the development of astrocytes ... 12

1.3.2 Astrocytes derived from embryonic or induced pluripotent stem cells .... 14

1.4 Astrocytes as stem cells ... 18

1.4.1 The de-differentiation of astrocytes to neural stem cells ... 20

1.4.2 The neurogenic potential of astrocytes ... 23

1.5 Specific aims of this study ... 25

2 Materials and methods ... 27

2.1 Materials ... 27

2.2 Methods ... 30

2.2.1 Maintenance of murine embryonic stem cells (mESC) ... 30

2.2.2 Differentiation of neural stem cells from mESC ... 30

2.2.3 Maintenance and selection of NSC from d7NSC ... 31

2.2.4 Astrocyte differentiation ... 31

2.2.5 De-differentiation of mAGES to NSC2... 32

2.2.6 Neuron differentiation ... 32

2.2.7 Preparation and maintenance of primary astrocytes ... 32

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2.2.8 Quantitative RT-PCR ... 33

2.2.9 Immunocytochemistry and EdU labeling ... 33

2.2.10 High throughput imaging ... 33

2.2.11 Microarray profiling and data analysis ... 34

2.2.12 Protein measurement and LDH release ... 34

2.2.13 Measurement of resazurin reduction ... 35

2.2.14 Glutamate uptake measurement... 35

2.2.15 Culture condition for metabolic flux analysis and metabolite extraction . 35 2.2.16 Measurement of glucose, lactate, and amino acids in supernatant ... 36

2.2.17 Quantification of mass isotopomers by GC-MS ... 36

2.2.18 ¹H-NMR spectroscopy for citrate determination in supernatant ... 36

2.2.19 Cell stimulation, NFkB translocation and IL6 ELISA ... 36

2.2.20 Measurement of CFSE-label dilution in adherent culture ... 37

2.2.21 Live imaging and single cell tracking... 37

2.2.22 Western blot ... 37

2.2.23 Statistical analysis ... 38

3 Results ... 39

3.1.1 Pivotal conditions for the generation of astrocytes from stem cells ... 39

3.1.2 Rapid generation of mature astrocytes from NSC ... 40

3.1.3 Factors affecting differentiation efficacy ... 42

3.1.4 Immunocytochemical phenotyping of NSC and mAGES ... 43

3.1.5 Whole transcriptome-based characterization of mAGES vs. NSC ... 47

3.1.6 Comparison of mAGES gene expression with primary cell populations . 50 3.1.7 Basic metabolic features of mAGES vs. NSC ... 51

3.1.8 Divergent biosynthetic metabolism of NSC and mAGES ... 53

3.1.9 Functional maturity of mAGES concerning innate immune response ... 56

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3.2 The stem cell potential of astrocytes – mAGES generate neurogenic NSC .... 61

3.2.1 The mAGES re-enter the cell cycle when exposed to growth factors ... 61

3.2.2 The mAGES give rise to neural stem-like cells ... 62

3.2.3 FGF2 signaling is required for cell cycle reactivation ... 64

3.2.4 NSC2 are multipotent stem cells ... 68

3.2.5 Functional and phenotypic resemblance between NSC2 and NSC ... 70

3.2.6 NSC2 are generated from mature astrocytes ... 73

3.2.7 Reactive astrocytes fail to de-differentiate ... 77

4 Discussion ... 81

4.1.1 Homogeneity of mAGES in contrast to primary astrocyte cultures ... 81

4.1.2 The mAGES are mature, non-proliferating astrocytes ... 82

4.1.3 Reproducibility and robustness of the mAGES generation ... 84

4.1.4 The generation of human astrocytes ... 85

4.1.5 Functional differences between NSC and mAGES ... 86

4.1.6 Conclusion ... 87

4.2 The stem cell potential of astrocytes ... 89

4.2.1 The mAGES vs. primary astrocytes to investigate de-differentiation ... 89

4.2.2 FGF2 converts mature astrocytes into neural stem cells ... 91

4.2.3 De-differentiated astrocytes resemble neural stem cells ... 93

4.2.4 Inflammatory stimulation inhibits the de-differentiation of astrocytes .... 93

4.2.5 Reactivation studies in human astrocytes ... 95

4.2.6 Conclusion ... 96

References ... 99

Supplementary figures ... 113

Acknowledgements ... 137

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A BSTRACT

Astrocytes are the most abundant cell type in the central nervous system, and they fulfill diverse vital roles under physiological as well as pathophysiological conditions. Recog- nition of new functions, such as the stem cell potential of astrocytes, or their ability to actively regulate neurotransmission, raised renewed interest for these cells in both basic and pharmacological research.

Primary monolayer cultures have become the dominating model system to study astrocyte functions in vitro. These cultures are often heterogeneous, resulting in contradictory studies, e.g., in inflammation, or the misinterpretation of data obtained on ‘mature’ astrocytes, e.g., in transcriptomic profiling.

An alternative approach is the generation of astrocytes from stem cells. We developed here a protocol for the generation of pure populations of mature, non-dividing astrocytes.

These ‘murine astrocytes generated from embryonic stem cells’ (mAGES) express markers specific for mature astrocytes, and perform astrocytic functions, such as the response to inflammatory cytokines, or the (metabolic) support of neurons. The absence of immature cells allowed a direct comparison of mAGES with the neural stem cells (NSC) from which they arise. It is known that astrocytes and NSC share several phenotypic and functional features, but a direct comparison proved difficult due the heterogeneity of primary cultures and the lack of markers, which clearly discriminate astrocytes from NSC. Tran- scriptome data of pure cultures of mAGES and NSC were used to re-evaluate known cell type-specific markers, and to identify new ones. Furthermore, studies on the metabolic profiles of these cells not only confirmed their close relationship, but also revealed clear differences regarding the glutamate/glutamine metabolism as well as citrate release.

In the second part of this study, the properties of mAGES (homogeneity, maturity) were used to study the de-differentiation of astrocytes to neural stem cells. This new research field emerged from the finding that neural stem cells of the adult brain are astrocytic cells, and that reactive astrocytes in pathological environments regain stem cells features. For studies on de-differentiation, homogeneous populations of mature astrocytes are desirable to investigate responsible signaling mechanisms and pathways. We demonstrated here that the addition of a single growth factor under strictly controlled conditions was sufficient to de-differentiate mAGES to neural stem-like cells (NSC2). The mAGES re-entered the cell cycle, when exposed to FGF2, and gave rise to cells expressing the neural

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stem cell marker nestin, while the astrocytic glial fibrillary acidic protein (GFAP) was downregulated. NSC2 were multipotent neural stem cells, which further differentiated into both astrocytes and neurons, and they resembled normal neural stem cells with regard to their phenotype and functions. Downstream of FGF2, the phosphorylation of ERK has been shown to be the main pathway responsible for the cell cycle re-entry of mAGES.

Under pathophysiological conditions, i.e., the exposure of mAGES to inflammatory cytokines, the de-differentiation to NSC2 was disturbed. IFNγ was shown to inhibit the cell cycle re-entry of mAGES by a phosphorylation of STAT1. Ruxolitinib, an inhibitor of the JAK/STAT pathway, was sufficient to recover the de-differentiation of mAGES, even in the presence of IFNγ and other cytokines. These findings may explain, why the de- differentiation is a rare event in pathology models, as the positive stimuli (FGF2) is, under such situation, always coupled with inhibitory, inflammatory events. Thus, mature astrocytes have been shown to de-differentiate into neurogenic neural stem cells, and inducing or inhibiting signals could be identified and characterized in this system.

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Z USAMMENFASSUNG

Astrozyten sind die am zahlreichsten vertretenen Zelltypen im zentralen Nervensystem.

Sie erfüllen eine Vielzahl an unterschiedlichen Funktionen sowohl im gesunden, als auch im erkrankten Nervensystem. Die Entdeckung neuer Funktionen, beispielsweise dem Potential dieser Zellen Stammzellen zu generieren oder ihrer aktiven Beteiligung an der Reizweiterleitung in Nervenzellen, hat erneutes Interesse an der Erforschung dieser Zellen geweckt.

Um die Funktionen von Astrozyten in vitro zu erforschen, werden hauptsächlich primäre Monolayerkulturen verwendet. Diese Kulturen enthalten allerdings häufig unterschiedliche Zelltypen, was zu widersprüchlichen Ergebnissen, wie beispielsweise in der Entzündungsforschung, oder einer Fehlinterpretation von Daten, wie zum Beispiel in Microarray-Studien, geführt hat.

Ein alternativer Ansatz ist die Generierung von Astrozyten aus Stammzellen. Diese Arbeit beschreibt ein Protokoll zur Herstellung von Reinkulturen bestehend aus reifen, sich nicht mehr teilenden Astrozyten. Diese ‚Maus-Astrozyten, generiert von embryonalen Stammzellen‘ (mAGES), zeigen Expressionsmuster sowie Funktionen, die typischerweise in reifen Astrozyten vorkommen, wie zum Beispiel Entzündungsreaktionen oder die (metabolische) Unterstützung von Nervenzellen. Da die Kulturen keine unreifen Vorläuferzellen enthalten, ist ein direkter Vergleich zwischen den reifen Astrozyten und den neuralen Stammzellen (NSC), aus denen sie differenziert werden, möglich. Beide Zelltypen ähneln einander in vielerlei Hinsicht. Ein direkter Vergleich war bisher allerdings schwierig, da Primärkulturen meist beide Zelltypen enthalten und nur wenige Merkmale bekannt sind, die eine eindeutige Unterscheidung beider Zelltypen erlauben. Eine Analyse des Transkriptoms dieser Reinkulturen von mAGES und NSC ermöglichte eine Neubewertung bisher bekannter zelltypspezifischer Marker sowie die Identifizierung neuer Merkmale zur besseren Unterscheidung. Darüber hinaus haben metabolische Untersuchungen nicht nur die nahe Verwandtschaft zwischen Astrozyten und NSC belegt, sondern auch klare Unterschiede, bezogen auf den Glutamat/Glutamine-Stoffwechsel sowie die Freisetzung von Citrat, aufgezeigt.

Im zweiten Teil dieser Arbeit wurden mAGES, aufgrund ihrer Homogenität und Ähnlichkeit mit reifen Astrozyten, dazu verwendet, die De-Differenzierung von Astrozyten hin zu neuralen Stammzellen zu erforschen. Dieses recht junge

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Forschungsfeld beruht auf den Entdeckungen, dass neurale Stammzellen im Erwachsenen-Gehirn astrozytäre Merkmale aufweisen, und dass reife Astrozyten unter bestimmten pathologischen Veränderungen Stammzellmerkmale wiedererlangen.

Studien zur De-Differenzierung erfordern homogene Kulturen von reifen Astrozyten, ohne eine Verunreinigung mit unreifen Zellen, um Mechanismen und Signalwege der Zellumwandlung zu erforschen. Es wurde gezeigt, dass die Zugabe eines einzelnen Wachstumsfaktors unter streng kontrollierten Bedingungen ausreicht, um mAGES zu neuralen Stammzellen (NSC2) zu de-differenzieren. Reife mAGES reaktivieren ihren Zellzyklus und teilen sich wieder, sobald sie mit FGF2 stimuliert werden. Sie exprimieren verstärkt den neuralen Stammzellmarker Nestin, während das astrozytäre ‚glial fibrillary acidic protein‘ (GFAP) herunterreguliert wird. NSC2 sind multipotente Stammzellen, welche sowohl zu Astrozyten als auch zu Nervenzellen differenzieren können. Außerdem gleichen sie neuralen Stammzellen in Bezug auf ihren Phänotyp und ihre Funktionen. Die Phosphorylierung von ERK, ausgelöst durch die Stimulation mit FGF2, ist hauptverantwortlich für das Wiederanschalten des Zellzyklus in mAGES. Unter pathologischen Bedingungen, in diesem Fall der Stimulation mit inflammatorischen Zytokinen, war die Umwandlung von mAGES zu neuralen Stammzellen gestört. IFNγ verhinderte das Wiederanschalten des Zellzyklus durch eine Phosphorylierung von STAT1. Die Zugabe von Ruxolitinib, einem Inhibitor des JAK/STAT-Signalweges, war wiederum ausreichend, um die Umwandlung von mAGES, selbst in Gegenwart von IFNγ und anderen Zytokinen, zu ermöglichen. Diese Ergebnisse könnten erklären, warum eine De-Differenzierung in Krankheitsmodellen nur selten vorkommt, da der positive Stimulus (FGF2) immer in Verbindung mit hemmenden, entzündlichen Faktoren auftritt.

Insgesamt wurde jedoch gezeigt, dass reife Astrozyten direkt in neurogene neurale Stammzellen umgewandelt werden können, und dass diese Umwandlung sowohl positiv, als auch negativ beeinflusst werden kann. Dadurch können künftig Mechanismen identifiziert und erforscht werden, welche eine De-Differenzierung von Astrozyten fördern oder hemmen.

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1 I NTRODUCTION

1.1 Astrocytes in general – functions and heterogeneity

Astrocytes are, as their name suggests, star-shaped glia cells, which were discovered more than 100 years ago (Kettenmann and Ransom 1995). They are the most abundant cell type in the brain (Cherniak 1990; Nedergaard et al. 2003) and fulfill diverse vital roles and contribute to normal CNS functions. Some of the well-known functions are the maintenance of the metabolic environment, neuronal support, and their involvement in inflammatory processes. However, astrocytes did not receive sufficient attention over a long period, based on the assumption of a primarily passive appearance in contrast to the excitability of neurons. The recognition of an active contribution of astrocytes to synaptic transmission and plasticity, and the increasing awareness of their pivotal role in almost all pathological processes in the brain, revived the interest in astrocytes for basic as well as pharmacological and toxicological research (Sofroniew and Vinters 2010).

The heterogeneity of astrocytes has long been underappreciated. They have been studied as a homogeneous cell population performing ’classical’ functions, such as potassium homeostasis, regulation of the blood brain barrier, or neurotransmitter uptake. Over the last decades, not only other functions have been discovered, e.g., the stem cell capacity of astrocytes, but also the heterogeneity of different subtypes of astrocytes and their functional specialization in different brain regions or within one region has been realized.

Nonetheless, the understanding of their diversity lags far behind that of neurons. This heterogeneity raises, of course, new problems regarding the definition of an astrocyte. In the following, astrocyte functions as well as their heterogeneity are summarized with the aim of establishing a new basis for the characterization of astrocytes in general.

1.1.1 Astrocyte functions

The term neuroglia derives from the greek word for ‘glue’ (γλοία), which indicates a structural as well as a supplying role (nutrients, oxygen) for these non-neuronal cells.

Astrocytes are in contact with different brain cells as well as the vasculature, thereby coupling neurons and other brain cells to blood supply through astrocytic endfeet. Their numerous processes are radially distributed and one astrocyte may contact several thou- sand synapses. Moreover, astrocytes themselves are coupled through gap junctions (con- nexins), forming highly regulated networks of complex cell interactions (Sofroniew and Vinters 2010).

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The housekeeping functions, including ion homeostasis, maintenance of the extracellular matrix, the metabolic support of neurons, or the maintenance of the blood brain barrier, can be considered the ‘classical’ functions due to their early recognition (Parpura and Haydon 2009). They are well described in the literature, and were responsible for the concept of astrocytes being passive supporter cells, which are not electrically excitable like neurons, but maintain the extracellular milieu. During neurotransmission, the levels of extracellular potassium increase, which would disturb the depolarization of neurons.

Astrocytes efficiently and rapidly take up potassium by several ion channels, e.g., Kir4.1, and distribute it throughout the cytoplasm and to neighboring cells via gap junctions. This potassium buffering is facilitated by a simultaneous flux of water through water channels (e.g., Aqp4) (Song and Gunnarson 2012). Furthermore, astrocytes express many different extracellular matrix proteins and adhesion molecules, thereby generating a dynamic environment, in which neurite outgrowth as well as migration is finely regulated (Thomas et al. 1996; Wiese et al. 2012). Another part of the housekeeping in the central nervous system is the formation and maintenance of the blood-brain-barrier (BBB), in which astrocytes play a crucial role by directly interacting with brain capillary endothelial cells.

This is not only the interface for nutrient supply, but a disruption of these interactions during disease may affect the BBB integrity, which is the case e.g., in Parkinson’s disease (Cabezas et al. 2014).

Besides these homeostatic and structural functions, astrocytes primarily support neurons in many different ways. First, they support neurons metabolically by providing important nutrients. The astrocytic endfeet, which contact blood vessels, express specific glucose transporters such as GLUT-1 (Morgello et al. 1995). Moreover, they are the predominant cell type in the brain to store glucose as glycogen, which is degraded under hypoglycemic conditions to maintain axon function or increased neuronal activity via lactate exchange (Brown and Ransom 2007). Astrocytes show high glycolytic rates producing lactate, which is released and taken up by neurons to meet their energy demand during neurotransmission (Waagepetersen et al. 1998; Westergaard et al. 1995). Second, astrocytes produce a large number of neurotrophic factors, which influence synaptogenesis and regulate synaptic activity and plasticity (Christopherson et al. 2005; Pfrieger and Barres 1997; Ullian et al. 2001). And third, they regulate neurotransmission by taking up neurotransmitters (Bak et al. 2006). The latter function has led to the concept of the tripartite synapse, in which the interactions between the pre- and the postsynaptic neuron with

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neighboring astrocytes are described (Araque et al. 1999; Perez-Alvarez and Araque 2013).

Fig. 1 describes the recycling mechanism of neurotransmitters with regard to glutamate uptake. Glutamate is released during synaptic transmission, binding to receptors at the postsynaptic membrane, and leading to a depolarization of the postsynaptic neuron. Dur-

ing synaptic transmission, the local extracellular concentration of glutamate can increase up to a millimolar range and an excessive or prolonged stimulation of postsynaptic receptors can lead to cell death of the postsynaptic neuron (Conti and Weinberg 1999). Thus, a rapid and efficient removal of remaining glutamate from the synaptic cleft is indispens- ible for terminating glutamate signaling and preventing excitotoxicity of glutamate

Fig. 1. The glutamate/glutamine shuttle between neurons and astrocytes. Astrocytes take up glutamate during synaptic transmission through the glutamate transporters GLT-1 and GLAST, and either release it as glutamine via the glutamine transporter SN1, store it in vesicles through vesicular glutamate transporters (vGLUT), or feed the tricarboxylic acid cycle (TCA). Conversion to glutamine is catalyzed by the glutamine synthetase (GS).

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(Schousboe and Waagepetersen 2005). Astrocytes express specific Na⁺-dependent transporters, such as the glutamate-aspartate transporter GLAST or GLT-1, which rapidly take up glutamate. In a knockout study, both transporters have been shown to be essential for preventing neurodegeneration by decreasing glutamate excitotoxicity (Rothstein et al.

1996). Astrocytes then convert the glutamate into glutamine by the glutamine synthetase, which catalyzes the ATP-dependent condensation of glutamate with ammonia, thereby also achieving ammonia detoxification (Rose et al. 2013). The newly formed glutamine is then released by the glutamine transporter SN1 and taken up by neurons, which convert it to glutamate again. Therefore, the tripartite synapse model describes a dynamic recycling mechanism of neurotransmitters, regulating extracellular levels and terminating synaptic transmission. Besides this recycling mechanism, astrocytes utilize glutamate as an anaplerotic substrate. It is deaminated by the glutamate dehydrogenase to form α-ke- toglutarate, which feeds the citric acid cycle (TCA) (Schousboe et al. 2013). Only recently, it has been proposed that astrocytes can also store glutamate in vesicles by the expression of vesicular glutamate transporters (vGLUT), and even release it in a calcium- dependent manner similar to neurons (Zorec et al. 2012). This process named gliotrans- mission is, however, still under debate (Sloan and Barres 2014). The exact mechanisms are not known and especially its relevance for and connection to neurotransmission remains to be clarified. A lot of current studies are focusing on calcium-signaling in astrocytes, which represents a kind of excitability of the cells and therefore replace the assumption of astrocytes being ‘passive’ cells (Rusakov 2015).

Besides their role in normal brain physiology, astrocytes perform several immune functions under pathophysiological conditions. During inflammation or injury, a lot of different factors are released by other brain cells like microglia, which drive astrocyte activation. These factors include inflammatory cytokines, such as TNFα or IL1β, which are mainly produced by activated microglia (Hanisch and Kettenmann 2007; Henn et al.

2011; Kreutzberg 1996). Growth factors such as FGF2 are mainly released by reactive astrocytes themselves or endothelial cells (Logan et al. 1992). Other factors like ATP or reactive oxygen species occur during stress responses of various cells (Fig. 2) (Sofroniew 2009).

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Fig. 2. Triggers and molecular regulators of reactive astrogliosis (Sofroniew and Vinters 2010).

All these factors trigger different responses in astrocytes, such as the activation of transcription factors NFκB and cJUN, or the production of the signal transducer cAMP, leading to molecular and functional changes in astrocytes (Sofroniew and Vinters 2010).

These changes may have beneficial as well as detrimental effects for the surrounding tissue depending on the severity and type of injury. These different activation states of astrocytes, leading to e.g., cell proliferation, are called astrogliosis. Astrogliosis occurs in almost all diseases and injuries of the CNS and results in mild, moderate, or severe man- ifestation (Sofroniew and Vinters 2010). With increasing degree of severity, the re-entry of astrocytes into the cell cycle and following proliferation leads to the generation of a glial scar around the lesion, accompanied by a tremendous reorganization of the tissue architecture. Since glial scar formation inhibits axon regeneration, it has been considered as a fully destructive phenomenon. However, beneficial effects of glial scar formation as well as the presence of beneficial reactive astrocytes has been described, e.g., the preven- tion of oxidative stress or the inhibition of the outspread of inflammatory and pathogenic cells and agents (Faulkner et al. 2004; Li et al. 2008; Macco et al. 2013; Sofroniew 2005).

Thus, therapeutic strategies regarding astrocyte activation have changed over the last years, from trying to prevent astrogliosis per se, to the inhibition of detrimental pathways

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and simultaneous activation of beneficial effects. However, the complexity of astrogliosis as well as the heterogeneity of reactive astrocytes aggravate the development of therapeutic strategies (Anderson et al. 2014; Sofroniew 2009). Therefore, many studies are currently performed to elucidate the identity of reactive astrocytes e.g., on transcriptomic level, and to identify involved pathways that lead to beneficial as well as detrimental effects (Anderson et al. 2014; Falsig et al. 2006; Zamanian et al. 2012).

1.1.2 Heterogeneity of astrocytes

Since the early 19^th century, a distinction is made between two major groups of astrocytes:

the protoplasmic astrocytes, usually found in the grey matter enveloping synapses, and the fibrous astrocytes, which are located in the white matter and contact nodes of Ranvier (Andriezen 1893; Kölliker 1989; Peters et al. 1976). Other classifications based on regional specialty were described with cerebellar Bergmann glia (Palay and Chan-Palay 1974) and retinal Müller glia (Bhattacharjee and Sanyal 1975). The full scope of astrocytic heterogeneity, however, has been realized over the last decades and is still rudimen- tary, especially when compared with the knowledge of neuronal diversity. This heterogeneity is not only of morphological nature, but bears also phenotypic, functional, regional, and local aspects, which are closely linked to each other. There are, for example, differences regarding the expression of the glial fibrillary acidic protein (GFAP) between grey matter (low) and white matter (high levels) astrocytes (Lein et al. 2007), although protoplasmic astrocytes also increase GFAP expression under pathophysiological conditions (Vijayan et al. 1990). There is further variety in phenotype between regional subtypes of grey matter astrocytes: hippocampal cells or Bergmann glia, in contrast to cells of the cerebral cortex and striatum, express high levels of GFAP under physiological conditions (Sergent-Tanguy et al. 2006). Nowadays, it is well described that there exists a subpopulation of mature, GFAP-negative astrocytes, which express astrocyte marker genes apart from GFAP and reveal functional inflammatory capacity (Cahoy et al. 2008; Kuegler et al. 2012; Pekny et al. 1995; Walz and Lang 1998). The functional consequence of this heterogeneous expression pattern is not yet understood, but might be engendered by different regional requirements. For example, connexin 30 (Cx30), which forms gap junction channels between neighboring cells, is expressed in grey matter, but not in white matter astrocytes (Nagy et al. 1999). These gap junctions are necessary for the fast propagation of signals, e.g., calcium waves, which also differ between white and grey matter astro-

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cytes (Haas et al. 2006). Even more interesting is the observation of a diversity of astrocytes within the same brain region. Such a local heterogeneity occurs, for example, in the cortex layer of rodents. The cortex layer IV is arranged in anatomical structures called barrels, consisting of neurons, which are highly coupled within one barrel, but very low connected to neurons of a neighboring one. The coupling of astrocytes in this region re- flects that of neurons, meaning that gap junctions are more or less restricted to individual barrels (Houades et al. 2008). The coupling correlates with the expression of Cx30 and Cx43 in astrocytes within one barrel, but not in astrocytes between those units (Rash et al. 2001). There are, thus, obvious phenotypic differences between astrocyte subtypes and these differences probably affect functionality of cells in distinct or within the same brain region, although the correlation is far from understood and much less experimentally investigated.

The origin of astrocyte variety is not clarified and could be a multifactorial mechanism.

One possibility is the descent from different progenitor cells during development (see also 1.3.1 Neural stem cells and the development of astrocytes). Another explanation may be the functional complexity and specification of astrocytes as an adaptation to local requirements. Astrocytes are found all over the CNS and are closely linked to each other, to neurons, and to endothelial cells, forming dense and complex networks of different composition. It is reasonable to expect that astrocytes adapt to the specific needs of their microenvironment. In contrast to developmental variety, which might be determined by intrinsic factors, the local heterogeneity might be the result of an adaptation to external factors. By regulating the expression of glutamate transporters, astrocytes might adapt to the local demand for glutamate uptake. GLT-1, for example, is higher expressed in grey matter astrocytes from the hippocampus and the cerebellum and the opposite expression pattern is found for the aspartate-glutamate transporter GLAST (Lehre et al. 1995). More- over, there exists a novel splice variant of the glutamate transporter GLT-1, which is primarily found in white matter astrocytes (Macnab and Pow 2007). However, these differ- ential expression patterns are not yet understood, and if astrocyte heterogeneity results from different developmental origins, or from functional specializations, or both together, has still to be clarified (Hewett 2009). Most of the studies addressing astrocyte heterogeneity have been done in vitro. In vivo studies of different astrocyte subsets, such as the transcriptomic profiling of freshly isolated astrocytes at different developmental stages or different brain regions (Cahoy et al. 2008; Lovatt et al. 2007), would certainly give

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more insights into the development of astrocyte subpopulations. Another promising alternative could be the investigation of stem cell-derived astrocytes. With this approach, it is possible to monitor astrocyte development in vitro and to investigate temporal changes in phenotype and functions. Furthermore, development can be manipulated with growth factors and other cytokines to generate different subpopulations of astrocytes, which can then be investigated with regard to their phenotypic and functional characteristics.

1.1.3 How to define a cell as an astrocyte?

The concept of astrocytes being a homogeneous cell population is not tenable any more, since there are obvious phenotypic and functional differences between astrocytes not only from distinct, but also from the same brain region. Primary astrocytes normally represent a mixture of cells from at least one brain section, which makes it even more difficult to investigate functions of a specific cell population. So there has to be a reconsideration of defining one cell as an astrocyte, and afterwards to investigate its specific function in a more precise context.

The phenotype of cells can be easily assessed by PCR on mRNA level and by immunocytochemistry on protein level. PCR provides an early response to changes in the environment, but does not allow single cell analysis. Immunocytochemistry is therefore crucial to examine the distribution of marker expression and protein localization in single cells of a possibly heterogeneous population. There are several specific astrocyte markers, which can be detected by one or both methods. The glial fibrillary acidic protein (GFAP) was and still is the ’pan-astrocyte’ marker (Zhang and Barres 2010), although it is nowadays well-accepted that it is not expressed in all astrocyte subtypes and that neural stem cells, which derive from radial glia, also express this intermediate filament protein (Liu et al. 2006; Walz and Lang 1998).

S100B, a calcium binding protein, is expressed in some mature astrocytes residing blood vessels and in NG2-positive cells, which are thought to be oligodendrocyte precursor cells (Deloulme et al. 2004; Hachem et al. 2005). There exist double-positive astrocytes for S100B and GFAP, but S100B-positive cells are often GFAP-negative (Raponi et al.

2007).

A very useful marker is the aldehyde dehydrogenase Aldh1L1, which has been discovered as highly specific RNA marker that is also expressed in GFAP-negative cells (Cahoy et al. 2008). Other markers concerning the glutamate metabolism are the transporters GLT-

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1 and GLAST and the glutamine synthetase. GLT-1 and GLAST are thought to be astrocyte-specific (Chaudhry et al. 1995), whereas GS can also be expressed in some oligodendrocytes (Damelio et al. 1990). Moreover, GLT-1 is often more abundant in mature astrocytes while GLAST is more prominent in immature cells and progenitors (Regan et al.

2007). Aquaporin 4 (Aqp4), a water channel distributed within astrocytic endfeet, is quite a reliable marker of mature astrocytes. Its expression overlaps especially with the glutamate transporter GLT-1, and it has been found in GFAP-positive and -negative cells (Vitellaro-Zuccarello et al. 2005). Taken together, these markers should only be used in combinations to define a cell as an astrocyte, since they are often not as astrocyte-specific as they have been reported earlier. Moreover, the lack of single markers does not exclude an astrocytic phenotype. Studies on transcriptome profiles of astrocytes referred to other cells like neurons or neural stem cells help to set up a more comprehensive set of markers (Cahoy et al. 2008; Gotz et al. 2015; Lovatt et al. 2007).

Even more important is a concomitant functional characterization of cells. The expression of, for example, GLT-1 and GS does not mean that the cells are actually able to take up and metabolize glutamate, although this can be easily assessed by measurement of GS activity or glutamate uptake. Thus, an overall concept of defining cells as astrocytes com- bines different approaches including the characterization of the phenotype, both on RNA and protein level, together with functional characteristics.

1.2 Research with primary astrocytes in vitro

Investigation of astrocyte phenotype and functions are normally performed with primary astrocytes in vitro. The use of primary cells, though, implies several disadvantages, both practical and transferring, which necessitates a suitable alternative. First, the handling of primary cells is time-consuming and expensive, while obtaining only a limited number of cells. Second, primary cell cultures are often heterogeneous, aggravating studies, which cannot be performed on single cell resolution. Nevertheless, new techniques like mag- netic cell sorting approaches greatly enhanced the isolation and preparation of relatively pure cultures of viable astrocytes, which are acutely analyzed as ex vivo populations (Feldmann et al. 2014). And third, the access to human material is restricted, especially for adult cells. This is particularly important for toxicological studies, in which a human cell system would be preferable due to its higher validity.

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For astrocytes, primary monolayer cultures have become the dominating model system.

However, contamination with other cell types such as microglia (Saura 2007) requires stringent controls, and the contribution of immature cells with high levels of nestin expression to standard primary cultures has often been neglected (Hansson 1986; Sergent- Tanguy et al. 2006; Stahlberg et al. 2011). In fact, most studies identify primary astrocytes only by the presence of GFAP. This may introduce errors in two directions, as the marker may be absent in a subpopulation of mature, quiescent astrocytes, while it is also expressed in immature cells that proliferate and have high levels of nestin. A quantification of negative markers, indicative of contaminating or immature cells, is often missing.

Moreover, many studies are not truly based on primary, but rather secondary or tertiary cultures. The associated selection and expansion of subpopulations, and cell activation may be the reason for contradictory studies on astrocytic inflammatory functions (Kuegler et al. 2012), and it makes it impossible to attribute specific metabolic functions unambiguously to primary mature astrocytes. An alternative for basic research is the use of astrocytic cell lines. However, these cell lines based on immortalized astrocytes often lose phenotypic or functional characteristics of mature cells due to their high proliferation rate, which has been described and discussed earlier (Schildknecht et al. 2012). Another approach to generate astrocytes devoid of contaminating cells like microglia is to generate astrocytes from embryonic stem cell derived neural stem cells (Kuegler et al. 2012) or primary neural stem cells (Crocker et al. 2008) cultured as neurospheres, but the resulting astrocyte population may still be heterogeneous containing nestin-positive precursors.

The same applies to the generation of human astrocytes from pluripotent stem cells. There exist several protocols that generate human GFAP-positive cells with high (> 70%) effi- ciency (Krencik et al. 2011; Roybon et al. 2013; Shaltouki et al. 2013), but they all still contain sizable subpopulations of immature or contaminating cells that might spoil results of immune activation studies, gene expression analysis, or identification of metabolic pathways.

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1.3 The generation of astrocytes from stem cells

The in vitro generation of astrocytes from pluripotent stem cells offers a promising alternative, and creates new possibilities to investigate not only fully differentiated cells, but also the development of astrocytes.

To date, the generation of astrocytes has been successfully accomplished from embryonic stem cells and primary neural stem cells. In this way, it is possible to produce large quan- tities of precursor cells without extended cultures of astrocytes themselves, and finally to generate pure populations of differentiated cells. A major problem, however, remains: It is uncertain whether these cells represent physiological homologues in vivo, or whether the self-renewing and multipotent state of stem cells in vitro resembles a synthetic phenomenon, based on contrived transcriptional reprogramming after exposure to growth factors (Conti and Cattaneo 2010). The aim should be to define the phenotype and function of generated astrocytes in more detail, while demonstrating their resemblance to in vivo cells, to finally establish them as a general or specific alternative to primary cells.

How important it is to match in vitro generated cells with their counterparts in vivo, and how the phenotype and function of cells is influenced by different culture conditions, has been summarized recently (Noble et al. 2011). Noble and others performed astrocyte- transplantation assays and could show that differently treated cells adopted distinct phe- notypes, resulting in opposite recovery effects in vivo.

Since astrocytes are involved in almost every process of normal and pathological physiology in the brain, the generation of functional and pure populations from stem cells could lead to a better understanding of their roles, and bears a promising approach for biomedical strategies.

Stem cells, including embryonic stem cells (ESC), induced pluripotent stem cells (iPS), and primary neural stem cells (NS), are defined as self-renewing cells, which can give rise to, in case of ESC and iPS, every cell type of the three embryonic germ layers (Takahashi and Yamanaka 2006; Thomson et al. 1998). This differentiation process in vitro follows, in an ideal situation, the developmental principles in vivo. Thus, it is de- pendent on our understanding of the embryonic development, which gives us the infor- mation how to guide stem cells and progenitor cells towards functional, fully differentiated cells. This complex process is based on both spatial and temporal factors and mechanisms, which already apply to the ’patterning’ of progenitor cells (Liu and Zhang 2011).

The potential of such systems is worth the expanse: First, they can help to recapitulate

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embryonic development, which is, at least in humans, experimentally inaccessible. Sec- ond, patient-derived iPS may even clarify cell-autonomous causes of developmental dis- orders. Third, these iPS may be utilized for drug screenings. And last, the differentiated cells may be used for regenerative therapies, in case of iPS actually personalized cell therapy.

This section describes the possibilities, by which astrocytes can be generated in vitro, and summarizes previous effort and success in this field.

1.3.1 Neural stem cells and the development of astrocytes

Neural stem cells (NS) are self-renewing cells in the embryonic nervous system, which continue to exist in some areas of the adult mammalian brain, maintaining a pool of multipotent cells that can give rise to neurons, oligodendrocytes, and astrocytes. During development, there are several types of neural stem cells, some of them representing only transient populations. The earliest NS population are neuroepithelial progenitors (NEPs), arising from the neuroectoderm, and forming the neural plate, which further folds into the neural tube (Götz and Huttner 2005). The initial symmetric division expands the neural tube, while following asymmetrical division increases the pool of NEPs in the ventricular zone (VZ), and gives rise to neurons that migrate outward. Radial glia, arising from NEPs in the VZ, form radial processes guiding migrating neurons, and also act as multipotent progenitors in the VZ and the subsequent subventricular zone (SVZ) (Malatesta et al.

2008). They undergo proliferative and asymmetrical divisions, generating initially neurons and later, at least some subsets, also glia cells (Costa et al. 2011; Malatesta et al.

2000). In the adult brain, neurogenesis is limited to two regions: the SVZ at the side of the lateral ventricle wall and the subgranular zone (SGZ) in the hippocampus (Doetsch et al. 1999; Kempermann and Gage 1999; Seri et al. 2001). Both regions are formed by radial glia descendants in the postnatal phase, which share some characteristics with astrocytes. There are, thus, many types of NS cells at any stage of CNS development, all with various differentiation capacities, giving an idea of the complexity of neural stem cells.

Gliogenesis occurs in the embryonic ventricular zone (VZ) and in the neo- and postnatal subventricular zone (SVZ) (Goldman 2007). Astrocytes in the postnatal brain have recently been reported to undergo symmetric division further increasing the pool of differentiated astrocytes in the cortex (Ge et al. 2012). Radial glia from the VZ can give rise both to neurons and astrocytes, and can also transform into Bergmann glia during the

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perinatal period (Fishell and Kriegstein 2003; Noctor et al. 2001). Glial intermediate progenitors from the neonatal SVZ migrate into the cortex, where they differentiate into oligodendrocytes and astrocytes (Ganat et al. 2006). SVZ cells have been shown to generate both grey and white matter astrocytes in vivo (Levison and Goldman 1993). Many of these progenitor cells are bi- or tripotent, but some cells in the neonatal SVZ are thought to be astrocyte-restricted precursors (Levison and Goldman 1997). There is, hence, a great variety in astrocyte lineages, and different origins of astrocyte populations could explain astrocyte heterogeneity.

There have been successful isolations and in vitro expansions of NS cells in different culture conditions and also their tripotency, at least in some cases, could be demonstrated.

The long-term maintenance of NS cells in their natural state originally faced problems concerning the simulation of their niche in vivo, which is determined by different factors and cell-cell contacts, which keeps them in their self-renewing state. Two different approaches have been established: the neurosphere culture and monolayer systems. Both systems used the epidermal growth factor (EGF) and the fibroblast growth factor 2 (FGF2) to trigger the self-renewal of cells with NS characteristics. Neurospheres are aggregates of progenitor cells, which float freely in low-attachment culture dishes. In this condition, supported by the addition of mitogens like EGF and FGF2, differentiating cells are supposed to die, leading to the expansion of NS populations. Reynolds and Weiss showed for the first time the in vitro expansion of NS cells in such neurosphere cultures (Reynolds et al. 1992). Surprisingly, they isolated NS cells not only from embryonic brain, but also from the striatum of adult mice, giving a first indicator of stem cells in the brain that exist beyond embryonic development (Reynolds and Weiss 1992). In both cases, these NS cells gave rise to neurons and astrocytes, indicated by a change in morphology and the upregulation of specific antigenic markers. The original protocol for NS propagation as neurospheres, published 2008 in Nature Protocols, describes the differentiation potential not only to neurons and astrocytes, but also to oligodendrocytes (Chojnacki and Weiss 2008), thereby demonstrating tripotency of the neural stem cells in vitro. In general, NS cells show multipotency, although they differ in their neurogenic and gliogenic potential. It has been shown that the neurogenic potential is temporarily regulated and declines during long-term expansion of the neurospheres in vitro (Grandbarbe et al. 2003). Neurospheres are no homogeneous cell population, and their potential to give rise to neurons or astrocytes can be influenced by growth factor exposure

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in vitro. It has been shown, for example, that exposure to growth factors such as EGF can induce multipotency of cells isolated from the SVZ, which has been neurogenic precursors in vivo (Doetsch et al. 2002). This heterogeneity and plasticity complicates, of course, the production of pure populations of astrocytes, a problem, which could be overcome by adherent propagation of NS cells. In the beginning, this system created cells of variable homogeneity, showing asymmetrical division that reduced the number of multipotent progenitors over time (Palmer et al. 1997). Nevertheless, these cells have been shown to generate neurons, oligodendrocytes, and astrocytes. Further progress has been made regarding the long-term expansion and the homogeneity of different adherently propagated NS cells. Conti, Pollard, and colleagues described in 2005 the adherent long-term culture of primary embryonic NS cells or ESC-derived NS cell lines as homogeneous populations (Conti et al. 2005). EGF and FGF2 have been shown to be sufficient for the maintenance of symmetrical self-renewal. These NS cell lines resemble radial glia in vivo, expressing precursor markers s nestin, vimentin, RC2, and GLAST, although GFAP was missing.

Upon exposure to bone morphogenetic protein 4 (BMP4) or serum, cells adopted an astrocyte morphology and expressed GFAP. They also kept the ability to differentiate into neurons. Functional analysis of the differentiated progenies, however, has only been done for neurons. In 2006, they extended these cultures to NS cells isolated from the SVZ of the adult mouse brain (Pollard et al. 2006). Sun and colleagues described in 2008 the generation of NS cells from human fetal tissue, which could also be expanded in a monolayer system with EGF and FGF2 (Ihrie and Alvarez-Buylla 2008). These cells expressed nestin, vimentin, GLAST, and GFAP, hallmarks for radial glia cells. They have been shown to be tripotent, but astrocyte generation has been demonstrated only due to morphological changes and the expression of GFAP.

1.3.2 Astrocytes derived from embryonic or induced pluripotent stem cells

Embryonic stem cells (ESC) are self-renewing, pluripotent cells, which can give rise, by definition, to all cell types of an adult organism. They are obtained from the inner cell mass (ICM) of the blastocyst. Murine ESC were the first time isolated in 1981 by Martin Evans, Matthew Kaufman, and Gail R. Martin (Evans and Kaufman 1981). In 1995, Mar- tin Evans and Austin Smith described the culture of murine ESC with leukemia-inhibitory factor (LIF), which keeps the cells in their pluripotent, self-renewing state. Human ESC have been isolated later in 1998 by James Thomson from the ICM of residual blastocysts

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of in vitro fertilizations (Thomson et al. 1998). They can be kept in a proliferative, non- differentiating state by the addition of FGF2. ESC are pluripotent, in contrast to the multipotent NS cells or adult stem cells, meaning that they can differentiate into cells of all three germ layers: ectoderm, endoderm, and mesoderm (Martello and Smith 2014). Cells of the nervous system in vivo arise from the ectodermal lineage, which forms the epider- mis and the neural plate. ESC can be differentiated into neural stem cells (NSC) in vitro by the withdrawal of growth factors. This withdrawal of factors, which keep the cells in a pluripotent state, leads to a spontaneous differentiation of the cells. This implies that they might not only form NSC, but also cells of other germ layers. However, NSC can be enriched by a switch to neural proliferation medium containing EGF and FGF2 to expand the desired progenitor cells. Neural stem cells have been generated from murine ESC (mESC) since 2001 (Tropepe et al. 2001). In 2003, a protocol for neural induction of mESC in adherent culture has been described, facilitating the generation of pure populations of NSC (Ying et al. 2003). Luciano Conti and colleagues showed in 2005 that these pure NSC can differentiate both into neurons and astrocytes, also after prolonged expansion in EGF and FGF2 (Conti et al. 2005). In 2007, the tripotential differentiation capacity of these cells could be shown by immunocytochemistry detecting oligodendrocytes (Glaser et al. 2007). Human embryonic stem cells (hESC) have been differentiated to neural precursors in 2001 by Su Chun Zhang and colleagues (Zhang et al. 2001). These neural precursors, obtained from neurosphere cultures, differentiated into neurons, astrocytes, and oligodendrocytes, although glia cells represented only a minor fraction. Their differentiation potential to astrocytes has been reported in 2011: Robert Krencik and colleagues generated NSC within 21 days from hESC and differentiated them into astrocytes within 160 days. The cells showed an astrocytic phenotype, expressed markers of immature and mature astrocytes, reacted to AMPA and glutamate, and supported neuronal sur- vival (Krencik et al. 2011). Gupta and colleagues also generated cells from hESC with astrocyte characteristics, and demonstrated neuroprotective effects of their cells (Gupta et al. 2012). The differentiation of human embryonic stem cells to astrocytes is much more complicated compared with mouse systems, since human development proceeds in a different time scale. Although human systems would be desirable, current protocols for the generation of astrocytes require long-term cultures and the differentiated astrocytes are in most cases heterogeneous and not fully mature (Roybon et al. 2013). Thus, for studies, which require pure populations of mature astrocytes, such as metabolic studies

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or microarray profiling, astrocytes generated from murine stem cells are still the best approach.

Induced pluripotent stem cells (iPS) are reprogrammed somatic cells, which regain stem cell characteristics by introducing pluripotency factors. This has been done first in 2006 with mouse fibroblasts by Shinya Yamanaka (Takahashi and Yamanaka 2006), who re- ceived, together with John Gurdon, the noble price for his study in 2012. Several transcription factors were introduced by retroviral transduction, and cells were cultured equivalent to embryonic stem cells on feeder layers in ESC medium. Oct3/4 (octamer- binding transcription factor 4), Sox2 (sex determining region Y box 2), and nanog have been known factors for maintaining pluripotency (Sun et al. 2006). However, only Oct3/4 and Sox2 to be essential for the generation of iPS. By introducing them together with c- myc and Klf4 (Kruppel-like factor 4), they got a self-renewing cell population expressing ESC markers, which could be differentiated into neural, muscle, and endodermal tissue cells. This finding has been revolutionary for stem cell research, and other iPS cell lines followed. In 2007, two human iPS lines have been generated. Takahashi and colleagues introduced the same factors in human dermal fibroblasts (Takahashi et al. 2007). At the same time, Yu and colleagues generated human iPS through four factors including Oct3/4, Sox2, and Nanog (Yu et al. 2007). Both cell lines regained embryonic stem cell characteristics, and were capable to differentiate into cells of all three germ layers. Now- adays, the classical reprogramming factors are Oct4, Sox2, Klf4, and c-Myc (Kim et al.

2011).

The generation of human iPS opened up new perspectives in biomedical research: iPS might be used for patient-specific cell-based therapy, reducing immunorejection after transplantation. Nevertheless, there are some limitations of the system, which need to be resolved before using iPS-systems in cell therapy. Some of the factors introduced into somatic cells are very potent oncogenes (e.g., c-myc), holding a risk of tumor formation in patients (Gutierrez-Aranda et al. 2010). Moreover, the integration of factors into the genome might engender genomic mutations (Lin and Wu 2015). And the reprogramming itself is debatable: In many cases, it is incomplete, giving rise to cells that are not equivalent to ESC (Jalving and Schepers 2009). Zhao and colleagues, however, showed in 2009 that murine iPS injected in blastocysts gave rise to viable mice (Zhao et al. 2009). There- fore, iPS actually might be suitable substitutes for ESC, although their safe application in cell therapy has still to be proven. In neuroscience, iPS and iPS-derived neurons or glia

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cells could be used for transplantation to substitute for injured cells in several CNS diseases such as Multiple Sclerosis, Alzheimer’s, or Parkinson’s disease. Therefore, iPS have been differentiated to a neural lineage since 2008. Wernig and colleagues generated tripotent NSC from murine iPS similar to ESC differentiation (Wernig et al. 2008). They could show that these precursor cells give rise to neurons and glia cells, when transplanted into fetal mouse brain. They also demonstrated therapeutic potential of dopaminergic neuron progenies, but were faced with problems concerning teratoma formation. Cai and colleagues used human iPS-derived dopaminergic neurons for transplantation assays in rats, but also observed tumor formation (Cai et al. 2010). To overcome the problem of teratoma formation, possibilities to enrich and increase purity of the desired cell type need to be developed (Lin and Wu 2015). Yao and colleagues used GFP constructs under the control of a neuronal promoter in mouse iPS to select for neurons prior to transplantation, without observing tumor formation (Yao et al. 2011).

Compared to the differentiation of iPS-derived NSC to functional neurons, the differentiation to glia cells lags far behind. A differentiation of iPS to functional oligodendrocytes has been reported in 2011 (Czepiel et al. 2011). The generation of mature astrocytes from iPS, at least as pure populations, has not been reported yet and tripotential differentiation capacity of iPS-derived NSC has mainly been demonstrated by the expression of GFAP (Conti et al. 2005). However, the differentiation of iPS, derived from Huntington’s disease patients, towards the astrocyte lineage revealed a vacuolation phenotype, indicating a cell-autonomous mechanism for astrocytes in this neurodegenerative disease (Juopperi et al. 2012). Moreover, the transplantation of iPS-derived astrocyte populations has been shown very recently to have beneficial effects in spinal cord injury (Li et al. 2015).

The generation of astrocytes presented here is mainly based on the protocol described in 2005 (Conti et al. 2005). This seminal work mainly focused on neurogenesis, while the generated astrocytes were hardly characterized. A reason may be that the original protocol requires adaptations and further specifications before high quality, pure astrocytes can be reproducibly obtained. As there is such a great, yet largely unrecognized, potential in the Conti et al. astrocyte generation procedure as basis for future astrocyte/NSC research, we set out in part 1 of this study: First, to define the critical steps of the procedure; Second, to characterize the resulting cells; And third, to demonstrate the usefulness of the new protocol for generating cell populations for inflammation and metabolism studies based on multiple stem cell lineages.

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1.4 Astrocytes as stem cells

A fundamental change in neuroscience occurred with the discovery of neural stem cells in the adult mammalian brain (Doetsch et al. 1999). The former dogma implied that the generation of newborn neurons was restricted to embryonic or postnatal development.

Thus, neurogenesis was thought to only occur in early development, but not in the adult brain. Nowadays, it has been observed in the adult brain in two regions: the subventricular zone (SVZ) around the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus (Doetsch et al. 1999; Seri et al. 2001). Stem cells in these areas continuously give rise to neurons of the olfactory bulb and the granule layer of the dentate gyrus throughout life. These stem cells can be isolated and expanded as neurospheres (non-adherent, free-floating aggregates of stem cells) in the presence of EGF and FGF2 (Reynolds and Weiss 1992). Nevertheless, the self-renewing state as well as their differentiation capacity in vitro differs from that in vivo. While the cells normally perform only some divisions and exclusively produce neurons in vivo, their self-renewing capacity as well as their potential to generate glial cells can be triggered in vitro by EGF and FGF2 exposure (Ortega et al. 2011). Thus, there is an obvious discrepancy of the lineage of stem cells in vivo and their differentiation potential in vitro (Gotz et al. 2015).

Even more interesting for this study is the fact that these self-renewing and neuropotent stem cells, have been shown to be astrocytic cells, present in these stem cell niches of the adult brain. Astrocytes and neural stem cells (NSC) share several phenotypic and functional features (Doetsch et al. 1999; Levitt and Rakic 1980; Seri et al. 2001). Some types of NSC, namely the radial glia or astrocytes from the subventricular zone, have been originally categorized as members of the heterogeneous group of astrocytes, due to their expression of GFAP (Eng et al. 2000; Ihrie and Alvarez-Buylla 2008; Steindler and Laywell 2003). Other markers, which can be found in both cell types, comprise proteins of the glutamate metabolism like GLAST (Liu et al. 2006; Ullensvang et al. 1997) and Glul (Hernandez et al. 1999; Monzon-Mayor et al. 1990). Thus, cells of an astrocytic identity act as neural stem cells in the adult mammalian brain.

The phenotypic and developmental relationship between mature astrocytes and neural stem cells gave rise to the idea that astrocytes outside the neurogenic niches might also bear the potential to act as stem cells. The response of astrocytes to injury or inflammation, i.e., the proliferation of cells in response to endogenous factors (Sofroniew and Vinters 2010), further ignited the discussion about mature astrocytes acting as stem cells.

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Under physiological conditions, mature astrocytes do not proliferate. However, reactive astrocytes present in a pathological environment (astrogliosis) not only re-enter the cell cycle, but also upregulate marker genes, which are normally found in neural stem cells (Sofroniew 2009). These observations might offer new possibilities regarding cell-based therapies for neurodegenerative and other diseases, in which excessive neuronal cell death occurs, e.g., stroke (Chouchane and Costa 2012). The idea of restoring neurological functions by generating new neurons from stem cells emerged over the last years. So far, two approaches were considered: First, transplantation of stem or progenitor cells expanded and pre-differentiated in vitro, which differentiate in vivo and integrate into neuronal networks (Benchoua and Onteniente 2011; Lindvall and Kokaia 2011). Second, the activation of endogenous neural stem cells in the neurogenic niches of the adult brain, which proliferate and migrate to the site of lesion (Leker et al. 2009). It has been shown that the newly generated neurons from the neurogenic stem cells niches actually migrated to lesion sites after focal ischemia in rats (Arvidsson et al. 2002; Parent et al. 2002). However, it is unclear whether the mobilization of endogenous stem cells can be transferred to a human system, since the migration distances in the human brain are much larger (Berninger 2010; Chouchane and Costa 2012). A relatively new, alternative approach is the use of local mature astrocytes for cell therapy. Astrocytes are highly abundant throughout the CNS and are therefore an attractive target within the lesion site. Moreover, some astrocytes are proliferative in response to injury or inflammation, even increasing their pool within the region of interest. Their proliferation is part of an astrogliosis, which has both beneficial and detrimental effects on the regeneration of neurons and axons.

Thus, in an ideal situation the targeting of astrocytes from glial scars might also help to reduce detrimental effects and restore an environment, in which neuronal growth and synaptic integration is possible (Berninger 2010; Chouchane and Costa 2012). Further- more, astrocytes also bear great plasticity regarding cell identity, facilitating the reprogramming or trans-differentiation into neurons. Despite direct conversion of astrocytes to neurons, a de-differentiation of astrocytes to neural stem cells, which further generate neurons, could be possible. Although the research on astrocytes as stem cells is a very young and evolving field, there is some evidence that mature astrocytes can in fact regain stem cell potential, and actually represent an attractive target for cell therapy. The basic principles regarding the stem cell potential of astrocytes are the objectives of the second

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part of this study, and previous efforts made on this topic are summarized in the following.

1.4.1 The de-differentiation of astrocytes to neural stem cells

Astrocytes, like other somatic cells, normally exit the cell cycle during maturation. How- ever, in certain pathological environments, in which a lot of different endogenous signals are released by surrounding cells or astrocytes themselves, they are able to re-enter the cell cycle (Sofroniew 2005; Sofroniew 2009). The proliferation of astrocytes in response to injury has been recognized already in 1970 (Cavanagh 1970). In 2013, in vivo live imaging by two-photon microscopy confirmed the proliferation of astrocytes contacting blood vessels close to the injury site, and excluded the migration of proliferating cells from other brain regions (Bardehle et al. 2013). Astrocytes in in vitro culture also generate a monolayer of cells, which exit the cell cycle due to contact inhibition. The re-entry of astrocytes into the cell cycle has been investigated mainly with primary astrocytes, exposed to different kinds of factors. However, the factors, which are responsible for the induction of proliferation in astrocytes, may differ, and the signaling mechanisms are not yet identified. So far, growth factors (EGF, FGF2, or VEGF), and nucleotides (adenosine, AMP, ADP, and ATP), as well as other factors such as sonic hedgehog (SHH), are suggested to be involved in proliferation induction of mature astrocytes.

The effect of extracellular nucleotides on proliferation of astrocytes has been investigated in chicken astrocytes. Aqueous extracts of the brain of chicken embryos have been used to induce proliferation of astrocytes. After purification and analysis of the extracts, which did not contain any amino acids, adenosine- 5'-monophosphate (AMP) was identified to be the mitogenic substance. Based on this finding, several other adenosine compounds were tested, and found to activate proliferation of astrocytes as measured by incorporation of [³H]-thymidine into newly synthesized DNA (Rathbone et al. 1992). The induction of proliferation in astrocytes by extracellular nucleotides or rather structural analogues, which activate P2 receptors, could be further confirmed in vivo by microinfusion into the rat nucleus accumbens (Franke et al. 1999).

Several other studies suggested a role for EGF in proliferation induction. It has been shown that EGF induced the proliferation in contact-inhibited rat cortical astrocytes. As- trocytes seeded at high density to ensure contact inhibition upregulated the postmitotic protein p27, a cyclin dependent kinase inhibitor, and exited cell cycle. When exposed to

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EGF, they re-entered the cell cycle as measured by BrdU incorporation, and formed several layers of cells. Although EGF did not alter p27 expression, it induced cyclin D1 expression, which supports the progression into S phase (Nakatsuji and Miller 2001). An- other study observed proliferation of astrocytes exposed to EGF, which was mediated by ERK activation. However, they also suggested a role for FGF2 in proliferation induction as EGF transactivated expression of FGF2 mRNA (Mayer et al. 2009). A study, in which proliferation of rat cortical astrocytes was directly induced by FGF2, confirmed again the involvement of ERK activation. Stimulation of cells with FGF2 induced the expression of cyclin D1/A and the proliferation of cells, which was even enhanced in the presence of ATP. ATP was shown to increase ERK activation by FGF2, which could be inhibited by U0126, an inhibitor of MEK, which is upstream of ERK (Neary et al. 2005). Further evidence for the involvement of FGF2 in proliferation induction of astrocytes was reported in an injury model of astrogliosis. Confluent primary astrocytes were scratch- wounded, and cell proliferation was quantified by BrdU incorporation. The addition of FGF2 increased the amount of proliferating cells around the scratch-wounded area. This effect was reduced, when cells were simultaneously treated with an anti-FGF2 antibody.

Interestingly, the neutralizing antibody itself decreased the proliferation of scratch- wounded astrocytes even in the absence of FGF2, indicating that endogenous levels of FGF2 induce proliferation of reactive astrocytes (Hou et al. 1995). The effect of FGF2 was also studied in vivo, by injecting the growth factor into different brain regions in rats.

After FGF2 injection, astrogliosis could be observed, involving strong upregulation of GFAP. However, only a minor part of the GFAP-positive cells were also positive for [³H]-thymidine, indicating that the increase in GFAP was not only due to the proliferation of astrocytes, but also due to an upregulation of the protein in non-proliferating cells (Eclancher et al. 1996). By contrast, EGF did not stimulate proliferation of astrocytes in vivo. Transgenic mice expressing TGFα, a member of the EGF family, displayed in- creased levels of GFAP in different brain regions, although proliferation of astrocytes could not be detected (Rabchevsky et al. 1998). Another signal for proliferation induction may be sonic hedgehog. SHH is mainly produced by reactive astrocytes themselves in response to neurodegeneration, and it was shown to induce the proliferation not only in astrocytes, but also in microglia and oligodendrocyte precursor cells (Pitter et al. 2014).

Thus, it is still not fully understood, which factors actually induce proliferation in mature astrocytes. A combination of different factors might be necessary, and transactivation of

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endogenous signals might be involved, complicating the identification of responsible signaling pathways. However, EGF, FGF2 as well as SHH are promising candidates, as they represent crucial signals also in the stem cell niches of the adult brain. Furthermore, EGF and FGF2 are standardly used in in vitro cell culture systems to expand and maintain neural stem cells in their proliferating state. In vivo, they show different effects on proliferation and differentiation of stem cells in the neurogenic niches of the adult brain. While both factors induced the proliferation of cells within the subventricular zone (SVZ) after intracerebroventricular administration, FGF2, but not EGF, favored the generation of newborn neurons (Kuhn et al. 1997). By contrast, EGF decreased the amount of newborn neurons, and favored the generation of astrocytes in the olfactory bulb. The increase in proliferation of cells in the subventricular zone by FGF2 could be further confirmed by measurement of [³H]-thymidine incorporation after FGF injection (Wagner et al. 1999), whereas injection of neutralizing antibodies against FGF2 inhibited the proliferation (Tao et al. 1997). SHH is another candidate signal, which regulates proliferation as well as neurogenesis of adult neural stem cells (Lai et al. 2003; Palma et al. 2005).

Several attempts have been made to isolate stem cells from outside the neurogenic niches.

Interestingly, there exist species-specific differences regarding the presence of stem cells in the adult brain. While stem cells can be isolated from several brain regions as well as the spinal cord in rats, the stem cells niches in mice seem to be restricted to the SVZ and the SGZ (Grande et al. 2013; Palmer et al. 1999; Sirko et al. 2013). Putative stem cells isolated from outside the neurogenic niches, however, could not be further passaged, and did not generate neurons. Furthermore, the origin of these stem cells, is still not clarified.

The only dividing cells outside the neurogenic niches are cells expressing the neural/glial antigen 2 (NG2), an integral membrane proteoglycan. These NG2-glia are slow-dividing cells supposed to be oligodendrocyte precursor cells (Nishiyama et al. 2014). It is known that these cells migrate to the site of lesion, and are able to proliferate to maintain their pool (Hughes et al. 2013).

Under pathophysiological conditions, astrocytes outside the neurogenic niches might regain the potential to generate neural stem cells. Besides re-entering the cell cycle, reactive astrocytes also adopt a more immature phenotype re-expressing markers, which are normally found in neural stem cells, and which are downregulated in mature astrocytes.

These markers include nestin, vimentin, the brain lipid-binding protein (BLBP), or the extracellular matrix protein tenascin-C (Pekny and Pekna 2004; Robel et al. 2011;