Comparative Analyses of the Neurogenic Capacity of Human Neuroprogenitor Populations Derived from Neural and Mesodermal Tissue

Abteilung für Neurologie

Leiter: Prof. Dr. A. C. Ludolph

Universität Ulm

Comparative analyses of the neurogenic capacity of human neuroprogenitor populations derived from neural and mesodermal tissue

Dissertation zur Erlangung des Doktorgrades

Dr. hum. biol.

der

Medizinischen Fakultät

der

Universität Ulm

vorgelegt von

Martina Wölfle, geb. Maisel

geboren in Böblingen

Ulm im Mai 2007

Amtierender Dekan: Prof. Dr. Klaus-Michael Debatin

1.Gutachter: Prof. Dr. Alexander Storch

2.Gutachter: Prof. Dr. Tobias Böckers

Tag der Promotion: 16. November 2007

Mach` immer was dein Herz dir sagt

Meiner Familie

Table of contents

1 INTRODUCTION 1

1.1 NEURAL STEM OR PROGENITOR CELLS 2 1.2 SOURCES OF NEUROPROGENITORS 3 1.2.1 FETAL MAMMALIAN BRAIN 3 1.2.2 NEUROGENESIS IN THE ADULT MAMMALIAN BRAIN 5 1.2.3 MESODERMAL STEM CELLS 7 1.2.4 NEUROECTODERMAL CONVERSION OF MESODERMAL STEM CELLS 8 1.3 DIFFERENTIATION CAPACITY OF NEUROPROGENITORS 9 1.3.1 DIFFERENTIATION POTENTIAL OF FETAL NEURAL PROGENITOR CELLS 9 1.3.2 DIFFERENTIATION CAPACITY OF ADULT NEUROPROGENITOR CELLS 10 1.4 AIM OF THIS THESIS 11

2 MATERIALS AND METHODS 13

2.1 ADULT HUMAN HIPPOCAMPAL NEURAL PROGENITOR CELL ISOLATION 13 2.2 FETAL HUMAN CORTICAL NEURAL PROGENITOR CELL ISOLATION 14 2.3 ADULT HUMAN MESENCHYMAL STEM CELL ISOLATION 15 2.4 NEUROECTODERMAL CONVERSION PROTOCOL 15 2.5 ADULT HUMAN HIPPOCAMPAL TISSUE ISOLATION 16 2.6 PA6-CELLS CULTURE CONDITIONS 16 2.7 MOUSE EMBRYONIC FIBROBLASTS CULTURE CONDITIONS 16 2.8 DIFFERENTIATION CONDITIONS FOR HUMAN NEUROPROGENITOR CELLS 16 2.9 FLOW CYTOMETRY 17 2.10 IMMUNCYTOCHEMISTRY 17 2.11 TELOMERASE ACTIVITY 18 2.12 ELECTROPHYSIOLOGY 18 2.13 CELL COUNTING AND STATISTICS 19 2.14 RNA EXTRACTION AND QUANTITATIVE REAL-TIME RT-PCR ANALYSIS 19 2.15 MICROARRAY ANALYSIS 20 2.16 DETERMINATION OF GABA, GLUTAMATE, DOPAMINE AND SEROTONIN PRODUCTION 21

Table of contents

3 RESULTS 22

3.1 CHARACTERIZATION OF ADULT HUMAN NEUROPROGENITORS 22 3.1.1 ISOLATION AND LONG-TERM EXPANSION OF NEUROSPHERE FORMING CELLS FROM THE ADULT HUMAN HIPPOCAMPUS 22 3.1.2 CHARACTERIZATION OF GENE EXPRESSION IN HUMAN ADULT NEUROPROGENITORS, COMPARED WITH FETAL NEUROPROGENITORS AND MESODERMAL STEM CELLS 26 3.1.3 DIFFERENTIATION CAPACITY OF HUMAN ADULT HIPPOCAMPAL NEUROPROGENITORS 45

4 DISCUSSION 51

4.1 ISOLATION AND CHARACTERIZATION OF HUMAN ADULT PROGENITOR CELLS 51 4.2 CHARACTERIZATION IN GENE EXPRESSION PATTERN OF ADULT NPCS, FETAL NPCS AND MESENCHYMAL HMSCS 52 4.3 DIFFERENTIATION CAPACITY OF HUMAN ADULT NPCS 57 4.4 THERAPEUTICAL POTENTIAL OF DIFFERENT NEURAL AND NEURAL-LIKE CELL POPULATIONS 59

5 SUMMARY 61

6 REFERENCES 62

7 APPENDIX A 76

8 APPENDIX B 78

9 DANKSAGUNG 89

List of abbreviations

AP action potential BrdU 5-bromo-2-deoxyuridine CD cluster of differentiation CFU-F colony forming unit fibroblasts CM conditioned medium CNS central nervous system DAPI 4´, 6 diamidin-2´-phenyl-indol-dihydrochlorid DG dentate gyrus DNA desoxyribonuclein acid EGF epidermal growth factor EGTA ethylene glycol tetraacetic acid ES-cells embryonic stem cells FACS fluorescence activated cell sorting (also flow cytometry) FN fibronectin GABA gamma aminobutyric acid GalC galactosylceramidase GDNF glial-derived neurotrophic factor GFAP Glial fibrillary acidic protein ICM inner cell mass IL interleukin LIF leukemia inhibitory factor LV laterale ventricle aNPCs adult human hippocampal NPCs hmNSCs human marrow-derived neural stem cell-like cells (also hMSC-NSC) hMSCs human marrow stromal cells HPLC high performance liquid chromatography MAP2 microtubule associated protein MAPCs multipotent adult progenitor cells MBP myelin basic protein MEFs mouse embryonic fibroblasts MRI magnetic resonance imaging mRNA messenger ribonucleic acid MSCs marrow stromal cells NCAM neural cell adhesion molecule NGFR NGF receptor NMDA N-Methyl-D-Aspartat NPCs neural progenitor cells NSCs neural stem cells NSE neuron specific enolase PD Parkinson’s disease PDGFR-B platelet derived growth factor receptor B PI-FACS propidium iodide FACS PLP proteo-lipid protein PSA-NCAM polysialic acid NCAM RMP resting membrane potential RMS rostro-migratory stream RNA ribonuclein acid RT-PCR reverse transcriptase polymerase chain reaction SGZ subgranular zone Shh sonic hedgehoc SVZ subventricular zone TH tyrosine hydroxylase Tuj1 neuron specific class III β-tubulin

Introduction

1 Introduction

Stem cells derived from embryonic, fetal or adult sources have great therapeutic potential, but much research is still needed before their clinical uses become commonplace. Not only Parkinson’s or Alzheimer’s diseases become more frequent with increasing lifespan, but stroke and traumatic brain injury are also responsible for a decline of neuronal functions. Furthermore, in chronic inflammatory diseases, such as multiple sclerosis, a loss of several types of neuroectodermal cells occurs. There is therefore a growing need for therapeutic approaches to restore neural cell loss or reconstitute their physiological functions. Cell transplantation is one of the strategies with a great potential for treatment of such neurological disorders, and many kinds of cells, including embryonic stem cells (ES-cells) and neural progenitor cells (NPCs), have been considered as candidates for transplantation approaches (for review see (McKay 1997; Gage 2000; Storch et al. 2002; Isacson et al. 2003; Hermann et al. 2004a)). As these cell systems fulfill many important requirements for the use of cells in regenerative treatment strategies (Lee et al. 2000; Hermann et al. 2004a), such as the generation of high yields of cells from a small starting population without losing their differentiation potential, on-demand availability of cells without major logistical problems, and the possibility to standardize the cell source in a clinical setting.

By definition a stem cell is an undifferentiated cell type that can produce daughter cells and either remains a stem cell in a process called self-renewal, or commits to a specific cell type via the initiation of a differentiation pathway leading to the production of mature progeny. Stem cells are classified according to their developmental potential as totipotent, pluripotent, multipotent or unipotent. A totipotent stem cell can give rise to a new individual if provided with appropriate maternal support. Thus the zygote and its immediate progeny around the blastula stage are totipotent cells, because each individual cell can give rise to all embryonic and extra-embryonic tissues required for mammalian development (Brook and Gardner 1997). After blastocyst formation occurs, the cells of the Inner Cell Mass (ICM) are pluripotent because they can give rise to all cell types of the embryo proper, including somatic and germ cells. During development, a stepwise commitment of cell-fate occurs, starting from the totipotent ES-cell, followed by germ-layer commitment (pluripotent stem cell with the potential to differentiate in all cell-types of the respective germ-layer) and further lineage restriction (multipotent stem cell), ending up in the terminal differentiated cell stage.

1 Introduction

Embryonic development and the subsequent adult life are viewed as a continuum of decreasing potencies. According to this classification, postnatal or adult stem cells are multipotent if they are able to differentiate into multiple cell types of a single tissue.

1.1 Neural stem or progenitor cells

Neural stem cells (NSCs) are tissue-specific stem cells. Their discovery in the nervous system was a major event in contemporary neurobiology (Gage et al. 1995). Compared with totipotent ES-cells, neural stem and progenitor cells have been categorized as multipotent tissue-specific stem cells producing all kinds of brain-specific cell types such as astroglia, oligodendroglia and neurons and may also replace or repair diseased brain tissue. Neural stem cells are characterized by the same main properties as stem cells in general, e.g. retention of the ability to divide (which is lost at the neuroblast stage) and pluripotentiality, which is the ability to differentiate in different directions. NSCs are single cells purified from neurogenic regions of the CNS. They were first seen in brain structures known for active neurogenesis throughout life: the subependymal, subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampal formation (Altman 1969; Cameron et al. 1993; Lois and Alvarez-Buylla 1993; Eriksson et al. 1998; Gould et al. 1999; Taupin and Gage 2002). Clonogenic NSCs can be directly isolated from fetal or adult nervous tissue, or derived from ES-cells (Cattaneo and McKay 1990; Kilpatrick and Bartlett 1993; McKay 1997; Ling et al. 1998; Svendsen et al. 1999; Vescovi et al. 1999; Gage 2000). Since the multipotent differentiation behavior could not be conclusively demonstrated in most cases (mostly due to problems with cloning), most authors call these cells neuroprogenitor cells (NPCs).

Various molecular markers identifying NSCs and their sequential differentiation stages are known (Gage et al. 1995). The first marker defining NSCs was the intermediate filament protein nestin, but some neural precursor cells are nestin- negative (Kukekov et al. 1997) and, on the other hand, nestin is expressed by other cell types, including non-neuronal cells (Selander and Edlund 2002) It should however be noted that these markers are arbitrary and their significance depends in part on the state and microenvironment in which the cells are located or isolated from. Thus, NSCs are usually identified operationally by their biological behavior after isolation. During expansion, they normally grow in floating, multicellular aggregates, so called ‘neurospheres’ (McKay 1997; Gage 2000). Further markers that define NSCs have recently been developed (Gage 2000; Uchida et al. 2000; Sawamoto et al. 2001; Vogel et al. 2003). Uchida and colleagues performed an

2 Introduction extensive analysis of surface markers of human neurospheres. They defined a subset of human NSCs as phenotypically CD133-positive, but negative for the hematopoietic stem cell marker CD34 and negative for CD45 (Uchida et al. 2000). They characterized commercial available cells as CD15, CD56, CD90, CD164, p75(NGFR), 57D2 and W4A5 positive, whereas the cells are negative for CD45, CD105 (endoglin), CD109, CD140b (PDGF-RB) and W8B2 (Vogel et al. 2003).

Recently the expression of various proneural genes like SOX1, Musashi1, Otx-1, Otx2, NeuroD1 and Neurogenin 2 was reported in mammalian NPCs, showing their neuroectodermal origin (Aubert et al. 2003; Cai et al. 2003). Although the field of NSC research is very young compared with e.g. bone marrow stem cells, there have been great advances in recent years in understanding neural development and the basic biology of NPCs.

1.2 Sources of neuroprogenitors

NPCs can be either extracted from fetal or adult nervous tissue and proliferated in culture (Cattaneo and McKay 1990; Kilpatrick and Bartlett 1993; McKay 1997; Ling et al. 1998; Svendsen et al. 1999; Vescovi et al. 1999; Gage 2000), or embryonic stem (ES) cells can be extracted, proliferated and differentiated into neural precursor cells (Fig. 1). Furthermore, there is evidence for multipotent adult stem cells from other tissues, which seem to have the potential to transdifferentiate into neuroectodermal lineages (Toma et al. 2001; Jiang et al. 2002b; Sanchez-Ramos 2002). ES-cells are totipotent and can be isolated from the inner cell mass of the preimplantation blastocyst, which give rise to all cells in the organism. ES-cells can be differentiated into neural stem cells and subsequently into neurons (Kawasaki et al. 2000; Lee et al. 2000; Kawasaki et al. 2002). However, their proliferative potential seems to be responsible for the high risk of tumor formation after transplantation of ES-cells (Amit et al. 2000; Bjorklund et al. 2002). In addition, there are immense ethical concerns regarding the use of human ES-cells and government restrictions that will, at least for the forthcoming years, render it unlikely that these cells will be therapeutically employed in many countries.

1.2.1 Fetal mammalian brain

In contrast to ES-cells, multipotent stem cells are also able to regenerate, but are believed to have a more restricted potential than ES-cells, and are often defined by the organ from which they are derived. NSCs have been categorized as multipotent stem cells derived from the nervous system with the capacity to regenerate and to give rise to cells belonging to all three major cell lineages of the

3 Introduction nervous system, namely neurons, oligodendrocytes and astrocytes (Cattaneo and McKay 1990; Kilpatrick and Bartlett 1993; McKay 1997; Ling et al. 1998; Potter et al. 1999; Svendsen et al. 1999; Vescovi et al. 1999; Gage 2000; Akiyama et al. 2001; Arsenijevic et al. 2001). Most studies involved fetal midbrain tissue taken from rodent embryos at embryonic day (ED)14 to ED15 (Ling et al. 1998; Potter et al. 1999; Storch et al. 2003) On human midbrain material, samples of 6-9 weeks post-fertilization were used (Storch et al. 2001). For the expansion of NSCs, the cells are plated in low-attachment culture flasks in serum-free media containing serum supplements, such as N2 or B27 (Vescovi et al. 1999; Rietze et al. 2001; Storch et al. 2001; Storch et al. 2003). The studies by Gensburger and co-workers (1987) describing that fibroblast growth factor-2 (FGF-2; formerly basic fibroblast growth factor) can induce proliferation of neural precursors in embryonic hippocampal cultures initiated a new field of cell culture experiments studying proliferation of neural precursors in vitro (Gensburger et al. 1987). Later, epidermal growth factor (EGF) was found to stimulate the growth of striatal precursors retaining their ability to differentiate into all major types of CNS cell types (Reynolds et al. 1992). Since these early studies, most protocols for the in vitro proliferation of embryonic, fetal and adult NSCs use both mitogens (FGF-2 and EGF) alone or in combination in serum-free-media (for review see (Svendsen et al. 1999)). Various other epigenetic approaches have been employed for in vitro cultivation of NSCs including erythropoietin (Shingo et al. 2001), and reduced atmospheric oxygen (Studer et al. 2000; Storch et al. 2001). For some of these proliferating factors it is not completely clear how these factors influence the differentiation potential into neurons and glial cells of the NSCs (Whittemore et al. 1999). Another established neuroproliferative substance is leukemia inhibitory factor (LIF): When added to the culture medium, LIF allows continuous cell growth in human NSC cultures (Carpenter et al. 1999). LIF acts through the gp130 signal transducing subunit and is also required for the continuous growth of mouse, but for example not human ES-cells. LIF appears to maintain these cultures in a proliferative state by preventing differentiation. Another strategy to enhance proliferation and to increase culture time is genetic manipulation by transduction of immortalizing genes into neural progenitors (for review, see (Martinez-Serrano and Bjorklund 1997; Whittemore et al. 1999)). Fetal stem or progenitor cells grow like adult stem cells in cellular aggregates called “neurospheres”, but additionally they can be expanded as adherent cell cultures on poly-L-ornithin/fibronectin-coated surfaces with both EGF and FGF-2 as mitogens. However, in contrast to precursor cells derived from human forebrain samples, which are easily expanded by using

4 Introduction

EGF/FGF-2 in 21% O2, precursors derived from human mesencephalon do not show any proliferation under these conditions or do not differentiate into dopaminergic neurons in vitro (Svendsen et al. 1996; Svendsen et al. 1997; Storch et al. 2001).

Under these conditions, EGF/FGF-2-responding NPCs with dopaminergic potential from rodent mesencephalic and human midbrain tissue of less than 20 weeks postfertilization can be successfully expanded for over 1 year. However, the way in which these proliferation factors influence the differentiation of NSCs into distinct subsets of neurons or glial cells is currently not completely clear (Whittemore et al. 1999).

Fig. 1: Schematic overview of various sources of neural stem cells (NSCs) with the capacity to differentiate into all major cell types of the central nervous system, namely astroglia, oligodendroglia and neurons. Most protocols produce NSCs growing in multicellular spheroid aggregates called “neurospheres”.

1.2.2 Neurogenesis in the adult mammalian brain

New neurons are continuously added to neural circuits in the adult vertebrate brain. In mammals, including humans, neurons are added to restricted brain regions. Neurogenesis has been reported to occur in various areas of the adult brain, including the neocortex, spinal cord and the striatum (Gould et al. 1999; Bedard et al. 2002; Gould and Gross 2002; Dayer et al. 2003) and in granule neuron populations in both the hippocampal dentate gyrus (DG) and the subventricular

5 Introduction zone (SVZ) of the lateral ventricles (Fig. 2). Neurons in the DG, area of the hippocampus are born locally in the subgranular zone (SGZ) (Cameron et al. 1993; Kornack and Rakic 2001; van Praag et al. 2002). Newly generated neuronal cells in the SVZ of the lateral ventricles (LV) migrate to the olfactory bulb (OB), through the rostro-migratory stream (RMS), where they differentiate into interneurons of the OB (Corotto et al. 1993; Lois and Alvarez-Buylla 1994; Kornack and Rakic 2001).

A model for adult mammalian neurogenesis in the subventricular zone was developed: neuroepithelial cells probably transform into radial glia, which transform into astrocyte-like adult stem cells, so called Type B cells, positive for GFAP, which give raise to transit amplifying cells, so-called Type C cells. Type C-cells (Dlx2+) can further become restricted to neuroblasts, so-called Type A cells (PSA-NCAM+ and Dlx2+) (Doetsch 2003). Neuroepithelial cells divide symmetrically to increase the pool of stem cells, but later divide asymmetrically, generating a stem cell that remains in the ventricular zone and a daughter cell that migrates radially outward (Haubensak et al. 2004). The accumulation of daughter cells thickens the developing brain and radially streches neuroepithelial cells. This division in the SVZ amplifies the number of cells produced by a given NPC division and may be an important determinant of brain size, since species with larger brains have a larger pool of intermediate progenitors (Martinez-Cerdeno et al. 2006). Environmental factors do indeed influence adult NPCs and intermediate progenitors. In the SVZ, neurotransmitters regulate neural progenitor (type B- and C-) cells. (Ever and Gaiano 2005; Guillemot 2005; Hagg 2005). Synaptically released dopamine stimulates C-cells via the D2 receptor (Hoglinger et al. 2004). On the other hand, type B-cells are inhibited by γ-aminobutyric acid (GABA) non- synaptically released from newborn neurons in the SVZ (Liu et al. 2005). The hippocampus is the other major site of neurogenesis in adult mammals. Granule neurons in the dentate gyrus are born locally in the subgranular zone (SGZ). In contrast to the extensive tangential migration undertaken by olfactory bulb neurons, hippocampal granule move only a short distance into the granule cell layer. The SGZ contains SGZ astrocytes (B-cells) and ultrastructurally dark GFAP- negative D-cells (Seri et al. 2001).

Curtis and colleagues reported (Curtis et al. 2007) migration of NPCs along a rostral migratory pathway to the OBs in the human brain. NPCs often produce intermediate progenitors that exist transiently and amplify the number of daughter cells produced by a NPC division. Curtis and colleagues used human OBs from patients had been administered 5´-bromo-2´deoxyuridine (BrdU) for the purpose of tracing the development of laryngeal and pharyngeal carcinomas after death. BrdU

6 Introduction is a thymidine analog and labels cells in the S- phase of cell cycle (Dolbeare 1995, 1996). They identified colocalization of BrdU and NeuN in the same cells in the perglomerular layer. This work verified the studies from Doetsch and colleagues in non-human mammals.

Furthermore, a few other recent studies demonstrated the isolation of neural progenitor cells (NPCs) from different regions of the adult human brain including cortex, amygdala, hippocampus and SVZ (Johansson et al. 1999a; Kukekov et al. 1999; Roy et al. 2000; Arsenijevic et al. 2001; Nunes et al. 2003; Westerlund et al. 2003; Moe et al. 2005), but there are only a few reports about human adult NPCs from hippocampus in vitro (Johansson et al. 1999a; Kukekov et al. 1999; Roy et al. 2000; Hermann et al. 2006d; Maisel et al. 2007).

Fig. 2: Sagittal schematic overview of neurogenesis in the adult rodent and avian brain. In mice, high levels of neurogenesis occur in the subventricular zone (SVZ) and the hippocampus (HP), where most neurons are incorporated into the dentate gyrus. Neuroblasts born throughout the SVZ of the lateral wall of the lateral ventricle (LV) migrate along the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate into inhibitory interneurons (red dots). The cerebellum (CB) does not appear to incorporate new neurons in adulthood from (Doetsch and Scharff 2001).

1.2.3 Mesodermal stem cells

Multipotent adult stem cells are of special interest with regard to autologous transplantation approaches, probably without immunological rejection. An important source of multipotent adult stem cells is bone marrow stromal cells (MSCs); also known as mesodermal stromal cells or mesenchymal stem cells. In contrast to human ES-cells or human brain-derived NPCs, human MSCs (hMSCs) are easy to isolate and can be expanded over a long period of time without serious ethical and technical problems. MSCs can be cultivated in vitro and contain progenitors capable of generating osteocytes, chondrocytes and adipocytes

7 Introduction

(Friedenstein et al. 1976; Reyes et al. 2001; Sekiya et al. 2002). MSCs are a homogenous population of fibroblast-like cells purified by density gradient centrifugation expanded in vitro and can be differentiated into multiple cell lineages, including bone, fat, tendon and cartilage (Pittenger et al. 1999). Several reports demonstrate that under specific experimental conditions, MSCs can also differentiate into cells that are not part of their normal repertoire: Skeletal and cardiac muscle, hepatocytes, glia and neurons (Azizi et al. 1998; Ferrari et al. 1998; Makino et al. 1999; Orlic et al. 2001; Sanchez-Ramos et al. 2001; Sanchez- Ramos 2002; Hermann et al. 2004b). Until now in hMSCs no functional tests have been reported (for review see (Hermann et al. 2006a)). Jiang and co-workers recently demonstrated a rare multipotent adult progenitor cell (MAPC) within MSC cultures from rodent bone marrow (Jiang et al. 2002b; Jiang et al. 2002a). These mouse MAPCs can be induced to differentiate in vitro into cells with biochemical, anatomical, and electrophysiological characteristics of neuronal cells by using a coculture system with astrocytes (Jiang et al. 2003).

1.2.4 Neuroectodermal conversion of mesodermal stem cells

Conversion between germ layer-committed or pluripotent stem cells is called transgerminal conversion, (e.g the conversion of MSCs into NSC-like cells (mNSCs) (Lee et al. 2003b; Hermann et al. 2004b)), whereas translineage conversion represents the conversion between lineage-restricted multipotent stem cells within one germ layer (e.g. the conversion of skin-derived progenitors into neuroectodermal stem cells (Joannides et al. 2004)). A novel approach for neuroectodermal specification of hMSCs is the two step technology, with initial conversion of hMSCs into immature NSC-like cells and subsequent terminal differentiation into mature neuronal and glial cell types (Lee et al. 2000; Hermann et al. 2004b; Bossolasco et al. 2005; Hermann et al. 2006b). Hermann and colleagues established a new protocol (Hermann et al. 2004b) to convert MSCs into cells with characteristics of immature neuroectodermal cells bearing all major characteristics of NSCs. The hMSCs were cultivated in uncoated flasks in serum- free medium supplemented with the mitogens FGF-2 and EGF at 3% atmospheric oxygen. These mNSCs proliferated with an estimated doubling time of 2.6 days in vitro for at least 10 weeks (~5-30 population doublings) neither changing phenotype nor morphology (Hermann et al. 2004b). The extensive marker expression analyses revealed that 90% of MSCs express high levels of nestin, but only a few cells still express low levels of the mesodermal marker fibronectin.

8 Introduction

1.3 Differentiation capacity of neuroprogenitors

NSCs or NPCs derived from mammalian embryonic/fetal and adult CNS tissue have been noted to generate oligodendrocytes, neurons and astrocytes after the removal of growth factors or mitogens in serum-free media on standard culture growth surfaces, such as poly-L-lysine or laminin (Gritti et al. 2002). This differentiation program can be influenced by exposure to various factors, such as growth factors or cytokines. Consequently, it has been postulated that stem cells and restricted precursors exhibit regionalization. Indeed, neurospheres generated from different CNS regions express region-appropriate markers and generate region-appropriate progeny. Spinal cord stem cells generate spinal cord progeny (Mayer-Proschel et al. 1997), whereas stem cells from the forebrain generate more gamma-aminobutyric-acid-containing neurons than dorsal stem cells cultures under identical conditions (Potter et al. 1999). Consistently, only NSCs isolated from the midbrain differentiate into functional mature dopaminergic neurons (Ling et al. 1998; Potter et al. 1999; Sawamoto et al. 2001; Storch et al. 2001).

1.3.1 Differentiation potential of fetal neural progenitor cells

NPCs retain the capacity to differentiate into oligodendrocytes, neurons and astrocytes (Cattaneo and McKay 1990; Kilpatrick and Bartlett 1993; Ling et al. 1998; Potter et al. 1999; Svendsen et al. 1999; Vescovi et al. 1999; Akiyama et al. 2001; Arsenijevic et al. 2001) in a ratio of approximately 1:5:25 (Gritti et al. 1996; Johe et al. 1996). Neurons generated from expanded populations of neural progenitor cells are to a large extend GABAergic, like cells isolated from the forebrain (Potter et al. 1999). However, it is possible to alter this differentiation program by exposure to different factors, such as growth factors or cytokines. Most of these studies on the induction of a specific neuronal phenotype focus on generating dopaminergic neurons as a source for transplantation in Parkinson’s disease. In the CNS, the salient patterning in the anterior-posterior and dorsal- ventral axes occurs early, concomitantly with neural induction.

Since NSCs have been suggested as a new source of tissue for regenerative therapy in PD (Dunnett and Bjorklund 1999; Bjorklund et al. 2003), several studies on midbrain-derived NPCs have focused on the induction of a dopaminergic phenotype. During the last few years, many aspects of the genetic and epigenetic control of the induction and phenotypic maturation of dopaminergic neurons have been characterized (for review see (Perrone-Capano and Di Porzio 2000). For example, transcription factors, such Nurr1 and Ptx3, have been discovered to play an important role in this process. Furthermore, Nurr1 is reported to be up-regulated

9 Introduction by the differentiation factor interleukin-1 (IL-1) in mesencephalic precursor cells from rat (Carvey et al. 2001). The over-expression of Nurr1 consistently induces the midbrain dopaminergic phenotype after the stimulation of NPCs with astrocyte- conditioned medium (Wagner et al. 1999; Kim et al. 2003a; Kim et al. 2003b). Consistently, Ling and co-workers demonstrated with rat-derived neural progenitor cells that IL-1 is able to induce the dopaminergic phenotype only in midbrain NSCs, with a significantly increased effect by adding other factors, such as IL-11, LIF, glial-derived neurotrophic factor (GDNF) and membrane fragments, but not in striatal NSCs (Ling et al. 1998). These dopaminergic cells show all the major morphological features and the functional properties of mature dopaminergic cells, such as dopamine production/release and the expression of sodium and hyperpolarization induced cation channels (Storch et al. 2003).

1.3.2 Differentiation capacity of adult neuroprogenitor cells

Traditionally, adult stem cells have been defined as tissue-specific cells, able to differentiate into a spectrum of cells restricted to their tissue of origin. Multipotent neural stem cells have the capacity in vitro, similar to progenitors from the adult spinal cord, have the potential to give rise to cells belonging to all three major cell lineages of the nervous system; neurons, oligodendrocytes, and astrocytes (Lie et al. 2002; Shihabuddin 2002). In addition, isolated cortical progenitor cells have the potential to give rise to glia and neurons in vitro (Palmer et al. 1999). Cells isolated from the substantia nigra, cultured in the presence of FGF-2 (Palmer et al. 1997; Vescovi et al. 1999) or FGF-8 (Lee et al. 1997), which is a mitogen for progenitor cells in the developing midbrain (Lee et al. 1997) expressed nestin and the glial precursor NG2, and the cells were able to generate cells from all three neural lineages (Lie et al. 2002) Adult hippocampal-derived neuroprogenitors showed the same ability (Palmer et al. 1997). Hermann and colleagues (Hermann et al. 2006c) differentiated NPCs from the mouse midbrain and investigated their dopaminergic differentiation potential: They established the isolation, expansion and in vitro characterization of adult mouse midbrain NSCs and their differentiation into functional nerve cells including dopaminergic neurons. In the presence of selected growth factors, midbrain NSCs differentiated into neurons, oligodendroglia and astrocytes with an approximate ratio of 1:1.5:14, respectively. Looking for neuronal subtype specification, midbrain NSCs could be differentiated into cells expressing the markers for cholinergic, GABAergic or glutamatergic neurons with similar amounts of the distinct neuronal subpopulations with respect to the various differentiation protocols. Interestingly, no serotonergic cells could be observed with

10 Introduction any of the differentiation protocols tested. Electrophysiological analyses revealed functional properties of mature nerve cells, such as TTX-sensitive sodium channels, action potentials as well as GABA-, glutamate and NMDA-induced currents, with the latter ones known to be exclusively expressed by neuronal cells (Verkhratsky and Steinhauser 2000). With regard to human adult neuroprogenitor cells (aNPCs), there are only a few reports. After removal of mitogens and plating human aNPCs on poly-L-lysine, they spontaneously differentiate into a neuronal (MAP2ab+), astroglial (GFAP+), and oligodendroglial (GALC+) phenotype (Westerlund et al. 2003; Moe et al. 2005; Hermann et al. 2006d). Differentiated aNPCs showed functional properties of neurons, such as sodium channels, action potentials and production of the neurotransmitters glutamate and GABA. To further characterize their in vitro differentiation potential co-culture experiments of aNPCs with astrocytes, MEFs and PA6 cells were reported: Interestingly, co-culture on all feeder cells increased neurogenesis of aNPCs, while only MEFs and PA6 cells also led to a morphological neurogenic maturation seen by elongated neurites. Remarkably, co-culture on astrocytes increased the differentiation of aNPCs towards astroglial lineages, which is controversial to data on mice SVZ-NSCs (Palmer et al. 1995), but could be explained by different cell sources for astrocyte cultures in the two studies. However, future studies are warranted to show detailed neuron subtype specification as well as to further define functional neuronal properties including action potentials accompanied by synaptic neurotransmitter release in fully differentiated aNPCs (Lois and Alvarez-Buylla 1994; Rakic 2002; Nunes et al. 2003).

1.4 Aim of this thesis

The overall scope of my thesis has been to identify possible adult human cell sources which fulfill important requirements for the use in autologous neurorestorative treatment strategies: (i) Generation of high yields of cells out of a small starting population without losing their differentiation potential, (ii) on-demand availability of cells without major logistical and ethical problems and (iii) the possibility to standardize the cell source in a future clinical setting. To date adult tissue-specific neural stem or progenitor cells might be a cell source which fulfills these characteristics.

Thus, I used two different starting populations of human adult neural progenitor cells to check for the mentioned properties by characterizing their biological properties during expansion and defining their neurogenic potential:

11 Introduction

1) Neural progenitor cells isolated from adult human hippocampus derived from epileptic surgery procedures (selective hippocampectomy or medio-temporal lobectomy).

2) Neural progenitor-like cells derived from human adult mesodermal stem cells from bone marrow by a two-step conversion protocol.

Both progenitor populations have been characterized with respect to their neurogenic potential in vitro and compared with a wide range of various neural and neural-like cell populations from different origins: Fetal neural stem cells, mesodermal stem cells as a multipotent adult stem cell control and tissue from adult hippocampus as well as various brain tissue samples from databases. The Affymetrix chip technology has been used to compare the complexities of the transcriptomes of these cell/tissue types to define a classification for the adult human neural progenitor cell populations.

12 Materials and Methods

2 Materials and Methods

2.1 Adult human hippocampal neural progenitor cell isolation

Adult human hippocampal tissue was obtained from routine epilepsy surgery procedures (selective amygdalo-hippocampectomy or anterior temporal lobectomy; 14 samples; age 18-55 years) following informed consent of the patients. All procedures were in accordance with the Helsinki convention and approved by the Ethical Committee of the University of Ulm (219/2001) and the Technical University of Dresden (EK 47032006). The tissue was taken from the removed hippocampi and stored in ice-cold Hank’s balanced salt solution (HBSS; Gibco BRL, Life Technologies; Tulsa, UK) supplemented with 11 mM glucose and 1% penicillin/streptomycin (Gibco BRL, Life Technologies; Tulsa, UK) for transporting the tissue from the operating theatre. All patients underwent high-resolution magnetic resonance imaging (MRI) excluding tumors and were screened for the presence of infectious disease. In all cases, MRI and neuropathological examination did not reveal evidences for tumor formation. For expansion of neurospheres consisting of human aNPCs, tissue samples were cut into small pieces with a scalpel, incubated in 0,1% trypsin (Sigma, St Louis, MO) for 30 min. at room temperature, incubated in DNase (40 mg/ml; Sigma, St Louis, MO) for 10 min. at room temperature and homogenized to a quasi single cell suspension by gentle triturating (Storch et al. 2001). The cells were added to 25 cm2 flasks (2×3 106 viable cells per flask) in knock-out DMEM (Gibco BRL, Life Technologies; Tulsa, UK), supplemented with 10% serum replacement (Gibco BRL, Life Technologies; Tulsa, UK), 0.5 mM glutamine, 1% penicillin/streptomycin (Gibco BRL, Life Technologies; Tulsa, UK) and 20 ng/mL of EGF and FGF-2 (both from

Sigma) at 5% CO2; 92% N2 and 3% O2 using an incubator equipped with an O2- sensitive electrode system (Heraeus, Germany). After 10-20 days, neurosphere formation was observed and these spheres were expanded for additional 5-8 weeks (in total 7 to 12 weeks, 5-12 passages) before differentiation was initiated. The medium was changed once a week, while the growth factors were added twice a week. For BrdU labeling, cells were incubated for 30 h with 10 µM BrdU (Sigma- Aldrich, Germany).

The patient for the genetically characterization with Affymetrix-chip-technology was a white female who was 15 years of age at the time of the temporal resection. She had a severe meningo-encephalitis at the age of 6 months, a delayed development and is still mildly mentally retarded. She suffered from pharmacoresistent, left-

13 Materials and Methods sided temporal lobe epilepsy with epigastric auras and complex focal seizures since 4 years of age. Carbamazepin, sultiam, vigabatrine, oxcarbazepin and levetiracetam were applied without sufficient success. Before the epilepsy surgery, she was treated with a combination of oxcarbazepine (1050 mg per day) and levetiracetam (2750 mg per day) and still had daily complex focal seizures. On MRI, she had left-sided widespread temporal lobe atrophy and hippocampal sclerosis. During the presurgical work-up using intracranial electrodes and video- EEG monitoring starting 12 days before surgery, levetirazetam was reduced and completely discontinued 7 days prior to surgery. Two days before surgery, she was put on 1500 mg oxcarbazepine daily as a monotherapy. The anterior temporal lobe including amygdala and hippocampus was then resected. The histology showed a scar of the temporal lobe with leptomeningofibrosis and signs of chronic and acute inflammation, which were attributed to a combination of a residuum from meningoencephalitis at 6 months of age and the meningeal irritation after implantation of subdural electrodes: and a depth electrode within the hippocampus 12 days before. Furthermore, reactive astroglial proliferation within the amygdala and hippocampal sclerosis was observed.

2.2 Fetal human cortical neural progenitor cell isolation

Human fetal brain tissue samples were harvested and supplied by Advanced Bioscience Resources Inc. (Alameda, CA, USA) according to NIH and local IRB guidelines. The cells were cultured in the research group of Johannes Schwarz, Leipzig. Only if there was no evidence for fetal pathology and informed consent was obtained, parts of the brain were transferred to our laboratory. The quality of the tissue was confirmed by initiating primary cultures and immunohistochemical characterization. NPCs were isolated from fetal mesencephalon and induced to proliferate as free-floating neurospheres. For that purpose the tissue samples were mechanically dissociated with a scalpel, sequentially incubated in 0.1% trypsin and 40 mg/ml DNase and expanded using EGF and FGF-2 as mitogens under reduced atmospheric oxygen (3%) as described previously (Storch et al. 2001). Fetal neurosphere formation was observed after 10-20 days. Neurospheres could be propagated up to 12 weeks. RNA was isolated after four weeks of propagation.

14 Materials and Methods

2.3 Adult human mesenchymal stem cell isolation

Adult human bone marrow samples were harvested from routine surgical procedures (pelvic osteotomies) following informed consent and in accordance with the terms of the ethics committees of the University of Ulm or the Technical University of Dresden. For cell culture, mesenchymal samples were rinsed twice with PBS (Invitrogen, Karlsruhe, Germany) supplemented with antibiotic solution (100 units/ml penicillin, 100 µg/ml streptomycin; Biochrom, Berlin, Germany) to eliminate contaminating blood cells, minced finely and digested with 0.4% collagenase (Roche, Mannheim, Germany) for 1 h at 37°C. After filtration through a 40µm pore membrane, the cells were washed twice in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Karlsruhe, Germany) containing 10% fetal calf serum (FCS; Biochrom, Berlin, Germany) and antibiotic solution (100 units/ml penicillin, 100 µg/ml streptomycin), counted and plated at low density containing 5×104 isolated cells/cm2. DMEM supplemented with 10% FCS was used as a medium during the proliferation phase. The cultures were incubated at 37°C in a humidified

5% CO2 atmosphere and media were changed three times a week. Cultures were split 1:2 by trypsin treatment (0.05% trypsin, 0.02% EDTA; Biochrom, Berlin, Germany) at 75% confluence. hMSC phenotype was proven by FACS analysis (see section 2.7 for details) with CD9+, CD90+, CD105+, and CD166+, as well as CD14-, CD34-, and CD45-, and by testing the potential to differentiate into osteoblasts, chondrocytes and adipocytes. MSCs were expanded up to passage 10 ( 50 population doublings) and used for RNA isolation as described in section 2.14.

2.4 Neuroectodermal conversion protocol

After passage 2-10 of hMSCs (»10-50 population doublings), conversion of hMSC into neurosphere-like structures was initiated. Specifically, cells were dissociated with 0.05% trypsin/0.04% EDTA and plated on low-attachment plastic tissue culture flasks (Nalge Nunc International, Rochester, NY, USA) at a concentration of 1-2´105 cells/cm2 in P4-8F medium (AthenaES, Baltimore, MD) supplemented with

20 ng/ml of both EGF and FGF-2 (both from Sigma, St Louis, MO) at 5% CO2, 92%

N2 and 3% O2. After 10-20 days, sphere formation could be observed. These neurosphere-like structures were expanded for an additional 2-10 weeks (2-4 passages; »5-30 population doublings) before glial or neuronal differentiation was started (section 2.6). The medium was changed once a week and growth factors were added twice a week.

15 Materials and Methods

2.5 Adult human hippocampal tissue isolation

Adult hippocampal tissue was obtained by surgical resection from male patient with epilepsy, 35 years of age following informed consent of the patient. High-resolution magnetic resonance imaging and neuropathological examinations did not reveal any evidences for tumor formation or infectious diseases. The tissue was stored immediately in RNA later (Qiagen, Hilden, Germany) until RNA isolation (see section 2.12).

2.6 PA6-cells culture conditions

For conditioned media experiments confluent PA6 cell layers were washed three times with the medium described above for coculture experiments and incubated further with the same medium. PA6-cells were cultured in α-MEM (Gibco BRL, Life Technologies; Tulsa, UK) with 10% FCS (Sigma, St Louis, MO). The conditioned media was D-MEM high glucose/F12 (50:50) containing 5% fetal calf serum, 5% horse serum; 1% penicillin/streptomycin., conditioned by exposure to PA6 cells for 72 h, then the medium was removed, centrifuged at 1800 g for 15 min, and the supernatant was used immediately or stored at - 20°C until further use. For these experiments, aNPCs were seeded on PDL at a concentration of 1.5–2.0x 105 cells/ cm2 similar to coculture experiments, but the media were supplemented with PA6 conditioned medium (50%). Medium change was performed on day 4 and every other day following that.

2.7 Mouse embryonic fibroblasts culture conditions

Mouse embryonic fibroblasts were cultured in DMEM-high-Glucose, 1% penicillin/streptomycin, 1% non-essential amino acids (Gibco ), 0.5 mM glutamine, 1% sodium-pyruvat (Sigma), 0,07% ß-mercaptoethanol and 15% fetal calf serum. Cells were seeded at a concentration of 1.5–2.0x 105 cells cm2. Medium change was performed on day 4 and every other day following that.

2.8 Differentiation conditions for human neuroprogenitor cells

Induction of neural differentiation was initiated by plating the cells on poly-D-lysine (PDL) coated glass cover-slips in Knock-out-DMEM, 10% serum replacement, 0.5 mM glutamine, 1% penicillin/streptomycin, 10 ng/ml rh-BDNF (Promega, Madison, WI), 100 µM di-butyryl-(db)-cAMP and 0.5 µM retinoic acid (both from Sigma). Cells were differentiated for 14 days. For co-culture analyses, various feeder cells (mouse embryonic fibroblasts (MEFs)), PA6 stromal cells as well as primary mixed cortical cultures (containing astrocytes and neurons) from E17 (embryonic day 17)

16 Materials and Methods mouse embryos were cultured on gelatine-coated cover-slips as described earlier (Kawasaki et al. 2000; Storch et al. 2001). In co-culture experiments, aNPCs were pre-stimulated with expansion media supplemented with 500 ng/ml Shh and 100 ng/ml FGF-8 for 48h prior to seeding the cells. When co-culture was started, confluent feeder layers were washed twice with PBS. The aNPCs were seeded at a concentration of 1.5-2.0×105 cells cm-2 in D-MEM high glucose/F12 (50:50) containing 5% fetal calf serum, 5% horse serum; 1% penicillin/streptomycin.

2.9 Flow cytometry

Human aNPCs were treated with Accumax® (Gibco) and washed with PBS. Dead cells were excluded from analysis by forward scatter gating. Samples were processed using a FACSCalibur flow cytometer and analyses were performed with the Cellquest software (both from Becton Dickinson, Franklin Lakes NJ). Antibodies for the aNPCs were used as follows: CD15-FITC 1:10, CD34-FITC 1:10, CD45-PE 1:10, CD133-PE 1:10 (all from Miltenyi Biotech, Bergisch Gladbach, Germany). A minimum of 10,000 to 12,000 events were acquired for each sample. In brief, the cells were dissociated by trypsin (Gibco) and washed with PBS. The cells were fixed with 80% ice-cold ethanol and incubated at -20°C for 30min. Then they were washed with PBS and PI stained (0.5mg/ml PI in 0.038 M sodium citrate buffer; pH 7.0) for 30min in the dark (37°C). Dead cells were excluded from analysis by forward-scatter gating. A minimum of 10,000 to 12,000 events were acquired for each sample.

2.10 Immuncytochemistry

Cell cultures were fixed in 4% paraformaldehyde in PBS (20min) or with 4% paraformaldehyde/PBS followed by ice-cold acidic ethanol and 2 N HCl for BrdU staining. Immunocytochemistry was carried out using standard protocols, blocking after fixation with 3% donkey-serum in PBS (0,2% Triton) for 2h at RT. Primary antibodies were cultivated over night in 4°C. Cells were washed with PBS, after this cultivated with a fluorescence-labeled secondary antibodies for 1h in the dark. Cell nuclei were counter stained with 4,6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: Mouse anti human nuclei 1:200; mouse anti-Nestin 1:500, rabbit anti-GFAP 1:1000, mouse anti-GalC 1:750, rabbit anti- Neurogenin2 1:500, rabbit anti-Musashi1 1:500, rabbit anti-NeuroD1 1:500 (all from Chemicon International, Temecula, USA); rabbit anti-Tuj1 1:2000, mouse anti-Tuj1 1:500 (both from Covance, Richmond, CA); rabbit anti-Ki67 1:500 (Novocastra, Newcastle, UK), rabbit anti-Olig2 1:2000 (kindly provided by Dr. Takebayashi) and

17 Materials and Methods secondary antibodies conjugated to Alexa 488, 567 or 648 1:500 (all from Invitrogen-Molecular Probes, Carlsbad, CA, USA), and rat anti-BrdU 1:40 with fluorescence labeled secondary antibody (both from Abcam, Cambridge, UK). Images were captured using a fluorescence microscope (Axiovert 135, Zeiss, Oberkochen, Germany) or a Leica TCS/NT confocal microscope equipped with krypton, krypton/argon and helium lasers.

2.11 Telomerase activity

A highly sensitive in vitro assay known as the quantitative real-time telomeric repeat amplification protocol (Emrich et al. 2002; Saldanha et al. 2003) has been used for detecting telomerase activity (OTD kit; Allied Biotech, Ijamsville, MD, USA). The telomerase activity in the cell or tissue extract is determined through its ability to synthesize telomeric repeats onto an oligonucleotide substrate in vitro. Telomerase from the cell extract or tissue adds telomeric repeats onto a substrate oligonucleotide and the resultant extended product are subsequently amplified by PCR. The PCR products are then visualized using DNA fluorochromes SYBR Green as described above. Mouse ES-cells (D3 ES cell line)- kindly provided from Thomas Seufferlein were used as positive control and cultured as described previously (Chung et al. 2002), whereas adult mouse cortical tissue was used as negative control (Milosevic et al. 2005).

2.12 Electrophysiology

Cells were investigated for membrane currents 3-10 days after initiation of differentiation on PDL using the standard whole cell patch clamp technique with an EPC-7 amplifier (List Electronics, Heidelberg, Germany) and pClamp data acquisition (Axon Instruments, Union City, CA, USA) essentially as described previously (Storch et al. 2003; Hermann et al. 2004b). For better sealing we added 5% FCS 5 hours prior to the patch clamp experiments. The extracellular solution -1 contained (in mmol l ): 142 NaCl, 8.1 KCl, 1CaCl2, 6 MgCl2, 10 HEPES, 10 D-

Glucose, pH 7.4. The pipette solution contained (in mmol/l): 153 KCl, 1 MgCl2, 5 EGTA, 10 HEPES, pH 7.3. Using these solutions, borosilicate pipettes had resistances of 6-10 MΩ. Seal resistances in the whole cell mode were between 0.5 and 1 GΩ. Data were analyzed using pClamp 8.0, Microsoft Excel 97 and Origin 5.0 software. Resting membrane potentials (RMP) were determined in the current clamp mode immediately after establishing the whole cell configuration. Action potentials (APs) were elicited by applying increasing depolarizing current pulses (300 ms, 10 pA current steps).

18 Materials and Methods

2.13 Cell counting and statistics

For quantification of the percentage of cells producing a given marker, in any given experiments the number of positive cells of the whole well surface was determined relative to the total number of DAPI-labeled nuclei. In a typical experiment, a total of 100 to 500 cells were counted per marker. In co-culture experiments, only cells positive for the anti-human nuclei antibody were determined in confocal microscopic images to avoid the counting of overlaying cells. Human nuclei/GFAP+ and human nuclei/Tuj1+ cells, respectively, were counted in sister cultures. Statistical comparisons were made by Dunnett`s t-test. If data were not normally distributed, a non-parametric test (Mann-Whitney U-test) was used for comparisons of results. Data presented are pooled from experiments performed on cells obtained from tissue of all 14 patients. All data are presented as mean  SEM.

2.14 RNA extraction and quantitative real-time RT-PCR analysis

Total cellular RNA was extracted from human aNPCs during expansion using the RNAeasy total RNA purification kit followed by treatment with RNase-free DNase (Qiagen, Hilden, Germany). Quantitative real-time one step RT-PCR was carried out using the LightCycler® System (Roche, Mannheim, Germany), and amplification was monitored and analyzed by measuring the binding of the fluorescence dye SYBR Green I to double-stranded DNA. 1 µl (approximately 50 ng) of total RNA was reversely transcribed and subsequently amplified using QuantiTect SYBR Green RT-PCR Master mix (Qiagen) and 0.5 µmol/L of both sense and antisense primers. Primers were designed with Primer Premier (PrimerBiosoft, Palo Alto, CA, USA) and ordered from Operon (Cologne, Germany). Tenfold dilutions of total RNA were used as external standards. Standards and samples were simultaneously amplified. After amplification, melting curves of the RT-PCR products were acquired to demonstrate product specificity. The results are expressed relative to the housekeeping gene HMBS (hydroxymethylbilane synthase), Quantified was with the 2^(CP (housekeeping gene)- CP (target gene)) method from Livak and colleagues (Livak and Schmittgen 2001). Primer sequences, lengths of the amplified products are summarized in Table A-1.

19 Materials and Methods

2.15 Microarray analysis

After RNA isolation first strand cDNA synthesis was completed with total RNA (5- 8µg) cleaned with RNAeasy columns (Qiagen, Hilden, Germany) and the superscript double stranded cDNA synthesis protocol (Invitrogen, Carlsbad, CA, USA), except that HPLC purified T7-promoter-dT30 primer (Operon, Cologne, Germany) was used to initiate the first strand reaction. Biotin labeled cRNA was synthesized from T7 cDNA using the RNA transcript labeling kit (Affymetrix, Santa Clara, CA, USA). The sample was cleaned with the Affymetrix GeneChip sample clean up module (Qiagen) to remove free nucleotides and quantitated spectrophotometrically. Biotin-labeled cRNA was fragmented and hybridized to human genome UA433 array according to the manufacturer’s manual with a final concentration of fragmented cRNA of 0.05 µg/µl. The arrays were scanned using a Hewlett Packard confocal laser scanner and analyzed using ArrayAssist Lite and Affymetrix® Microarray Suite version 5.0 software. The p-value for comparison of each probe set was computed using the Wilcoxon's Signed Rank test. Additional expression data sets in form of cel-files were obtained from the web (http://www.ncbi.nlm.nih.gov/geo/) or kindly provided per DVD (http://wombat.gnf.org/ suppl.html#reqdata_geneatlas). Datasets included the NCI60-dataset (http://wombat.gnf.org/ samples/NCI60_sample_info.htm), the GNF- SymAtlas (Su et al. 2002) and the “breadth of expression”-dataset (Ge X 2005). Own experimental data and external data were put together and low-level corrected using the RMA-algorithm (Irizarry et al. 2003b). Expression data was then exported in log2-scale. Additionally, individual expression profiles were fitted by means of median polish to a reference profile in regard to intensity distribution and RNA-quality. Over-expressed genes were determined to exceed a minimal difference of 2.5 (in log2-scale) between their individual intensity and the median intensity of all analyzed 201 tissues. For further analysis we decided to focus on neural tissues only. Hierarchical clustering was performed using the Genesis software package (Sturn et al. 2002).

20 Materials and Methods

2.16 Determination of GABA, glutamate, dopamine and serotonin production

These experiments were done in cooperation with Manfred Gerlach, University of Würzburg. For determination of dopamine production, media were supplemented with 100 µmol/L tetrahydrobiopterin and 200 µmol/L ascorbate 2 days prior to medium harvest. Dopamine levels were determined in medium stabilized with EGTA/glutathione solution as reported previously (Storch et al. 2001), and stored at -80°C until analysis. Dopamine was assayed by reverse-phase HPLC with an electrochemical detector as previously described (Gerlach et al. 1996). GABA, glutamate and serotonin were assayed by HPLC with fluorescence detection (Gerlach et al. 1996), employing pre-column derivatisation with ortho- phtaldialdehyde and an automatic HPLC system (Kontron Instruments, Neufahrn, Germany). The excitation and emission wave-length of the fluorescence detector were set at 330 and 450 nm, respectively. These experiments were done in cooperation with Manfred Gerlach (University of Würzburg, Germany).

21 Results

3 Results

3.1 Characterization of adult human neuroprogenitors

3.1.1 Isolation and long-term expansion of neurosphere forming cells from the adult human hippocampus

In the present section the human hippocampal neurospheres are characterized on protein and RNA level to give an outline of adult neural stem cells in regard of their regeneration potential. After 10–20 days in vitro, the cells formed small spheres with all typical morphological properties of neurospheres (Fig. 3). FACS analysis revealed that the phenotype of these neurosphere-forming NPCs was CD15/Lex1low/–, CD34–, CD45–, CD133– (Fig. 4). Extensive analyses of the expression of early and late neuroectodermal marker genes using quantitative real- time PCR and immunocytochemistry (Fig. 3 and Fig. 5 for complete names of genes and encoded proteins refer to Table A-1) revealed that aNPCs expressed high levels the early neuroectodermal or CNS precursor cell markers, such as Nestin and Musashi1 (Cattaneo and McKay 1990; Reynolds et al. 1992; Kaneko et al. 2000) on both the mRNA and protein level. Furthermore, adult aNPCs showed detectable expression levels of the proneural genes NeuroD1 and Olig2 on the mRNA and/or the protein level (Fig. 3 and Fig. 5). Consistent with the FACS analysis data, only a few cells were weakly positive for CD15 protein (Fig. 3). Quantitative real-time PCR additionally showed the expression of several early neuroectodermal marker genes, such as NTRK1, OTX1/2 and SOX1,2,10 as well as surface receptors important for neural development (NOTCH1, NTRK1, FZD1, p75(NTR); Fig. 5). This phenotype is similar to that of NPCs derived from adult mouse SVZ or hippocampus (Doetsch et al. 1999; Rietze et al. 2001; Hack et al. 2004). The aNPCs could be passaged and cultured as secondary and tertiary neurospheres without changing morphology and phenotype for up to 12 weeks (8– 10 passages). To confirm the proliferation potential of aNPCs, the proliferation marker Ki67 was used as well as BrdU incorporation to identify DNA-synthesizing cells (Fig. 3). Ki67 is detected in the nucleus of proliferating cells in all active phases of cell cycle from the late G1 phase through the M-phase but is absent in non-proliferating and early G1- phase cells and cells undergoing DNA repair (Gerdes et al. 1984; Key et al. 1994; Gerdes et al. 1995). As expected, total Ki-67- staining (representing late G1- through M-phase) was higher at each time point (67 ± 8% of cells were Ki67+ n = 3) than total BrdU staining (11 ± 1% BrdU+ cells; n = 3), which only marks cells within the S-phase of the cell cycle. After 4–7 weeks, an

22 Results average number of 7 ± 6% of aNPCs were found as spontaneously apoptotic (chromatin condensation and margination, apoptotic bodies in some cells) by DAPI staining. These cells were negative as well as for most other markers tested such as Nestin and Olig2 (Fig. 3).

Fig. 3: Characteristics of adult human hippocampal NPCs during in vitro expansion. Morphology and marker expression of adult NPCs during expansion in the presence of EGF and FGF-2. Spheres (a) were cultured for 2 to 3 h to allow attachment and then stained for Nestin (b), Ki-67 (c), Musashi1 (d), Nestin and NeuroD1 (e) or Olig2 (f), MAP2ab (g), GFAP (h) and Lex1/CD15 (i). Nuclei were counterstained with DAPI. Scale bars 100 µm (a-c, f-i) or 50 µm (d,e).

23 Results

Telomerase is inactive in most somatic cells, but present in various stem cell populations (Meyerson et al. 1997; Ostenfeld et al. 2000; Mattson and Klapper 2001). Quantification of telomerase activity in aNPCs in comparison to adult human differentiated tissue using the telomeric repeat amplification protocol (Emrich and Karl 2002; Saldanha et al. 2003) showed significant telomerase activity in aNPCs compared to human cortical differentiated tissue as negative control without changes over the three months expansion period (Fig. 6). Together, investigations of the proliferation characteristics revealed the typical pattern of slowly dividing cells with a small amount of spontaneous apoptosis within the aNPC neurospheres.

Fig. 4: Flow cytometry of aNPCs cultured for 4 to 12 weeks (5 to 10 passages). Cells were labeled with fluorescence-coupled antibodies against CD15, CD34, CD45, CD133 or immunoglobulin isotype control antibodies. Cells were analyzed using a FACSCalibur flow cytometer. Black line, control immunoglobulin; red line, specific antibody.

24 Results

Fig. 5: Quantitative transcription profile of aNPCs. Results of real-time RT-PCR analysis of the NSC markers Nestin and Musashi1 (NES, MSl1), proneural genes (NeuroD1, NGN2), genes early expressed in the development (Frizzled 1 (FZD1)), Notch1, OTX1, OTX2, SOX1, SOX2 and SOX10) and tyrosin-receptor kinases like p75NGF and NTRK1 are displayed. Expression levels are expressed relative to the housekeeping gene HMBS. For primers complete names of the genes and accession numbers see Table A-1 Results are mean values  s.e.m. from at least three independent experiments.

Fig. 6: Telomerase activity in human aNPCs after 3 to 8 weeks as well as adult cortical tissue and mouse ES-cells during expansion measured by the quantitative real-time telomeric repeat amplification protocol. Telomerase activity was normalized to protein content. Results are mean values ± s.e.m. from three independent experiments. # indicates p < 0.05, ## represents p < 0.01 when compared to adult cortical tissue; + indicates p < 0.05 when compared to ES-cells.

25 Results

3.1.2 Characterization of gene expression in human adult neuroprogenitors, compared with fetal neuroprogenitors and mesodermal stem cells

This part is focused on the transcriptome analysis of human adult and fetal NPCs propagated in neurospheres to provide (i) knowledge of the molecular mechanisms of governing self-renewal and multipotent differentiation capacity restricted to the neural cell fate and (ii) a framework for a standardized comparative gene expression analysis of human NPCs with other stem cell populations or differentiated tissues. Therefore the Affymetrix gene chip technology was applied. RNA derived from human hippocampal aNPCs, human cortical fNPCs, converted mesencymal stem cells into neural-like stem cells (hmNSCs) and multipotent adult human mesenchymal stem cells (hMSCs) as an established adult stem cell control were used. Experiments were performed with human hippocampal tissue as reference differentiated tissue. In addition, expression profiles of adult and fetal neural tissues available in public databases were correlated. With this approach a large number of genes which delineate the phenotypical differences of adult and fetal NPCs could be defined.

3.1.2.1 Global transcriptome complexity of human neural and mesenchymal progenitors

The dataset consisted of 37 individual chips from three different sources. Eight tissues were represented in replicas processed from different labs. All these eight pairs were clustered together, respectively, thus demonstrating the performance of our fitting procedure (Fig. 10). Furthermore, multiple microarray analyses of one respective progenitor cell type from different tissue samples/patients (n=2-3) showed a 93 to 94% overlap of specifically over-expressed genes. The gene expression of adult hippocampus was profiled initially to evaluate the comparability between the chip and the data obtained from the databases (Fig. 10). After completing the fitting procedure a correlation coefficient (R2) of 0.9935 was reached between the hippocampus data and the hippocampus data retrieved from literature (Ge X 2005), providing a strong evidence that the used experimental protocol is valid. The two NPC subtypes did not fall into the same cluster and showed rather limited overlap in gene expression. Fetal NPCs clustered together with their tissue of origin, the fetal brain. In contrast, adult NPCs clustered together with corpus callosum and spinal cord mainly consisting of white matter tissue and thus did not resemble their respective tissue of origin, the hippocampus.

To further capture an overall estimate of the similarities and differences between the analyzed tissues, the transcriptome of aNPCs, fNPCs, hMSCs and adult

26 Results hippocampal tissue on a global scale in which cell types were compared in a pair- wise manner (Fig. 7). The transcriptomes of all these cell/tissue types were significantly different with lower correlation coefficients of 0.612 to 0.664. These data were confirmed by analyzing the expression of enriched genes in all cell types showing only a small overlap of gene expression in all analyzed cell/tissue samples (see Fig. 11 for Venn diagrams).

Fig. 7: Global comparison of adult neural progenitor cell (aNPC), fetal NPC, hippocampus and human mesenchymal stem cell (hMSC) transcripts. Displayed are the scatterplots of intensities (log2 intensity scale) for all 22.215 probesets. The corresponding correlation coefficients are shown in the panels.

Fig. 8 and Table 1 show that most previously known stem cell markers were enriched in their respective progenitor cell population providing additional validation of the protocol. The human brain-derived aNPCs showed high expression of the neural stem cell marker NES (nestin), as well as of OLIG2, MSl1, NKX2-2 and CD44. The expression of NES and MSI1 were already reported in aNPCs by Hermann and colleagues (Hermann et al. 2006d). Additionally, the expression of

27 Results various SOX genes was detectable, such as SOX2 and SOX10. NTRK1 and the P75 (NGFR) genes were as well highly expressed as revealed by both the gene array and on RT-PCR level (Hermann et al. 2006d). GPR17 was on RNA level highly expressed only in the adult NPCs, no expression was detectable in differentiated aNPCs, fetal NPCs or hMSCs (Fig. 9B).

On the microarrays the fNPCs showed a high expression e.g. of NES, PROM1 (prominin1, CD133), Olig2, MSI1 as well as SOX-genes like SOX2, SOX3, SOX4, SOX9 and SOX11. In contrast, hMSCs expressed various extracellular matrix proteins, such as various collagen isoform and other genes related to connective tissue and its metabolism (LOXL1, LOXL2, LUM). Fully differentiated neural tissue like the hippocampus expressed neuronal and glial genes like NTRK2, NTRK3, MBP and GFAP. The presence or absence of gene expression of the most prominent genes (CCR7, EDNRB, GPR17, LAMA4a, NES, P75 (NGFR), PDGFRα and TNC) was confirmed by real-time RT-PCR in all cell types (Fig. 9A and C).

28 Results

Fig. 8: Transcriptional profiling of human neural progenitor cells (aNPCs). Human fetal cortical NPCs and adult hippocampal aNPCs, as well as adult human mesenchymal stem cells, (hMSCs) as a multipotent adult stem cell control and tissue from adult human hippocampus as a differentiated tissue control, were isolated and their cRNAs were hybridized to Affymetrix U133A-DNA chips. The arrays were analyzed by a combination RMA software, followed by public database searches and functional annotation. Indicated at the bottom are genes that were previously described to be specifically expressed in each of the two stem cell types and tissue.

29 Results

Fig. 9: Quantitative transcription profile of aNPCs, fNPCs, hMSCs and for GPR17 differentiated aNPCs were used. A: Receptors found in chip analysis: p75(NGF), PDGFR- α, CCR7 and EDNRB are displayed.B: Results of real-time RT-PCR analysis of the receptor found in chip analysis: GPR17- only high expressed in aNPCs. C: RT-PCR- Validation of expression of extracellular matrix genes found in chip analysis.Expression levels are expressed relative to the housekeeping gene HMBS. For primers complete names of the genes and accession numbers see Table A-1. Results are mean values  s.e.m. from at least three independent experiments.

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Table 1: Genes expressed in aNPCs, fNPCs, human hippocampus or hMSCs. Number of plus signs reflects the relative transcriptional activity in quartiles per gene. No plus signs indicate absent or low expression within the first quartile and three plus signs denote very strong expression within the fourth quartile.

adult Cate Gene Gene name Probeset aNPCs fNPCs hippo- hMSCs gory Symbol campus Kinases/ Phosphatases epidermal growth factor EGFR 201983_s_at +++ +++ receptor fibroblast growth factor FGFR1 210973_s_at ++ ++ receptor 1 v-yes-1 Yamaguchi sarcoma LYN viral related oncogene 210754_s_at +++ + + homolog maternal embryonic leucine MELK 204825_at +++ + zipper kinase platelet-derived growth factor PDGFRA 203131_at +++ +++ receptor, alpha polypeptide serum/glucocorticoid regulated SGK 201739_at +++ ++ kinase tribbles homolog 2 TRIB2 202479_s_at +++ (Drosophila) dual specificity phosphatase DUSP10 221563_at +++ ++ 10 DUSP4 dual specificity phosphatase 4 204014_at + +++ DUSP6 dual specificity phosphatase 6 208892_s_at + +++ + + Stem cell markers NES Nestin 218678_at +++ +++ + polycomb group ring finger 4 PCGF4 202265_at +++ + + ++ (BMl1) PROM1 prominin 1 (AC133, CD133) 204304_s_at +++ sprouty homolog 1, antagonist SPRY1 212558_at +++ of FGF signaling (Drosophila) [ chemokine (C-X-C motif) CXCL12 ligand 12 (stromal cell-derived 203666_at + + +++ factor 1; SDF-1) chemokine (C-X-C motif) CXCR4 receptor 4 (CD184, fusin, 217028_at ++ +++ + SDF-1 receptor) Proliferation CDC28 protein kinase CKS2 204170_s_at + +++ regulatory subunit 2 denticleless homolog DTL 218585_s_at +++ (Drosophila) insulin-like growth factor IGFBP3 210095_s_at +++ + +++ binding protein 3 antigen identified by MKI67 monoclonal antibody Ki-67 (Ki- 212023_s_at +++ 67) Myeli n MBP myelin basic protein 209072_at +++ +++ Myelin-oligodendrocyte MOG 214650_x_at +++ ++ glycoprotein proteolipid protein 1 PLP1 (Pelizaeus- Merzbacher 210198_s_at +++ + +++ disease protein) GPM6A glycoprotein M6A 209470_s_at + +++ +++ Surface markers CD44 CD44 antigen 204489_s_at +++ ++ chondroitin sulfate CSPG2 215646_s_at + +++ +++ proteoglycan 2 (versican) chondroitin sulfate CSPG4 204736_s_at +++ ++ + +++ proteoglycan 4 (NG2)

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adult Cate Gene Gene name Probeset aNPCs fNPCs hippo- hMSCs gory Symbol campus Modulation of transcription achaete-scute complex-like 1 ASCL1 209988_s_at + +++ ++ (Drosophila; HASH1, MASH1) DLL3 delta-like 3 (Drosophila) 219537_x_at +++ DLX2 distal-less homeobox 2 207147_at +++ ++ empty spiracles homolog 2 EMX2 221950_at +++ + +++ (Drosophila) forkhead box M1(FKHL16, FOXM1 202580_x_at +++ + HFH-11, HNF-3) hairy and enhancer of split 1, HES1 203394_s_at + +++ (Drosophila) hairy/enhancer-of-split related HEY1 44783_s_at + +++ ++ with YRPW motif 1 hypoxia-inducible factor 1, HIF1A 200989_at + +++ + +++ alpha subunit HOP homeodomain-only protein 211597_s_at +++ +++ LHX2 LIM homeobox 2 206140_at +++ + Meis1, myeloid ecotropic viral MEIS1 integration site 1 homolog 204069_at + +++ (mouse) Meis1, myeloid ecotropic viral MEIS2 integration site 1 homolog 2 207480_s_at + +++ + (mouse) MYST histone MYST4 acetyltransferase 4 (querkopf, 212462_at +++ + MORF) neurofilament, heavy NEFH 33767_at ++ polypeptide 200kDa NEUROD1 neurogenic differentiation 1 206282_at + NEUROD2 neurogenic differentiation 2 210271_at ++ NK2 transcription factor NKX2-2 206915_at +++ + + related, locus 2 (Drosophila) oligodendrocyte lineage OLIG2 213825_at +++ +++ + transcription factor 2 SRY (sex determining region SOX2 213721_at ++ +++ Y)-box 2 SRY (sex determining region SOX3 214633_at + +++ + Y)-box 3 SRY (sex determining region SOX4 201417_at ++ +++ ++ Y)-box 4 SRY (sex determining region SOX9 202936_s_at +++ Y)-box 9 SRY (sex determining region SOX10 209842_at +++ ++ Y)-box 10 SRY (sex determining region SOX11 204914_s_at +++ Y)-box 11 SRY (sex determining region SOX13 38918_at +++ + Y)-box 13 TCF4 transcription factor 4 212382_at +++ ++ ++ ++ TCF8 transcription factor 8 210875_s_at +++ TCF12 transcription factor 12 (HTF4) 208986_at + +++ + topoisomerase (DNA) II alpha TOP2A 201292_at +++ 170kDa ID1 inhibitor of DNA binding 1 208937_s_at + + ++ ID2 inhibitor of DNA binding 2 213931_at +++ +++ ID3 inhibitor of DNA binding 3 207826_s_at + ++ +++ ID4 inhibitor of DNA binding 4 209292_at +++ + musashi homolog 1 MSI1 206333_at +++ +++ ++ (Drosophila) Transmembrane

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adult Cate Gene Gene name Probeset aNPCs fNPCs hippo- hMSCs gory Symbol campus receptors chemokine (C-C motif) CCR7 206337_at +++ receptor 7 chemokine (C-X-C motif) CXCR4 217028_at ++ +++ + receptor 4 endothelin receptor type B EDNRB 206701_x_at +++ + (Hirschsprung disease 2) frizzled homolog 3 FZD3 219683_at +++ + (Drosophila) GPR17 G protein-coupled receptor 17 206190_at +++ + GPR37 G protein-coupled receptor 37 209631_s_at +++ ++ integrin beta 1 (fibronectin Ib1 211945_s_at ++ + + +++ receptor beta) interleukin 6 signal transducer IL6ST 211000_s_at +++ +++ (gp130, oncostatin M receptor) neuropilin (NRP) and tolloid NETO2 218888_s_at +++ + (TLL)-like 2 nerve growth factor receptor NGFR (p75 NTR, TNFR superfamily, 205858_at +++ member 16) PTCH patched homolog (Drosophila) 209815_at + TSPAN15 tetraspanin 15 218693_at +++ Miscellaneous BMP and activin membrane- BAMBI bound inhibitor homolog 203304_at +++ ++ (Xenopus laevis) doublecortex; lissencephaly, DCX 204850_s_at ++ + X-linked (doublecortin) DHFR dihydrofolate reductase 202534_x_at +++ Dicer1, Dcr-1 homolog DICER1 213229_at +++ + +++ (Drosophila) ENAH enabled homolog (Drosophila) 217820_s_at + +++ + +++ GAS1 growth arrest-specific 1 204457_s_at +++ + LMNB1 lamin B1 203276_at +++ MYO1D myosin 1D 212338_at +++ ++ MYO1E myosin 1E 203072_at +++ + ++ neurofilament 3 (150kDa NEF3 205113_at ++ medium) neurofilament, light NEFL 221916_at +++ polypeptide 68kDa

RELN reelin 205923_at +

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3.1.2.2 Adult neuroprogenitors cluster together with white matter tissues

Hierarchical clustering of aNPCs and various tissues from different sources showed strong similarities of human neural progenitor cells mainly with tissue from white matter, such as spinal cord and corpus callosum (Fig. 10). White matter tissue is characterized by a strong expression of MHCII-genes and myelin- components such as peripheral myelin protein 2 (PMP2), myelin associated glycoprotein (MAG) or the myelin-oligodendrocyte glycoprotein (MOG). The over- expression of CD44 and GDF1/LASS1 might point to still existing but restricted stem cell potential. A number of genes specific for neurons were transcribed only on reduced levels: the synaptosomal-associated protein, 25kDa (SNAP25), neurogranin (NRGN) and others. When looking for genes specifically expressed in aNPCs, a large number of such genes can be retrieved. The mRNA for the low affinity nerve growth factor receptor precursor (p75, NGFR) and for Nestin (NES) was expressed in a very high abundance. Also the dual specific phosphatase 10 (DUSP10), the CC-motif containing chemokine receptor 7 (CCR7), the transcription factor 4 (TCF4), the G protein-coupled receptor 17 (GPR17) and the chondroitin sulfate proteoglycan 4 (CSPG4) were specifically highly expressed in the aNPCs (Table 1 and Fig. 9). The gene CSPG4 encodes for the epitope NG2 which is reported to be specifically expressed on oligodendrocyte precursor cells. Interestingly, only three genes were exclusively expressed in aNPCs, namely NGFR, GPR17 and CCR7.

The transcriptome of fNPCs was very different from adult CNS tissue and shared the closest relation with fetal brain. Both tissues lacked the mRNA for myelin components and a number of SOX- genes were highly expressed in comparison to adult brain like SOX4, SOX11 and SOX12. Additionally in both tissues the up- regulation of prominin 1 (PROM1 or CD133) was detectable.

3.1.2.3 In total adult hippocampus are no adult neuroprogenitor markers expressed

Unlike the fetal NPCs, adult NPCs showed little overlap with their tissue of origin (Fig. 11A). It was not possible to detect elevated expression of specific markers for adult NPCs in hippocampus. Instead the expression of such markers in olfactory bulb and spinal cord tissue (e.g. chromosome 14 open reading frame 139 (C14ORF139), BMP and activin membrane-bound inhibitor homolog (BAMBI) or the myelin associated glycoprotein (MAG) could be identified. This is likely demonstrating the very low frequency of NPCs in the adult brain.

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Fig. 10: Hierarchical clustering of 31 neural tissues. Expression data (i.e. the 1,000 most variant genes) were clustered using Pearson’s correlation as similarity measure and average linkage as agglomeration rule. External sources of chip data are denoted in brackets: gnf from SymAtlas (Su et al. 2002); tp from ‘breadth of expression’ dataset (Ge et al. 2005). Own experimental datasets are marked with colors. All biological replicates are clustered together illustrating the performance of the chosen fitting procedure. The hippocampus array forms a branch together with hippocampus (tp) and the two amygdala chips (gnf, tp). The fetal NPCs fall in one cluster along with fetal brain tissues (gnf, tp). The adult NPCs cluster together with corpus callosum (gnf) and spinal cord (gnf, tp).

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3.1.2.4 Fetal neural progenitors maintain an expression pattern of undifferentiated cells

The expression pattern of fNPCs was clearly distinguishable from those cells of the peripheral nervous system (Fig. 10). It was different from adult CNS tissue, but in close relation with fetal brain. Both tissues lacked the mRNA for myelin components of mature oligodendrocytes: Myelin basic protein (MBP), proteolipid protein 1 (PLP1), myelin-associated oligodendrocyte basic protein (MOBP) and others. The neurofilament heavy chain (NEFH) or the GABA-receptor, alpha 1 (GABAR1) were not expressed. Instead a number of SOX- and homeobox genes were highly expressed in comparison to adult brain: SOX4, SOX11, SOX12 and the LIM homeobox gene 2 (LHX2), or the distal-less homeobox gene 2 (DLX2). Additionally, an up-regulation of prominin 1 (PROM1 or CD133) could be detected in both tissues. Important genes uniquely over-expressed in fNPCs are predominantly associated with cell proliferation like topoisomerase II alpha (TOP2A), histone acetyltransferase 1 (HAT1) (Guidez et al. 2005) or cell division cycle 2 (CDC2). Additionally, the strong expression of the “hairy and enhancer of split 1” gene (HES1) is expected due to active notch signaling maintaining a proneural undifferentiated state of the cells. The observed over-expression of transcription factor 8 (TCF8) and SOX2 supports this finding. Other genes exclusively found in fNPCs included the dual specificity phosphatase 6 (DUSP6) (known to inactivate MAPK1 [mitogen-activated protein kinase 1]), extracellular signal-regulated kinase 2 (ERK) (Tarrega et al. 2005), the endothelin receptor type B (EDNRB), the myeloid ecotropic viral integration site 1 homolog (MEIS1) and the tissue factor pathway inhibitor (TFPI).

3.1.2.5 Comparison of adult neuroprogenitors with fetal neuroprogenitors

Despite the global differences between transcriptomes of aNPCs and fNPCs, a few similarities could be observed (Fig. 13A, B and Table 1). Most of these similarities could be attributed to genes known to be associated with cell cycle progression, such as a disintegrin and metalloproteinase domain 9 (ADAM9), HAT1-, protein kinase-, DNA-activated, catalytic polypeptide (PRKDC), or RNA binding motif protein 3 (RBM3). Both cell types over-expressed other genes commonly associated with a neural stem/progenitor cell fate: Jagged 1 (JAG1) (Nyfeler et al. 2005), SOX2 (Episkopou 2005), SOX4 (Kuhlbrodt et al. 1998), Nestin (NES) (Wiese et al. 2004), the oligodendrocyte lineage transcription factor 2 (OLIG2) (Takebayashi et al. 2002), the G protein-coupled receptor 56 (GPR56) (Liu et al. 1999a), the vascular endothelial growth factor (VEGF) (Brill et al. 1992) and others.

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The stem cell marker Musashi1 (MSl1 (Kaneko et al. 2000)) was also over- expressed in both adult and fetal NPCs (Table B-1). The categorization of the genes by Gene Ontology terms were similar in both NPC populations (Fig. 13A and Fig. 13B), while the genes themselves overlapped only occasionally (Fig. 13A, B, Table B-5 and Table B-6) for genes showing greater than 10-fold differences between aNPCs and fNPCs.

Fig. 11A: Overlaps between genes enriched in aNPCs compared to various neural tissue and cell types. Venn diagrams of the number of genes enriched in each aNPC population and adult hippocampus.

3.1.2.6 Comparison of adult neuroprogenitors with mesenchymal stem cells hMSCs were included in the expression analysis as a multipotent adult stem cell control in the the expression analysis. This cell-population over-expressed a number of genes commonly found in connective tissues (decorin (DCN), fibronectin (FN1) and various collagen proteins), similar to PNS tissues. These genes are likely responsible for the observed association of hMSCs with tissues of the PNS upon cluster analysis, being completely separated from all neural tissues including adult and fetal NPCs. However, there are various genes expressed by both aNPCs and hMSCs, mainly extracellular matrix proteins (COL1A1, COL1A2, COL3A1, LOXL2) and genes like insulin-like-growth-factor 3 and 5 (IGFBP3 and IGFBP5)

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(refer to Table 1). Three of the four genes expressed in aNPCs, fNPCs and hMSCs were also extracellular matrix components, namely laminin-4 (LAMA4), tensacin C (TNC) and integrin-α7 (ITGA7). The other gene was pleiotrophin (PTN) involved in various developmental processes (Fig. 11B). In contrast, there was no relevant overlap of gene expression with respect to cell cycle, proliferation, stem cell markers or genes known to maintain an undifferentiated cell fate (Fig. 13C, D, Table B-2, Table B-7Table B-8 and Table B-8) for genes showing greater than 10- fold differences between aNPCs and hMSCs).

Fig. 11B: Overlaps between genes enriched in aNPCs compared to various neural tissue and cell types. Venn diagrams of the number of genes enriched in each NPC population and adult human mesenchymal stem cells

3.1.2.7 Genomic comparison of adult neuroprogenitors with neural-like stem cells

Despite the global differences between transcriptomes of aNPCs and mNSCs, a few similarities could be observed (Fig. 12, Table B-4, Table B-11 and Table B-12). Most of these similarities could be attributed to genes known to be associated with genes responsible for regulation of neuropeptides like secretory granule neuroendocrine protein1 (SGNE1) and the neuroendocrine convertase 1 (PCSK1)

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(Cadet and Paquin 2000). Genes involved in metabolism like apolipoprotein E and D (APOE and APOD) were also upregulated in aNPCs and mNSCs and were not expressed in the hMSCs. Brain and acute leukemia gene (BAALC) and SNAP25, a synaptic gene were as well upregulated in the mNSCs and the aNPCs, such as neuroectodermal markers the endothelian receptor one (EDNRA) and Stathmin- like 2 (SCGN10). Stmn2 plays an important role in the development of the mature oligodendrocyte morphology (Zhang et al. 2006). Table B-4 shows both the expression of genes in the aNPCs and the mNSCs. Some neuronal and neuropeptides-related genes were expressed by both cell populations, like secretory granule neuroendocrine protein 1 (SGNE1), Brain and acute leukemia gene (BAALC); and a neuronal Glycoprotein 6B (GPM6B). GPM6B is a membrane glycoprotein, related to proteolipid protein (PLP) on oligodendrocytes and neurons (Werner et al. 2001).

Differences were detected in gene expression concerning the neural direction of the aNPCs, like Nestin (NES), Enolase (ENO2), and Synuclein-alpha (SNCA) (Fig. 14C, D and Table B-12). These genes were highly expressed in aNPCs in comparison to mNSCs. However mNSCs expressed e.g. growth-associated protein (GAP43) and the monoaminooxidase A (MAOA), other genes involved in neuronal development.

3.1.2.8 Genomic comparison of neural-like stem cells with their origin mesenchymal stem cells

The mNSCs lost some of the markers highly expressed in hMSCs such as decorin (DCN), fibronectin and various collagen proteins and expressed neuroectodermal markers like e.g. stathmin-like 2 (SCGN10) (Zhang et al. 2006) as well as genes involved in metabolism of neuropeptides like e.g. neuroendocrine convertase 1 (PCSK1) cutting neuroendocrine hormones (Cadet and Paquin 2000). Genes associated with connective tissue fates, such as lysyloxidase (LOX), COL4A1, a disinthegin-like and metalloproteinase with thrombospondin-type 1 (ADAMTS1) were downregulated in mNSCs in comparison to the expression pattern in hMSCs. Different genes associated with the mesodermal direction, like the latent transforming growth beta-binding protein 2 (LTBP2), hematopoietic killer lineage protein (KLIP1), muscle (ACTA2, THBS1), cartilage (COMP) and osteoklast (TNFRSF11B) differentiation were also downregulated whereas neuroectodermal genes were upregulated (Table B-9 and Table B-10). Interestingly various immune- system-related genes, such as different CXC-motif chemokines, like CXCL1,2,3,5,6 and IL8 (CXCL8), on one chromosome-location (4q12-13) were

39 Results significantly down-regulated. These genes are growth-related oncogenes, the genes belonging to the Gro-Family and regulate downregulation of genes encodes connective tissue fates (Unemori et al. 1993). Another interesting point was the expression of various genes involved in fat-metabolism (Fig. 14A, B and Table B- 9), like alcohol- dehydrogenase 2 (ADH1B), carbonic anhydrase 9 (CA9), phospholipid-transfer-protein (PLTP) and thiamine transporter protein 1 (SLC19A2). The mNSCs expressed neural genes in comparison to the hMSCs like growth-associated protein (GAP43), monoamine-oxidase (MAOA), the neuronal adhesion molecule (NRCAM) and stathmin2 (SCGN10). Table B-3 shows the common genes expressed in hMSCs and hmNSCs like various collagen isoforms (COL1A1, COL5A1 and COL6A1) and different components of the extracellular matrix (TNC, PCOLCE and TPM2).

Fig. 12: Overlaps between genes enriched in aNPCs compared to hMSCs and mNSCs. Venn diagrams of the number of genes enriched in each NPC population and adult human mesenchymal stem cells.

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Fig. 13A, B: Global comparison between adult NPCs and fetal NPCs based on at least stringent criteria of >10-fold differences. The categories of genes are derived by the gene ontology classification and the genes are shown with the respective percentage distribution. The adult and fetal NPCs differ in a wide spectrum of genes, with the largest cause of difference being due to “signaling” and “metabolism” genes.

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Fig. 13C, D: Global comparison between adult neural progenitor cells (aNPCs) and hMSCs based on at least stringent criteria of >10-fold differences. The categories of genes are derived by the gene ontology classification and the genes are shown with the respective percentage distribution. The adult NPCs and hMSCs differ in a wide spectrum of genes, with the largest cause of difference being due to “signaling”, “metabolism” and extracellular matrix genes.

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Fig. 14A, B: Global comparison between hMSCs and hmNSCs based on at least stringent criteria of >10-fold differences. The categories of genes are derived by the gene ontology classification and the genes are shown with the respective percentage distribution.

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Fig. 14C, D: Global comparison between hmNSCs and aNPCs based on at least stringent criteria of >10-fold differences. The categories of genes are derived by the gene ontology classification and the genes are shown with the respective percentage distribution.

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3.1.3 Differentiation capacity of human adult hippocampal neuroprogenitors

Adult human hippocampal NPCs differentiated into functional neuronal cell types. After 14 days, 36 ± 8% acquired morphologic and phenotypic characteristics of astrocytes (GFAP+), 31 ± 13% of oligodendrocytes (GalC+), 29 ± 7% of early neurons (Tuj1+) and 11 ± 8% that of mature neurons (MAP2ab+ n = 7; Fig. 15A, Fig. 15B). MAP2ab+ cells were not found during expansion of aNPCs within the neurospheres (Fig. 3); all MAP2ab+ neurons must therefore been derived from progenitor cells not expressing the marker for mature neurons. GFAP and Tuj1 were never found in the same cell (Fig. 15A). Very few cells expressed markers for dopaminergic (tyrosine hydroxylase) or serotoninergic (5-hydroxytyramine, 5-HT) cells.

Fig. 15A: In vitro differentiation capacity of adult human hippocampal NPCs: aNPCs differentiated by plating onto poly-D-lysine (PDL), mitogen withdrawal and incubation with BDNF (10 ng/ml) db-cAMP (100 µM) and retinoic acid (0.5 µM) for 14 days. The cells were stained against markers for astrocytes (GFAP), oligodendrocytes (GalC), and/or neurons (Tuj1; MAP2ab), respectively. In the right panel, arrows mark GFAP+ astrocytes and arrowheads mark Tuj1+ neurons. Nuclei are counterstained with DAPI (blue). Scale bar 30 µm.

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Fig. 15B: Quantification of differentiation capacity of 14-day cultures of aNPCs. Data shown are mean values  s.e.m. from three to seven independent experiments.

Pre-stimulation of aNPCs with expansion media supplemented with Shh (500 ng/mL) and FGF-8 (100 ng/mL) for 48 h prior to seeding the cells did not lead to significant changes in their differentiation behavior (compare Fig. 15 with Fig. 18A). To analyze the functional properties of the differentiated aNPCs, whole-cell voltage-clamping was performed to record voltage-gated sodium and potassium currents and current-clamping to search for the capacity to generate action potentials after differentiation for 3–7 days. The resting potential of the cells, determined in the current clamp mode immediately after establishing the whole cell conformation, was 41 ± 12 mV (n = 8). The majority of differentiated adult NPCs (5 out of 8 recorded cells) expressed a sustained outward current ranging from a few hundred pA to up to 4 nA (Fig. 16A). These currents showed a voltage dependence and kinetics characteristic for delayed rectifier K+ channels (Fig. 16B). In a few cells, inward currents with voltage dependence and kinetics typical for voltageactivated Na+ channels were identified and could generate action potentials (Fig. 16C).

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Fig. 16: Electrophysiological recordings on aNPCs differentiated without feeder layers as described in 2.8 A: For voltage-clamp measurements, cells were held at -70 mV and hyper- or depolarized in 10 mV steps to +100 mV for 50 ms. B: Example of a fast inward current and a sustained outward current (inset: only currents for depolarizing steps to -60, -50, 0, 20 and 60 mV are shown). C: Current-voltage relationships of the sustained outward currents (upper diagram) and the inward currents (lower diagram) of the cell displayed in (B). Representative voltage traces of current-clamp recordings of differentiated aNPCs in response to depolarizing current pulses of increasing amplitude. The resting membrane potential was ~ -65 mV and the action potential peaked at ~ +30 mV.

Fig. 17: Neurotransmitter production of differentiated aNPCs. Glutamate (A) and GABA (B) production were measured in aNPCs differentiated on poly-D-lysine (PDL), MEFs or PA6 cells for 14 days (see 2.11) using a HPLC-based assay. Quantification of the neurotransmitters was performed in medium conditioned for 2 days. Feeder cell layer did not produce detectable amounts of glutamate or GABA. # indicates p < 0.05; ## represents p < 0.01 when compared to cells differentiated on PDL.

Neurotransmitter (glutamate, GABA, dopamine and serotonin) production was studied in corporation with Manfred Gerlach on aNPCs differentiated for 14 days using an HPLC-based method. Significant production of glutamate and GABA by differentiated aNPCs was found (Fig. 17), but no generation of dopamine and serotonin.

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3.1.3.1 Stromal cell types induce neurogenesis from adult human hippocampal NPCs

To investigate the neuronal differentiation potential of adult NPCs the neuroprogenitor cells were plated on feeder layers of several cell types known to promote neuronal differentiation of ES-cells and/or NPCs including fetal mixed cortical cultures, mouse embryonic fibroblasts (MEFs) and PA6 stromal cells (Kawasaki et al. 2000; Perrier et al. 2004; Kitajima et al. 2005; Hermann et al. 2006d). The human origin of immunostained cells was confirmed by costaining the cultures with a human-specific antibody and investigating the cells using a confocal microscope. As shown in Fig. 18 and Fig. 19, co-culture of aNPCs with primary cortical cells, MEFs or PA6 stromal cells for 14 days resulted in a significant increase of Tuj1+ neurons, while only astrocytes and MEFs produced more GFAP+ glial cells compared to control conditions (PDL-coated culture surface).

The potential of different feeder cells to induce neurogenesis of adult aNPCs could be ordered as follows: PA6 cells > MEFs > cortical cultures > PDL, whereas the ranking order for the promotion of an astroglial phenotype was: Cortical feeder cells > MEFs > PA6 cells = PDL. Consistently, morphological analysis of the Tuj1+ neurons derived from adult NPCs by measuring the length of neurites revealed that co-culturing the aNPCs with MEFs or PA6 cells promoted neurite outgrowth of differentiating aNPCs compared to cultures on PDL or mixed cortical cultures (Fig. 20). Media conditioned by PA6 stromal cells for 72 h did not show any relevant effects on glial or neuronal differentiation of aNPCs (no significant changes of GFAP+ or Tuj1+ cell counts compared to aNPCs differentiated in normal media; Fig. 18 and Fig. 19).

Furthermore, aNPCs on MEFs or PA6 cells produced significant higher amounts of the neurotransmitters glutamate and GABA compared to cells cultured on PDL or mixed cortical cultures (Fig. 17). Unfortunately no dopamine or serotonin production under all culture conditions could be detected.

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Fig. 18: Differentiation capacity of adult hippocampal aNPCs on various co-culture systems including mouse fetal mixed cortical cultures, mouse embryonic fibroblasts (MEFs), and PA6 stromal cells, as well as by PA6 cell conditioned media (CM). Differentiation was initiated after an expansion phase of 3 to 10 weeks as described in 2.8. Confocal microphotographs of triple immunostainings of aNPCs for markers of astroglia (GFAP) or neurons (Tuj1) as well as human cells (human nuclei) were done. Nuclei were counterstained with DAPI (blue). Scale bar 75 µm.

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Fig. 19: Quantification of GFAP+ or Tuj1+ derived from aNPCs (counterstained with human nuclei antibody) by differentiation on PDL, mixed cortical feeder cell layers, MEFs, PA6 cells, or on PDL with PA6 cell conditioned media (50%), # p < 0.05, ## p < 0.01, ### p < 0.001 in comparison to PDL and PA6 CM, respectively; + p < 0.05, ++ p < 0.01 in comparison to cortical cocultures; * p < 0.05 in comparison to MEFs.

Fig. 20: Length of the neurites of Tuj1+/human nuclei+ neurons derived from aNPCs under coculture conditions. Results are mean values ± SEM from at least three independent experiments. # p < 0.05, ## p < 0.01, ### p < 0.001 in comparison to PDL and PA6 CM, respectively; + p < 0.05, ++ p < 0.01 in comparison to cortical cocultures.

50 Discussion

4 Discussion

4.1 Isolation and characterization of human adult progenitor cells

In my thesis, the isolation, long-term in vitro expansion and the differentiation potential of multipotent adult human hippocampal NPCs derived from epilepsy surgery is described. Noteworthy, diseased hippocampal tissue was used to isolate aNPCs that most likely containing mesangial sclerosis reported to include an increased number of neural progenitor cells in vivo (Blumcke et al. 2001; Crespel et al. 2005). These brain-derived NPCs were expanded in neurosphere cultures similar to fetal human NPCs (Svendsen et al. 1998; Svendsen et al. 1999; Caldwell et al. 2001; Storch et al. 2003) and expressed several markers for early neuroectodermal cells or NPCs as well as significant telomerase activity.

Since there are only a few reports on isolation and short-term propagation of adult hippocampal NPCs from human origin (Johansson et al. 1999b; Kukekov et al. 1999; Roy et al. 2000), detailed data on their morphological properties and marker expression during expansion as well as their differentiation capacity are missing. NPCs are often defined in vitro by the presence of the fetal and adult CNS stem cells marker Nestin (Cattaneo and McKay 1990; Lendahl et al. 1990; Dahlstrand et al. 1995), and the recently recognized NPC marker Musashi1 (Kaneko et al. 2000). Consistently, Nestin and Musashi1 are highly expressed in adult human hippocampal NPCs on both mRNA and protein levels. Another family of genes expressed in early neuroectodermal cells are the proneural genes encoding transcription factors of the basic helix-loop-helix (bHLH) class such as NeuroD1, Neurogenin2 and Olig2 (Franklin et al. 2001; Nieto et al. 2001; Simmons et al. 2001; Hack et al. 2004). Adult hippocampal NPCs expressed these proneural genes, which are necessary and sufficient to promote the generation (proliferation and self-renewal) of progenitors that are committed to neural differentiation (Bertrand et al. 2002; Bylund et al. 2003; Graham et al. 2003). The early neurogenic markers OTX1/2, several SOXB1 transcription factors (SOX1, 2, 10) and surface receptors important for neural development (NOTCH1, NTRK1, FZD1, p75(NTR)) were also found on the mRNA level in adult hippocampal NPCs. FACS analysis revealed that the aNPCs are CD15low/–, CD34–, CD45– and CD133–. This phenotype is similar to that found in fetal human and adult mouse NPCs (Capela and Temple 2002; Vogel et al. 2003; Capela and Temple 2006). The stem cell marker CD133 (or prominin-1) is expressed on several primitive cells such as hematopoietic stem or progenitor cells, embryonic neuroepithelial stem cells and

51 Discussion endothelial stem cells (Weigmann et al. 1997; Uchida et al. 2000; Bhatia 2001), but there is no systematic report on CD133 expression in adult NPCs. However, in neuroepithelial stem cells, CD133 is mainly localized at the apical cell membrane facing the ventricular wall (Weigmann et al. 1997; Corbeil et al. 1999; Corbeil et al. 2000). Consistently, the adult NPCs isolated from the hippocampus region without ventricular contacts were CD133–. Together, human adult hippocampal NPCs expressed a typical pattern of early neuroectodermal and/or NPC marker genes, including major factors for proliferation/self-renewal and neurogenic differentiation. Due to a very slow growing behavior of the adult aNPCs (approx. 8–14 days doubling time), single cell (clonal) analysis of their differentiation capacity was not performed. However, aNPCs were negative for MAP2ab+ during expansion within the neurospheres demonstrating that all MAP2ab+ neurons after differentiation must be de novo generated from MAP2ab-negative progenitor cells. Finally, showing proliferation by Ki67 and BrdU immunostaining as well as the expression of telomerase (Mattson and Klapper 2001), these adult NPCs fulfill the major characteristics of NPCs during in vitro expansion.

4.2 Characterization in gene expression pattern of adult NPCs, fetal NPCs and mesenchymal hMSCs

For the first time, global gene expression profiles of human adult and fetal NPCs are compared. Noteworthy, only diseased hippocampal tissue was used to isolate both aNPCs and RNA that most likely containing mesangial sclerosis reported to include an increased number of neural progenitor cells in vivo (Blumcke et al. 2001; Crespel et al. 2005). Both cell types were propagated as neurosphere cultures in vitro for at least 8 weeks ( 6-8 cell doublings) under similar conditions using EGF and FGF-2 as mitogens. Gene expression profiles show that both NPC subtypes express core neural stem cell makers, but neither share global expression profile with each other nor with multipotent hMSCs or adult brain (hippocampal) tissue. In contrast, aNPCs share expression profiles with white matter brain tissue, while fNPCs show closest relation with fetal brain. The data strongly suggest that adult and fetal human NPCs use divergent paths to maintain the neuroprogenitor cell state.

Gene chip technology is a powerful tool to generate global gene expression profiles of cells and tissues. This technique is exceptionally valuable when looking for novel genes associated with a defined biological process. However, despite standardized protocols and technical equipment the deduced results display a

52 Discussion varying degree of specificity. The analysis algorithm (i.e. MAS5) specified by Affymetrix is very basic and delivers rather noisy data which makes it especially challenging to interpret low intensity signals. The inherent difficulties have led to number of third party software approaches to improve the situation (Li and Wong 2001; Irizarry et al. 2003a). Low level signals close to background level remain difficult to interpretate. ‘No signal’ could have a biological background meaning that is the gene is not expressed or worse; the probe set is not properly designed to detect a high signal. The latter is often caused by underestimation of the length of 3’-UTR of genes placing the probeset very far from the polyA-tail and thus exceeding the average length of the reverse transcribed sequences. Therefore, an ‘absence’ call should only be issued when there is positive knowledge that the probeset allows for detection at all. The U133A-chip contains up to 3,000 (out of 22,215) ‘dead’ probesets not capable to deliver a signal. This assumption is based on the observed lack of an appropriate signal in all 385 analyzed chips, which covered a wide range of normal and cancerous tissues, stem cells and cell lines. Multiple gene array analyses of a respective progenitor cell type derived from different tissue donors revealed a > 90% overlap of gene expression demonstrating high similarity of transcriptomes within one progenitor cell type independent of the donor. However, to i) make this necessary positive control available and ii) to provide an internal reference the individual chips required extensive scaling and normalization. Finally the technical variance was smaller than the biological. This goal could be achieved by showing that hierarchical clustering group biological replicas regardless of the lab conducting the experiments (Fig. 10).

Two previous studies investigated the gene expression profile of human fetal NPCs (Wright et al. 2003; Cai et al. 2006). The data from the fetal cortical NPCs could be compared with data from Wright and colleagues, but unfortunately the data were not available in the database. Instead they showed gene-lists with expressed genes from their cell population. Their findings are in good general agreement with the expression data derived from the fetal neural stem cells. The levels of similarity exceeded former attempts to compare different expression profiles from neural stem cells (Ivanova et al. 2002; Ramalho-Santos et al. 2002). In contrast to Wright and colleagues, expression of SOX2 and SOX13 was detected in fetal neuroprogenitor cell cultures. It was possible to extend the list of stem cell biology related genes including the transcripts of MELK (maternal embryonic leucine zipper kinase) or EMX2 (empty spiracles homolog 2) and others (Table 1; (Wright et al. 2003)). Furthermore, the data on fetal NPCs generated by the MPSS (Massively

53 Discussion parallel signature sequencing profiling) (Cai et al. 2006) showed a similar expression pattern.

In general, adult and fetal NPCs display a level of overlap comparable to any other pair of the analyzed neural tissues retrieved from databases. The expression profile of the analyzed populations of adult NPCs resembled more the patterns obtained from corpus callosum and spinal cord, but this similarity was not based on the shared expression of markers for adult progenitor cells (e.g. NGFR, GPR17). Therefore, it is not a clear sign for a noteworthy subpopulation of NPCs in spinal cord or corpus callosum. The mentioned NGFR gene was expressed in high abundance in the adult hippocampal derived neurospheres. NGFR plays a central role for the decision between survival and death in neural cells. The confusingly complex pattern of signal transduction involving a wide range of co-receptors and various ligands was so far mainly of interest for the delineation of the molecular decision governing the fate of neurons. However, for the oligodendroglial lineage a clear assignment as pro-mitotic or pro-apoptotic signal remains to be resolved. There are reports on NGFR expression in post-mitotic oligodendrocytes in vivo and in vitro often associated with apoptotic activity (Yoon et al. 1998; Bonetti et al. 1999). Despite these findings there is evidence that high level NGFR expression is a marker for oligodendroglial precursors mostly associated with sclerotic lesions (Chang et al. 2000; Petratos et al. 2004). The reports provide varying information in regard to the frequency of NGFR-positive cells found outside of sclerotic lesions. Chang and colleagues (Chang et al. 2000) noted a widespread appearance throughout the brain whereas Petratos and collegues (Petratos et al. 2004) found these cells rather concentrated within and adjacent to the subventricular zone. Both sources reported a characteristic co-expression of NGFR along with NG2. The NG2 epitope is encoded by the chondroitin sulfate proteoglycan 4 gene (CSPG4) which is specifically over-expressed in the adult NPC population (CSPG4 expression was detected in lower amounts also in the fetal precursor cells and in olfactory bulb). NG2-proteoglycan expressing cells have been identified as a unique class of glia, separated from astrocytes, oligodendroglia and microglia and comprising 5-8% of all glial cells (Levine et al. 1993; Reynolds and Hardy 1997). They have been identified as precursors of mature oligodendrocytes with a subpopulation which remains as a pool of proliferating and immature precursors. Additionally it has recently been demonstrated that NG2+ cells are capable of giving rise to neuronal cell types, as well, and thus contributing to adult hippocampal neurogenesis (Belachew et al. 2003). Compared to fetal NPCs PDGFRA was only detected in reduced amounts. Despite this we found other

54 Discussion highly expressed markers commonly referred to NG2+ progenitors: CSPG4, NES (nestin), CNPase and S100beta. The adult NPC population was slowly dividing but capable to give rise to the three major differentiated neural cell types (Hermann et al. 2006d). In addition to the previously known set of known markers of NG2+ progenitor cells, the list could be extended with e.g. the putative P2Y receptor-like gene GPR17. There is not much known about its function yet, but it was initially detected while screening a human hippocampal cDNA library and identified to be expressed only in brain by Northern blotting (Blasius et al. 1998). G-protein- coupled receptors (GPRs) have a broad repertoire of activating ligands ranging from photons to chemokines and induce an array of cellular events in virtually every physiological system. GPR17 belongs to the P2Y-like receptor family. The P2Y receptor specificity was originally thought to be restricted to purine (adenine) nucleotides has been extended to pyrimidine nucleotides (uridine) and more recently to a receptor with specificity for UDP, but only when conjugated to glucose (Lee et al. 2003a). Ciana and colleagues now identified GPR17 as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor in brain or organs in human and rats typically undergoing ischemic damage (Ciana et al. 2006). This receptor reacts on UDP and it is possible that GPR17 induces proliferation. It will be interesting to show the in vivo localization of GPR17 in comparison with, for example, nestin. Furthermore it was important to find genes specifically over-expressed in adult NPC compared to any other adult neural tissue. A short list of such genes include nestin (Dahlstrand et al. 1995), Tenascin C (Irintchev et al. 2005), semaphorine 5a (Fiore et al. 2005) and Olig2 (Zhou and Anderson 2002), all involved in stem cell and brain development. The fetal NPC population was grouped together with whole fetal brain by hierarchical clustering and hence likely indicating that the fetal brain at week 12 contains a large proportion of uncommitted stem cells. By looking for genes which are specifically over-expressed in fetal neurospheres but not in whole fetal brain could retrieved a large list was retrieved containing genes associated to mitosis CDC2; MCM2; PCNA or CDKN3 which are critical for maintaining an undifferentiated state, such as SOX2, SOX3, SOX4 and SOX9. This category also includes the transcription factor 8 (TCF8, also named Zfhep/deltaEF1), which was found to be expressed in early neural progenitors in the rodent brain (Yen et al. 2001). In addition, over-expression of TCF8 was detected in vascular smooth muscle cells, human vascular endothelial cells and in hMSCs, which points to a more general role for this gene in the development of human stem cells. As another example, the MEIS1 gene was over-expressed in fetal neurospheres but not in whole fetal brain. Like other homeobox-genes this gene plays a crucial role

55 Discussion in normal development. MEIS1 is crucially involved in the limb formation (Mercader et al. 1999) but also important for leukemic transformation (Thorsteinsdottir et al. 2001). A dominant negative MEIS1 splice variant yielded cell clones with impaired cell proliferation, gain of differentiated phenotype, increased contact inhibition and cell death in human neuroblastoma lines (Geerts et al. 2003). The MEIS1 expression pattern fits to that observation and suggests a SOX2-like role for the gene in human neural development.

The mNSCs lost some of the markers highly expressed in hMSCs like decorin (DCN), fibronectin and various collagen proteins, other markers for connective tissue like CTGF (Luo et al. 2004) were downregulated as well. mNSCs expressed neuroectodermal markers like e.g. stathmin-like 2 (SCGN10) (Zhang et al. 2006) and genes involved in metabolism of neuropeptides like e.g. neuroendocrine convertase 1 (PCSK1) (Cadet and Paquin 2000), which cuts neuroendocrine hormones. Brain and acute leukemia gene (BAALC) and SNAP25, a synaptic gene was also upregulated in the mNSCs and the aNPCs, such as neuroectodermal markers like the endothelian receptor one (EDNRA) and Stathmin-like 2 (SCGN10). Zhang and collegues (Zhang et al. 2006) reported about ES-cells, cultivated with IL6R1IL6 (soluable IL6-Receptor fused to IL6), enhancing their differentiation potential into oligodendrocytes and with a high expression of stathmin-like 2. It would be interesting to cultivate mNSCs with IL6R1IL6 to show the oligodendroglial potential of these cells for transplantations in animal-models for myelin-degenerative diseases. This cell-population has an advantage because of its autologous transplantation potential.

Indeed, there is much more common expression of genes in hMSCs and hmNSCs than in hmNSCs and aNPCs. Mainly various collagen isofoms, like COL1A1, COL1A2 and COL11A1 were expressed in both the hMSCs and in the hmNSCs. Interesting was the downregulation of various immune-system-related genes, such as different CXC-motif chemokines (like CXCL1,2,3,5,6 and IL8 (CXCL8)) on one chromosome-location (4q12-13). These genes are growth-related oncogenes, which belong to the Gro-Family and lower expression encoding connective tissue fates (Unemori et al. 1993). This was shown in the gene expression with downregulation of various collagen-isoforms.

56 Discussion

4.3 Differentiation capacity of human adult NPCs

The adult human NPCs are able to differentiate into all major cell types of the brain, namely astroglia, oligodendroglia and neurons. Differentiation of adult aNPCs was initiated by removal of mitogens and plating the cells onto poly D- lysine and incubation for 14 days with BDNF, db-cAMP and retinoic acid with or without pre-stimulation with Shh and FGF-8 for 48h. This mixture of differentiation factors was reported to induce neurogenic effects in adult hippocampal and tegmental NPCs from mouse without affecting the feeder cell layers (Hermann et al. 2006c). The preincubation of adult NPCs with Shh and FGF-8 did not induce significant changes of the neuro-astroglia differentiation behavior of the adult NPCs. Overall, aNPCs differentiated into all major cell types of the CNS, namely mature (MAP2ab+) neurons with long neurites (350 µm after 14 days of differentiation), oligodendrocytes and astrocytes. Functional analyses of the differentiated NPCs revealed obligate functional properties of neurons including the expression of voltage-gated sodium and potassium channels, the generation of action potentials, and the production of neurotransmitters such as glutamate and GABA. The central finding of the study was that non-neuroectodermal cells derived from the mesoderm (mouse embryonic fibroblasts [MEFs] and PA6 stromal cells) promote neurogenesis and maturation of neurons from adult human hippocampal NPCs.

The majority of these cells expressed sustained outward currents with the typical properties of delayed rectifier potassium channels, but no detectable sodium channels. A few cells showed properties of early neurons, such as expression of sodium channels and generation of immature action potentials with a long duration and no relevance after hyperpolarization. This in vitro differentiation capacity is similar to that previously described for adult human SVZ and hippocampal NPCs (Johansson et al. 1999b; Kukekov et al. 1999; Roy et al. 2000; Westerlund et al. 2003; Moe et al. 2005). Roy and colleagues described potassium and sodium channels, but no action potentials in neuronal cells derived from adult hippocampal NPCs expanded for only a short period of time (2 weeks) (Roy et al. 2000). In contrast, Westerlund et al. 2003 and Moe et al. 2005 reported detailed electrophysiological analysis of adult SVZ NPCs showing not only sodium channels and action potentials in differentiated NPCs, but also functional neuronal networks (synaptic connections) from single differentiated aNPCs (Moe et al. 2005). However, these cells were generated from the ventricular wall and not from hippocampal aNPCs and it seems to be important to distinguish well between NPCs from different brain regions as shown for mice counterparts (Liu et al. 1999b;

57 Discussion

Doetsch 2003; Kempermann et al. 2004). In addition to these previous results, it could be demonstrated that differentiated aNPCs produce various neurotransmitters, such as glutamate and GABA.

There is a growing body of evidence that cell-to-cell contacts and/or soluble trophic factors produced by feeder cells are important for neuroectodermal specification of fetal and adult NPCs (Kawasaki et al. 2000; Song et al. 2002; Storch et al. 2003). Various cell types derived from neuroectodermal tissue including cortical, striatal and mesencephalic astrocytes support not only neuronal differentiation, but also neuronal subtype specification of fetal and adult brain-derived NPCs (Kawasaki et al. 2000; Perrier et al. 2004; Kitajima et al. 2005; Hermann et al. 2006c). Additionally, several mesoderm-derived cell types, such as fibroblasts and PA6 stromal cells, are reported to significantly alter the differentiation capacity of ES- cells as well as various fetal and adult NPC types (Kawasaki et al. 2000; Perrier et al. 2004; Kitajima et al. 2005; Hermann et al. 2006c). In contrast to the data from Song and co-workers (Song et al. 2002) showing that adult astrocytes derived from neurogenic regions (hippocampus) promote neurogenesis from adult mouse aNPCs, mixed cortical cultures from E17 mouse embryos containing mainly astroglial did not alter neurogenesis from adult human NPCs in the present study. This discrepancy most likely resulted from differences of the developmental stage and the brain region from which the primary cultures were isolated. In contrast, both mesoderm-derived cell types (MEFs and PA6 cells) induced neurogenesis of adult human NPCs with over 80% Tuj1+ neurons in cocultures of aNPCs together with PA6 cells. Moreover, MEFs and PA6 cells promote morphological and functional maturation of adult NPC-derived neurons. These neurons showed longer neurites with more branches and produced higher amounts of neurotransmitters. On the other hand, trophic support provided by PA6 conditioned media did not lead to significant changes of glial and neuronal differentiation of aNPCs. This is in agreement with studies on mouse and human ES cell derived NPCs showing that fibroblasts and PA6 cells promote neurogenesis from these NPCs with co-culturing being much more effective than PA6 conditioned media (Kawasaki et al. 2000; Zeng et al. 2004). Interestingly, PA6 feeder cells did not influence the glial potential of aNPCs, whereas MEFs and particularly mixed cortical cultures increased the number of astrocytes differentiated from aNPCs. Together, these results demonstrate a neurogenesis inducing activity of mesoderm-derived cell types not only in ES-cells as reported previously (Kawasaki et al. 2000; Zeng et al. 2004), but also in adult human NPCs. The mechanisms or factors of this neurogenic activity are largely unknown, but most likely include cell-to-cell contacts and/or cell

58 Discussion

(membrane)-bound factors, such as the Wnt ligands (Kawasaki et al. 2000; Storch et al. 2001; Song et al. 2002; Schmidt and Patel 2005). However, due to the different neuronal-glia induction potential of the investigated feeder cell types, it seems to be most likely a combination of different trophic factors and cell surface interactions which influence the neuro-glia fate determination. Rodent feeder cell layers were used to analyze the neural differentiation capacity of adult NPCs and therefore it is unclear whether human feeder might have different trophic effects.

The described technique for long-term expansion of these adult human NPCs allows the generation of high yields of cells for further characterizations by molecular biology or protein biochemistry, such as gene array analysis or proteomics. This study provides therefore a framework for standardized comparative analysis of brain-derived adult NPCs with adult NPCs derived from other human cell sources, such as bone marrow stromal or skin cells (Toma et al. 2001; Hermann et al. 2004b). However, future studies are warranted to further define the functional (electrophysiological) properties including synaptic neurotransmitter release of differentiated adult human NPCs and compare these data with data obtained on primary human astrocytes and neurons. Furthermore, it was demonstrated that mesoderm derived cell types, such as MEFs and PA6 stromal cells, not only induced neurogenesis from adult human NPCs, but also promoted morphological and functional maturation of neurons derived from these progenitor cells. The analysis of the underlying molecular mechanisms in the future might help to better understand neurogenesis and neuronal maturation in the adult human brain.

4.4 Therapeutical potential of different neural and neural-like cell populations

My work describes new adult human cell sources which fulfills important requirements for the use in autologous neurorestorative treatment strategies. The major advantage of hMSCs is the possibility of an autologous approach in stem cell therapy without the need of therapeutic cloning. Furthermore, for the neuroectodermal converted NPCs exclusively epigenetic factors were used without genetically engineering the cells. This expands their potential use in humans by reducing the risk for tumourgenesis, as well as for unwanted gene transfer to the host organism.

Adult neuroprogenitor or stem cells are suited to use for transplantation strategies in regard of the lack of need for immunosuppression. Another important point is the amount of cells which is needed. In a typical experiment, from 1x106 hMSCs,

59 Discussion routinely 1.6x108 marrow-derived NPCs, and subsequently 0.1x108 neurons, 0.7x108 astroglial and 0.4x108 oligodendroglial cells were obtained. Considering all parameters of proliferation, cell death and selective differentiation throughout the multiple conversion and differentiation steps, at least 7x107 hmNSCs, or 0.5x107 neurons, 3x107 astroglial and 2x107 oligodendroglial cells, respectively, could numerically be produced from one hMSC harvested from the bone marrow sample. These cell quantities are sufficient for most transplantation protocols as well as for extensive characterizations of adult NPCs by molecular biology or protein biochemistry, such as gene array analysis or proteomics. Thus, the gained knowledge about hmNSCs could lead to a better understanding of the molecular mechanisms of neural differentiation in adult human NSCs. In contrast to most previous protocols for the conversion of MSCs into neuroectodermal cells generating mature neuronal and/or glial cells attached to the culture surface (Sanchez-Ramos et al. 2000; Hofstetter et al. 2002; Jiang et al. 2002b; Kim et al. 2002a; Woodbury et al. 2002; Jiang et al. 2003), here the possibility is reported to generate undifferentiated NPCs or premature neural cells from human origin. Those are commonly used and more suitable for neurotransplantation compared to fully differentiated neural cells (Bjorklund and Lindvall 2000; Carvey et al. 2001; Kim et al. 2002b; Pluchino et al. 2003), as differentiated neuronal cells are well known to poorly survive detachment and subsequent transplantation procedures. Although studies in animal models of neurodegenerative diseases are needed to assess the function and safety of hmNSC-derived neural cells in vivo, the method provides the means to study autologous approaches in neurotransplantation using adult human NPCs as well as adult human NPCs derived from bone marrow, an accessible tissue in every individual. The first transplantation studies using neuroectodermally converted immature cells derived from human bone-marrow showed that these cells survive and migrate to diseased brain areas (Lee et al. 2003b). However there is a hypothetical risk that the patients own cells might carry genetic information that increases their vulnerability to the pathogenetic progress underlying the patient`s disease.

In comparison to the brain-derived NPCs, the hMSCs are an easily accessible and on-demand available cell source in every individual. Using routine bone-marrow punction to isolate these cells for further in vitro proliferation and subsequent sorting or priming towards the lineage of interest would revolutionize stem cell therapy.

60 Summary

5 Summary

The neurobiological dogma that the adult mammalian brain is not able to produce new neurons, was finally proven to be wrong about a decade ago. Ongoing neurogenesis in the adult mammalian brain is now widely accepted, but the mechanisms controlling development and differentiation of adult neural stem cells are still largely unknown. The importance of understanding the basic mechanisms of neurogenesis in the adult brain becomes obvious in the view of possible treatment strategies of neurological diseases and injuries, which damage CNS neurons. My thesis aims to characterize the phenotype and differentiation capacity of human adult hippocampal NPCs (aNPCs) derived from patients who underwent epilepsy surgery. Their differentiation potential was tested on various feeder cells including fetal mixed cortical cultures, mouse embryonic fibroblasts (MEFs) and PA6 stromal cells. Isolated aNPCs were cultured in clonal density by transferring the cells to serum-free media supplemented with FGF-2 and EGF in 3% atmospheric oxygen. These aNPCs showed neurosphere formation, expressed high levels of early neuroectodermal markers, such as the proneural genes NeuroD1 and Olig2, the NPC markers Nestin and Musashi1, the proliferation marker Ki67 and significant activity of telomerase. The phenotype was CD15low/–, CD34–, CD45– and CD133–. After removal of mitogens and plating them on poly D-lysine, they spontaneously differentiated into a neuronal (MAP2ab+), astroglial (GFAP+), and oligodendroglial (GalC+) phenotype. Differentiated aNPCs showed functional properties of neurons, such as sodium channels, action potentials and production of the neurotransmitters glutamate and GABA. Co-culture of aNPCs with fetal cortical cultures, MEFs and PA6 cells increased neurogenesis of aNPCs in vitro, while only MEFs and PA6 cells also led to a morphological and functional neurogenic maturation. A global gene expression profiling was performed using RNA from hippocampus-derived aNPCs and multipotent frontal cortical fetal NPCs in comparison to adult human mesenchymal stem cells (hMSCs) as a multipotent adult stem cell control, converted neural-like stem cells derived from hMSCs, and adult human hippocampal tissue, to define a gene expression pattern which is specific for the human NPCs. The results were compared with data from various databases. Hierarchical cluster analysis of all neuroectodermal cell/tissue types revealed a strong relationship of adult hippocampal NPCs with various white matter tissues, while fetal NPCs strongly correlate with fetal brain tissue. However, adult and fetal NPCs share the expression of a variety of genes known to be related to signal transduction, cell metabolism and neuroectodermal tissue. In contrast, aNPCs and hMSCs overlap in the expression of genes mainly involved in extracellular matrix biology. For the first time a detailed transcriptome analysis of human adult NPCs suggesting a relationship between hippocampal NPCs and white matter-derived precursor cells is presented. This provides further a framework for a standardized comparative gene expression analysis of human brain-derived NPCs with other stem cell populations or differentiated tissues.

61 References

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75 Appendix A

7 Appendix A

Table A-1: Human Primers used for quantitative real-time RT-PCR

Genes Accession no.

-TUBB4/III 5´-GGC CTC TTC TCA CAA GTA CG-3´ NM_006086 5´-CCA CTC TGA CCA AAG ATG AAA-3´ (ß-tubullin-4 class 3) CCR7 5´-CGATACCTACCTGCTCAACCTG-3´ NM_001838 (chemokine,cc-motif, 5´-AAAGTGGACACCGAAGACCC-3´ receptor7) EDNRB 5´TGG TTC TGG CTG TCC CTG A-3´ NM_000115 5ÁAG CAG AAA TAG AAA CTG AAT AGC C-3´ (endothelin receptor type B) FZD1 5´-GGA TTG GCA TTT GGT CAG TG-3´ NM_003505 5´-CTT GTC ATT ACA CAC CAC TCG G-3´ (frizzled 1) GFAP 5´GAG GCG GCC AGT TAT CAG GA-3´ NM_002055 5´GTT CTC CTC GCC CTC TAG CA-3´ (glial fibrillary acidic protein) GPR17 5´GGA GAC GCC ACT GGA GAA C-3´ NM_005291 (G-protein-coupled-receptor 5´-ATG AAA AGC CAC AGA GCC AG -3´ 17) HMBS 5´TCG GGG AAA CCT CAA CAC C-3´ NM_001024382 (hydroxymethylbilane 5ĆCT GGC CCA CAG CAT ACA T-3´ synthase) LAMA4 5´ GAC CCGCCTGAGACGAGC-3´ NM_002290 5´ TACAGTCGCAGGGCACACAT-3´ (laminin-α4) MBP 5´-CGG CAA GAA CTG CTC ACT ATG-3´ NM_001025101 5´GTG CGA GGC GTC ACA ATG-3´ (myelin-basic protein) MSI 5´GCC CAA GAT GGT GAC TCG-3´ NM_002442 5ÁTG GCG TCG TCC ACC TTC-3´ (musashi 1) Nestin 5´-CGA GCC CTA CAC CTC CTA TCA C-3` NM_006617 5´-TCT TGAA GGT GTG CCA GTT GC-3´ (nestin)

NeuroD1 5´-CGC TGG AGC CCT TCT TTG-3´ NM_002500 5´-GCG GAC GGT TCG TGT TTG-3´ (neurogenic differentiation 1)

Neurogenin2 5´-CGC ATC AAG AAG ACC CGT AG-3´ NM_024019 5´-GTG AGT GCC CAG ATG TAG TTG TG-3 (neurogenin 2) Notch1 5´-GCG ACA ACG CCT ACC TCT-3´ NM_017617 (notch homolog 1 5´-GCA CAC TCG TAG CCA TCG-3´ translocation associated) NTRK1 5´-CTA CAG CAC CGA CTA TTA CCG-3´ NM_001012331 (neurotrophic tyrosine kinase, 5´-CGA TTG CCT CCG TGT TG-3´ receptor, type 1) OTX1 5´-CAC TAA CTG GCG TGT TTC TGC-3´ NM_014562 5´-AGG CGT GGA GCA AAA TCG-3´ (orthodenticle homolog 1)

76 Appendix A

Genes Accession no.

OTX2 5´-CAC TTC GGG TAT GGA CTT GC-3´ NM_021728 5´-CGG GTC TTG GCA AAC AGT G-3´ (orthodenticle homolog 2) P75(NGFR) 5´-CTTTTGGGGTATCCATAGCAGT-3´ NM_002507 5´-CCACGGGACCCTTCATTC-3´ (p75 NGF-recepor) PDGFRα 5´CCTGGAGAAGTGAAAGGCAAA-3´ NM_006206 (platelet- derived- growth 5´CTTTGACCTCCCTGGTAGCC-3´ factor receptor-α) SOX1 5´-GCC CAG GAG AAC CCC AAG-3´ NM_005986 (sex determining region Y- 5´-CGT CTT GGT CTT GCG GC-3´ box 1) SOX2 5´-CAG GAG AAC CCC AAG ATG C-3´ NM_003106 (sex determining region Y- 5´-GCA GCC GCT TAG CCT CG-3´ box 2) SOX10 5´-GCA AGG CAG ACC CGA AGC-3´ NM_006941 (sex determining region Y- 5´-GTC CAA CTC AGC CAC ATC AAA G-3´ box 10) TNC 5´GCGGTCGGGGCAACTT-3´ NM_002160 5´GACAGTTGCCTGGACATTCG-3´ (tenascin C)

77 Appendix B

8 Appendix B

Table B-1: Genes enriched in both adult NPCs and fetal NPCs

Category Genes Signaling MyoD family inhibitor (MDFI); Transcription factor NFIB; Protein-Tyrosine-Phospatase (PTPNS1); Ras association domain family protein (RASSF2); TTYH1 Cell membrane Glycoprotein 6A; Glycoprotein 6B Axon guidance Fasciculation and elongation protein zeta1 (FEZ1) Brain Brevican (BCAN); Brain specific angiogenesis inhibitor 2 (BAI2); Tubulin- (TUBA3) Cytoskeleton integrin-7 (ITGA7); Coronin 2B (CORO2B); Kinesin 5C (KIF5C); Extracellular Matrix proteins Collagen9A3 (COL9A3); laminin-4 (LAMA4); Tenascin C (TNC) Metabolism Fatty acid binding protein 5 (FABP5); GABA Transferase (ABAT) ; ATPase (ATP1B2); Potassium voltage-gated channel (KCND3); Reticulon1 (RTN1) Development S100B; Semaphorine 5a (SEMA5A); Pleiotrophin (PTN); JAG1; SOX2; SOX4 Adhesion NCAM1 Stem cell markers Nestin, Olig2, Musashi (MSI) Astroglial markers GFAP Neural markers Brain and akute leukemia gene (BAALC) Receptors Glutamate receptor 2 (GRIA2), GPR56 Other GLT25D2; QKI; RIPX; SCRG1: SPARC-like Protein 1 (SPARCL1); Synaptotagmin 11 (SYT1); VEGF Unknown KIAA0523

Table B-2: Genes enriched in both adult NPCs and adult hMSCs

Category Genes Signaling Insulin-like growth factor-binding protein 5 (IGFBP5); IGFBP3 Extracellular matrix TenascinC (TNC); collagen type I -1 (COL1A1); COL1A2; COL3A1; lysyl-oxidase-like2 (LOXL2); laminin-4 (LAMA4); matrixmetalloproteinase 2 (MMP2); tissue inhibitor of matrixmetalloproteinase 1 (TIMP1); Chitinase3 (CHI3L1) Cell Membrane Glycoproprein NMB (GPNMB); Integrin-α7 (ITGA7) Cytoskeleton Caldesmon 1 (CALD1) Receptors Neurotrophic Tyrosine Kinase type2 (NTRK2) Metabolism Cathepsin Z (CTSZ); La ribonucleoprotein family member 6 (LARP6); Plasminogen Activator Tissue (PLAT) Development Dickkopf 1 (DKK1); DKK3; Pleiotrophin (PTN) Adhesion Thrombospondin II (THBS2) Apoptosis Cytrochrom 450 (CYP1B1) Others Phosphodiesterase 2 (ENPP2); Inhibin-1 (INHBA); Plasminogen activator inhibitor1 (Serpine1), Endothelial Differentiation Gene2 (EDG2); Phosphodiesterase 4D- interacting protein (PDE4DIP)

78 Appendix B

Table B-3: Genes expressed both in hMSCs and hmNSCs

Category Genes Signaling EPS8 (Epidermal growth factor receptor pathway substrate 8); IGFBP3 (Insulin growth factor binding protein); IGFBP4; IGFBP5; LY96; RND3 (RHO family GTPase 3); TGFB1 Structur/ Cellmembrane CAV1; CDH11; CSPG2; DCN (Decorin); FAP (Fibroblast activation protein); FBN1 (Fibrillin 1); FER1L3; FN1; GJA1; GPNMB (Glycoprotein NMB); ITGBL1 (integrin-β-like 1); LUM; MMP2 (Matrixmetalloproteinase 2); NID1 (Entactin); NID2; POSTN (Integrin-Ligand); PRG1 (Proteoglycan 1); SDC2 (Heparan sulfate Proteoglycan); SGCB (Sarcoglycan-β); SRPX (Sushi repeat containing protein X); STC2 (Glycoprotein hormone); SYNC1 (syncoilin); TFPI (Tissue factor pathway inhibitor); TMEM45A (Transmembrane protein 45A); TMEM47 (Transmembranprotein 47) Extracellular Matrix COL11A1; COL1A1; COL1A2; COL3A1; COL5A1; COL5A2; COL6A1; COL6A2; COL6A3; EFEMP2; FBLN1; LAMA4; LAMB1; LAMC1; PLOD2; TIMP3; TNC; MYL9 (Myosin-lght-chain); MYO1B; P4HA2 (Prolyl 4- hydroxylase); PCOLCE (Procollagen type 1); PLOD2 (Procollagen lysine); SPARC; TPM2 (Topomyosin2) Immune System related IL1R1 Cell cycle CCND1(Cyclin D1) CDs CD73 Metabolism CALU; CSTA (Cystatin A); CTSK (Cathepsin K); GALNT1 (GalNAc) Transferase 1); HEPH (Hephaestin); KCTD12 (potassium channel tetramerisation domain containing 12); KDELR3 (endoplasmic reticulum protein retention receptor 3); LOX (Lysyl-oxidase); LOXL1; LOXL2; NNMT (Nicotinamide N-methyltransferase); PAPSS2; PLEKHC1 (Pleckstrin homologydomain-containing protein) ; PPP1R3C (Protein-Phosphatase1); PROS1 (Protein S); PSD3 (Proteasom 26 subunit); RCN1; SLC1A1 (Glutamat-transporter 1); SLC2A10 (Glucose- Transporter 10); STCH; WIPI1 Adhesion THBS2 (Thrombospondin 2) Transcription/ Translation HRB2 Development EDG2 (Endothelial differentiation Gene); FZD1; GREM1; INHBA (Inhibin-β1); PDLIM4 (PDZ and LIM domain protein 4); Ptx3 (pentraxin 3); SNAI2 (Neural crest tarnscription factor slug); WISP2 (Wnt1 inducible pathway protein); WNT5A; Apoptosis/Cancer/ CYP1B1 (Cytochrom P450); EMP1 (Epithelial membrane Proliferation protein 1); GAS1 (Growth arrest- specific); GAS6; RECK; SART2; TUSC3 (Tumor supressor candidate 3) Receptors PDGFRA; Myeloid HOXA9 Mesencymal Genes PDGFC; WWTR1 (WW Domain-containing transcription regulator 1) Other CBLB; FKBP11; GLIPR1; FKBP11; LHFP; OLFML3; PAPPA; PXDN; STEAP1; XIST; SERPINE 1; SERPINE 2; VEGFC Unknown LOC51334

79 Appendix B

Table B-4: Genes expressed both in hmNSC und aNPCs

Category Genes Signaling IGFBP3 (insulin-growth-factor-binding-protein 3); IGFBP5; PHLDA1* (Pleckstrin homology like domain family A, member 1) Structur/ Cellmembrane CALD1 (caldesmon 1); GPNMB (glykoprotein NMB); PDPN* (prodoplanin-glykoprotein 36) Extracellular Matrix COL1A1 (collagen1A1); COL1A2; COL3A1; LAMA4 (laminin A4); THBS2 (thrombospondin II); TNC (tenascin C) Immune System related IL8 (interleukin 8*) Metabolism APOD (apolipoprotein D) *; LOXL2 (lysyloxidase 2); MMP2 (matrix metalloproteinase 2) Development ANGPTL4 (Angiopoietin-like 4) *; INHBA (Inhibin-βA); Sema 5a (semaphorine 5A) * Apoptosis/Cancer/ CYP1B1 (cytochrom P450); Proliferation Neural BAALC (Brain and acute leukemia gene); Neuronal GPM6B (Neuronal Glykoprotein 6B) Neuroendocrine SGNE1 (Secretory granule neuroendocrine protein 1) * Endothelial EDG2 (Endothelial differentiation gene 2) Mesencymal Genes SPP1 (Osteopontin) * Other Serpine 1 * no expression in hMSCs

80 Appendix B

Table B-5: Genes upregulated in aNPCs versus fNPCs (genes showing greater than 10- fold differences)

Category Genes Signaling RAPGEF5; PLEKHB1; DUSP10; PCTK3; CAPN3; PHLDA1; SH3BP5; SYNJ2; RAPGEF5; GNG7; PCAF; ALK; WNK1; IGFPB3; IGFBP5; PIK3R1; CTGF; IL6ST; EPAS1; KLF2; PACE4; FLRT3 Structur/ Cellmembrane HCN2; ANK3; MAPT; AIP1; ITM2A; MCAM; EPB41L1; CDH19; ITM2A; ELMO1; LDB3; GPNMB; DNM3; PRG- 3; PTGDS; SORL1; TM4SF11; CHN2; KCNK10; MAP7; MAP1B; DCTN1; SPOCK; MOX2; WASL; DCTN1 Extracellular matrix CHI3L1; COL1A1; COL1A2 Immune System related HLA-DRA; HLA-DQA1; CCL2; RNF128; HLA-DRB3; HLA- DMA; HLA-E; IFI16; TAP1; ISG20; LY96 Cell cycle CDC14B CDs CD9; CD74; CD44; CD59 Cytokines CCR7; IL8; IL11 Metabolism TF; GLS; PACE4; DNAJC6; ENPP2; HEPH; FOLH1; SGNE1; SLC23A1; CHI3L1; GPD1; APOD; ASPA; ITPKB, SLC31A2; RPS6KA2; MT1K; ENO2; ABCA2; PDE4DIP; GSTA4; ATP8A2; ATP8A1; GSN; LOXL2; ME1; CTSD, APOC1; ARHI; TIMP1 Transcriptional regulation HDAC9; PADI2; HSPA2; ZNF536; ZNF365; ERBB3 Translation DDX17 Development MAL; DKK3; NCDN; CLU; PCDH9; SOX10; DKK3; WARP; FGF1; Apoptosis/Cancer/ CYP1B1; FAIM2; DLG1; BACE2; ANGPTL2; ALK; MET; Proliferation ERBB3; DLG1 Cell adhesion/Migration NFASC; THBS2 RNA processing RNASE1; NAP1L3; DICER1 Receptors GPR17; GPR37; NGFR; GPRC5B; EDG2; NTRK2; PLAUR; TNFRSF21 Synaptic genes SYNGR2; KIF1A; VAMP3 Muscle DMN; CAPN3 Brain KLK6; APLP1; GAS7; SEPT4; TPPP Oligodendrocytes NKX2-2; MOG; PMP2; PLP1; MBP; MAG; MOBP; OMG; UGT8; GALC; PMP22 Neural Genes KLK6; APLP1; DDR1 Neuronal Genes TUBB4; NPTX1; NEFL Other BAMBI; SPP1; DSCR1; KIAA0367; ABHD2; KIAA0420; LHPP; FA2H; MAN1C1; SLC22A17; GRSP1; CA2; STMN4;ABHD2; EFHD1, KIAA0877; TRIM2; DSCR1L1; BCAS1; SLCO3A1; BTBD14A; T1A-2; RAB40B; SLC1A4; PKP4; LYZ; MVP Unknown C20orf35; C11orf9; MGC29643; C14orf139; C3orf4; C14orf78

81 Appendix B

Table B-6: Genes upregulated in fNPCs versus aNPCs (genes showing greater than 10- fold differences)

Category Genes Signaling TCF8; CKS2; LRRC17; ID4; ID2; LYN; RIS1; MARCKS; HMGB2; FARP1; TRIB2; FOXM1; ZNF22; MEIS2; TCF7L2; LRRN3; PXMP2; PIK3R3; MELK; IGFBP2; SPRY1; CYR61; DUSP6; PSIP1; DUSP4; CRMP1; SFRS10; ARHGAP19, TRAF4; NFIB; TSPAN6; PLEKHC1; PDLIM1; PRKCN Structur/ Cellmembrane TMSNB; SPON1; ETV1; KIF11; LMNB1; TFPI; KIF15; MYO5C; FXYD6; GPM6A; CSPG5; FARP1; CHRDL1; PRG4; ADD2; EPB41L3 Immune System related TCF7L2; FKBP1B Cell cycle PRC1; CDC2; CCNB1; MCM6; MCM7; WEE1; CENPF; TPX2; RRM2; MCM2; PCNA; CDKN3; MCM4 Extracellular Matrix proteins ADAMTS3; TNC Metabolism SLC4A4; PDK; SLC15A2; IER5; SNCAIP; FABP7; ELOVL2; DDAH1; RFC3; ATP1A2; KCND2; TYMS; ZFP36L1; CHST11; DHFR; PECI; KCNIP1; CAP2; NUDT11; SCN1A; RRM1; RTN3; UBE2C Transcriptional regulation LHX2; IMP-3; H2AFZ; ZNF505; HEY1; HES1 DNA repair ETV1; H1FX; MSH2; CHC1L Development HOP; SOX11; GAS1; SOX9; DCX; ZIC1; FZD3; DLL3; CTBP2; PAX6; ZFPM2; NETO2; NR2E1; ASCL1; EMX2; NELL2; PEG10; UCP2; NEDD9; PUM2; SFRS1; SOX2; NOTCH3 Apoptosis/Cancer/ TOP2A; LMO2; RRM2; DAPK1; TCF7L2; BIRC5; KLIP1; Proliferation PNMA2; HN1; SPRED2; MCL1; APC; STK17A; MN1 Cell adhesion/Migration GJA1; CHL1; KAL1; CDH4 RNA processing RBPMS; PAIP1; IMP-3; ELAVL1 Receptors EGFR; EDNRB; PDGFRA; PTPRZ1; GPR51; GRIA1; F2R; FGFR3; Synaptic genes SYNE2; SNAP25 Brain MLC1; TOX Neuronal Genes GAD1; LRRN3; SYNE2; SNAP25; TUBB Other SIAT7E; NUSAP1; LHX2; TNRC9; Pfs2; ZWINT; SEPT11; TU3A ; ZNF423; FHL1; SMC4L1; FNBP1L; SETBP1; SNN; FAM3C; RAFTLIN; DCP2; BZW1; LSM5; MAC30 Unknown C11orf8; C5orf13; C6orf62; C1orf29

82 Appendix B

Table B-7: Genes upregulated in aNPCs versus hMSCs (genes showing greater than 10- fold differences)

Category Genes Signaling ITPKB; CIT; CHN2; RASSF2; PRKCZ; RAPGEF5; ALK; RAPGEF5; ERBB3; SH3GL3; ISG20 Structur/ Cellmembrane TUBB4; ITM2A; KIF5C; TM4SF11; ELMO1; PRG-3; T1A- 2; DNM3; KCNK10; CORO2B; BCAN; FUT9; SEPT8 Extracellular Matrix COL9A3 Immune System related HLA-DRB1; MAL; HLA-DRA ; HLA-DRB3 ; CCR7 ; HLA- DMA; HLA-DQA1; TGFA; CXCL14; IL1B; IL11 Cell cycle PPP2R2B; KIF1A CDs CD74; CD9 Metabolism TF; APOD; FOLH1; SORL1; RNF128; GATM; CA2; GPD1; FABP5; FA2H; GATM; APOC1; ABHD1; ATP8A2; PACE4; CYB5R2; SLC31A2; ASPA; GLUL; B3GAT1; ATP8A2; CHI3L1 Adhesion PCDH9 ; NCAM1 ; NRCAM ; SEMA4D; TM4SF2; L1CAM; CDH19; PCDH17 Transcriptional regulation TTYH1 ; ZNF536 ; CROC4 Translation PADI2 Development GPM6B; SOX10; SEPT4; GAS7; GDF1; ITM2A; GAS7; SOX2; TGFA Apoptosis/Cancer/Proliferation FAIM2; BCAS1 RNA processing RNASE1 Receptors NGFR; GPR17; GPRC5B; TNFRSF21; GPR56; GRIA2; GPR37 Muscle CAPN3 Endothel SPARCL1 Brain KLK6; MTUS1; SNCA; FABP7; PTGDS; BAALC Neural Genes S100B; NES; DDR1 Oligodendrocyte MBP; PLP1; MAG; PMP2; MOG; UGT8; OMG; OLIG2; NKX2-2; MOBP Glia GFAP Neuronal Genes NPTX1; PLEKHB1; MAPT; SGNE1; SYT11; VGF Neurotransmitter ABAT Other SEPP1; CXADR; LRRC15; CITED1, PHLDA1; KIF5C; RTN1, CAPN3; CaMKIINalpha; GNG7; NDRG2; NOVA1; ATF3; SIAT9, CLU; PTPRE Unknown C11orf9

83 Appendix B

Table B-8: Genes upregulated in hMSCs versus aNPCs (genes showing greater than 10- fold differences)

Category Genes Signaling IGF2, IGFBP4; ISLR; DAB2; CDC42EP3; PLCB4; LRRFIP1; STAT1; CYR61; PDLIM1; TRAF5; PLCB4; IGFBP7; STAT6; FGF2; MARCKS; EGFL3, SMAD6; IGFBP7; TCF7L2; FOSB; RCN3 Structur/ Cellmembrane MFAP5; ITGBL1; KCTD12; ELN; ACTA2 ; ANXA1; TPM1; LMOD1, TPM2; DSP, MYL9; PRG1, TIMP3; PRG1; TM4SF10; MYO1B; FN1; AGC1; CNN1, FAP; TM4SF1; NID2; CDH11; AGC1; LAMC1; PDLIM7; ADCY7; NUCB2, FXYD5; ENG; CLIC3; PLCB4; PHACTR2; PDLIM7; OLFML3 Keratin KRT7; KRT14; ; KRT18; KRT19; KRTAP1-1; KRT16 Extracellular Matrix proteins COL1A1; FBN1; LUM; COL3A1; THBS1; LOX; EFEMP1; FBLN1; LAMC1; COL5A1; COL5A2; BGN; COL14A1; DCN; HSPG2; LTBP2 Immune System related CXCL12; IFI27; CRLF1; IFITM1, GDF15; SART2; VCAM1; TGFBI Cell cycle G0S2; PLK2 CDs CD268 Metabolism PTGIS; SLC1A1; NNMT; SLC7A11; PAPSS2; ASS; RBP4; DDAH1; PPIC; PPP1R3C; TIMP3; ADAMTS1; GFPT1; SULF1; UGCG; SEC23A; GALNT1; ATP10A; AKR1C2; AKR1C1; SLC2A10; PAPSS2; MTHFD2; ADARB1; TFPI; TPST2; SEC24D; MTHFD2 Epidermis FLG Granulocytes CSTA Transcriptional regulation PTX3; HOXC6; RECK; LDB2; KLF4; HOXC10; FOXD1; ZNF423; CTNNAL1; ID2; TCF7L2; FOSB; GATA6 Translation PAPSS2 Development WNT5A; SLIT3; NOTCH3; EN1; FGF2; FZD2; EVC; FZD7; EMX2 Apoptosis/Cancer/Proliferation TPD52L1; GAS6, DAPK1; D2S448; PAWR; CYP26B1; TRIM22 Angiogenesis VEGFC Hormone PENK; STC2 RNA processing RBPMS Receptors NPR3; TNFRSF11B; LEPR; OXTR; EGFR; PDGFRA; TNFRSF6; ROR1 Cell Adhesion/Migration GJA1 Osteogenesis TNA Adipocyte DF Chondrocyte EXT1 ; FARP1; COMP Muscle TAGLN; COX7A1; CALD1 Brain PLSCR4 Neural Genes GREM1; SNAI2 Neuronal Genes CRIM1 Other FGF7; PSG5; FSTL1; CITED2; KCTD12; CRIP1; KDELR3; RCN3; HFL1; SULF1; PARG1; DEPDC6, PAPPA; CBS; LOC51334 Unknown C10orf116; C1orf24

84 Appendix B

Table B-9: Genes upregulated in hmNSCs versus hMSCs (genes showing greater than 10- fold differences)

Category Genes Signaling BTBD3; CAMK2N1; DIRAS3; DUSP6; GEM; IGFBP1; RGS2; RIS1; TCF8 Extracellular matrix COL18A1; COL21A1; COLEC12; DPT; LAMA2; MMD; MMP3; MMP13; SPON1; TIMP4 Cell Membrane/Structure AREG; F3; GPM6B; MME; SVIL; TNFAIP6; TNFSF11 Cartilage CHI3L1 Muscle CDCP1 Adhesion SPARCL1 Hormones STC1 Immune-system-related C3; C7; CCL20; IFI44L Gro-family CXCL1; CXCL2; CXCL3; CXCL5; CXCL6; IL8; Receptors AGTR1; BDKRB2; EDNRA; NETO2; TNFRSF21 Neuropeptide-Metabolism PCSK1; QPCT; SGNE1; TAC1 Cell Cycle CCND2 DNA/RNA Processing EIF2S3 Transcriptional/Translation ZBTB16; ZNF395 Metabolism ADH1B; ALDOC; CA12; CA9; GFPT2; GK; HSD11B1; KYNU; PLTP; SAT; SLC19A2; SOD2 Fat-Metabolism ANGPTL4; APOD; APOE; PTGS1 Development BMP2; EREG; HGF; PBEF1 Chromosome Remodeling HMGA2 Brain BAALC Neuronal GAP43; MAOA; NRCAM; SLC6A15; SNAP25; STMN2 Others AIM1; FLJ00133; FLJ10159; FLJ20323; FLJ20701; FLJ23091; SPAG4; SPP1; SRPX; T1A-2, VEGF

85 Appendix B

Table B-10: Genes upregulated in hMSCs versus hmNSCs (genes showing greater than 10-fold differences)

Category Genes Signaling CDC42EP3; IGF2; KLF4; LRRC15; PTTG1; RGS4; STK6; Extracellular matrix ADAMTS1; COL4A1; LTBP2; MFAP5; LOX; SULF1 Cell Membrane/Structure AGC1; ANK3; ELN; KRT14; KRT7; KRTAP1-1; KRTHA4; PODXL; TPM1 Gro-family CTGF; CXCL12; Cartilage COMP; Muscle ACTA2; ACTC; LMOD1, TAGLN; THBS1; Osteoklast differentiation TNFRSF11B; Hematopoetic KLIP1; Mesodermal Genes LTBP2; Immune-system-related ALCAM; CRLF1; CRYAB Receptors AMIGO2; HMMR; LEPR; NTRK2 Cell Cycle BIRC5; CCNB1; CCNB2; CDKN3; CENPF; DACT1; KIF20A; NEK7; PRC1; RRM2; WFDC1 DNA Processing TOP2A; Chromosomal Genes HMGB2; NUSAP1; Metabolism ATP1B1; CPT1A; CRIP1; DSCR1L1; KCNE4; PDK; PENK; PTGIS; RBP4; RPS4Y1; SEPP1; SLC14A1; SLC1A4; SMURF2 Development DKK1; DKK3; DLG7; DLX2; JAG1; MID1; NOV; PTN; SLIT3 Transcriptional Regulation FOXM1; ID1; ID3; ID4; Brain ASPM; PEA15; Apoptosis/Cancer/ DAPK1; OAS1; Proliferation Others AMOTL2; CRIM1;DEPDC6; DLC1; EFHD1; FAM38B; FST; KIAA0101; KIAA0830; KIAA1199; KIAA1644; LBH; LOC51063; OLFML1; TACC3; Unknown C10orf116; C10orf3; C1orf24; C9orf3

86 Appendix B

Table B-11: Genes upregulated in mNSCs versus aNPCs (genes showing greater than 10- fold differences)

Category Genes Signaling RGS2; DUSP6; RIS1; TNFAIP6; BMP2, CAMK2N1; IGFBP1; DIRAS3; ZBTB16, TCF8 Structur/ Cellmembrane AREG; GPM6B; SPON1; T1A-2; DPT; F3; SVIL Extracellular Matrix proteins COL21A1; TIMP4; LAMA2; COLEC12; COL18A1 Immune System related PBEF1; MMD; C7; GEM; C3 Cell cycle CCND2 Fat-Metabolism APOD; PTGS1; PLTP; QPCT; APOE

Metabolism GFPT2; SOD2; MME, ADH1B, GK; HSD11B1; SAT; CA12; MMP3; SLC19A2; KYNU; ALDOC Neuropetides TAC1; PCSK1 (Neuroendokrine genes) Gro-family IL8; CXCL6; CXCL5; CXCL2; CXCL1; CCL20 Translation EIF2S3 Osteoclast differentiation SPP1 Receptors TNFRSF21; NETO2, BDKRB2; EDNRA; AGTR1 Cell Adhesion/Migration NRCAM Myeloid Genes TREM1 Hormones STC1 Chondrocyte CHI3L1 Mesenchymal genes HMGA2 Neural Genes BAALC Neuronal Genes GAP43; SNAP25, MAOA; SLC6A15; SGNE1 Other HGF; AIM1; EREG; SRPX; VEGF; ANGPTL4; BTBD3

87 Appendix B

Table B-12: Genes upregulated aNPCs versus mNSCs (genes showing greater than 10- fold differences)

Category Genes Signaling ARHI; ATF3; CALM1; CIT; E2F1; HEY1; IGFBP2; ISG20; ITPKB; KHDRBS3; LDB3, MDFI; MLP; NDRG2; PCTK3; PHLPP; PIK3C2B; PLEKHA1; PRKCZ; QKI, RAB33A; RAPGEF5; RNF128; SGK; SH3BP5; SH3GL3; TNIK, TRIM14; TRIM2; ZNF536 Structur/ Cellmembrane ANPEP; ATP1B1; ATP1B2; ATP8A2; BCAN; CHN2; CLCN6; CLU; CORO2B; CSPG5; DNM3; EPB41L1; EPN2; FXYD6; FXYD7; GJB1; GPM6A; HCN2; ITGB4; ITM2A, KCND3; KCNJ10; KCNJ2; KCNK10, KCNMB4, KIF13B; KIF1A; KIF5C; L1CAM; MAPT; MOX2; MPP2; MYH14; NLGN1; NPTX1; PRG-3; SEPT8; SEPT4; SLC11A2; TM4SF11, TPPP Extracellular Matrix proteins ADAM19; ADAMTS8; COL9A2; COL9A3; FLRT1; FLRT3; HYAL1; MMP15; MMP9 Immune System related CXCL14; HLA-DMA; HLA-DMB, HLA-DPA1; HLA- DPB1; HLA-DQA1; HLA-DQB1; HLA-DRA; HLA- DRB3; HSPA2; IL11; IL1B; IL1RAPL1; MAL; RGC32; TGFA Cell cycle CCNE2; CDKN1C; CYFIP2 Fat-Metabolism APOC1; BTN3A3 Metabolism A2M; ABCA3; ABHD1; ABHD2; ABHD6; AMD1; AMPD3; ASPA; CA14; CA2; CAPN3; CTSC; CYB5R2; FABP5; FABP7; FUT9; GAL3ST1; GATM; GLDC; GPD1; INPP5F; LYZ; MTUS1; PACE4; PHLDA1; PPAP2C; PTGDS; SEPP1; SIAT8E; SIAT9; SLC31A2; SLCO3A1; UBL3; UGT8; Neuropetides(Neuroendokrine PCSK1N genes Apoptosis/Cancer/ ARC; CYP2J2; FAIM2; GLUL Proliferation Translation/Transcription CUGBP2 Development ALK; COBLM; DLG1; GDF1; PEG10; PEG3; PLXNB3; POU3F2; PTN; SEMA4C; SEMA4D; SOX10; SOX2; TTYH1 Receptors CCR7; CELSR2; CXADR; CXCR4; FCER1G; GPR17; GPR37; GPR56; GPRC5B; GRIA2; NGFR, PTPRD; PTPRE; PTPRZ1; RARRES2; RARRES3; RASSF2; SORL1; TYRO3 CD-Markers CD74; CD9 Cell Adhesion CDH19; NCAM1; PCDH9 Oligodendoglial Genes CNP; MAG; MBP; MOBP; MOG; NKX2-2; OLIG2; OMG; PLP1; PMP2 Chrosomale Genes APC2 Brain APLP1; BAI2; BCHE; GAS7; MT3; PDE2A; STXBP6; VGF Neural Genes DDR1; NES Neuronal Genes ABAT; ENO2; S100B; SNCA; SV2A; SYT11; TUBB4 Glial genes GFAP Other BCAS1; BEXL1; CECR1; CITED1; DTR; KLK6; PADI2 Unknown C11orf9; C14orf139; C18orf1; C20orf35; C3orf4

9 Danksagung

Eine Vielzahl von Menschen hat beim Durchführen und Vollenden meiner Doktorarbeit einen kleineren oder größeren Beitrag geleistet. Ohne ihren Beitrag wäre das Gelingen bedeutend schwerer, wenn nicht unmöglich gewesen. Ich danke daher...

…Prof. Dr. Alexander Storch für die Überlassung des Themas, die engagierte Betreuung und dass er mir viel Geduld und Vertrauen entgegengebracht und mich in schwierigen Situationen mit seiner Diskussionsbereitschaft und seinen wertvollen Ratschlägen motiviert hat.

…Herrn Prof. Dr. Ludolph für die Durchführung der Arbeit in seiner Abteilung.

...Prof. Dr. Tobias Böckers für die Übernahme des Zweitgutachtens.

… meinen ehemaligen Labor- Kollegen und -Freunden in Ulm Vera, Stefan, Hans-Jörg, Nancy und Thomas für die Hilfsbereitschaft, Unterstützung und die freundliche Atmosphäre.

Prof. Dr. Rolf Brenner und Dr. Hans-Jörg-Habisch für die Bereitstellung der mesenchymalen Zellen. Alexander Kleger und Tobias Busch möchte ich für die Bereitstellung der MEFs (Mouse embryonic fibroblasts) danken.

… Andreas Hermann für die vielen gemeinsamen Stunden am Mikroskop mit Klonierungen, und Färbungen in Ulm und Dresden. Mit ihm wurden die Projekte der humanen und der murinen Zellen durchgeführt.

…Florian Wegner und Oana Popa für das Erlernen der Elektrophysiologie. Ausserdem möchte ich mich bei Prof. Dr. Holger Lerche, Dr. Yvonne Weber, Prof. Dr. Gregor Antoniades und Dr. Matthias Kirsch für die Bereitstellung des humanen Gewebes in Ulm und Dresden bedanken.

… Prof. Dr. Johannes Schwarz und Dr. Sigrid Schwarz in Leipzig für ihre Unterstützung, Dr. Javorina Milosevic für die Bereitstellung der humanen fetalen Stammzellen in diesem Projekt.

…Prof. Dr. Manfred Gerlach für die Kooperation und Durchführung der Neurotransmitterdetektion.

…Dr. Alexander Herr, der mich bei der Auswertung der Micro-Arrays unterstützt hat.

….den „Labormädels“ in Dresden, Anne, Annet, Carmen, Jule, Silvia und Susi für die nette Atmosphäre und natürlich auch Anke für die „organisatorische“ Unterstützung.

.. dem „Umzugsteam“ und den besten Freunden die man haben kann: Carmen, Heike, Peter und Tobias. Danke euch, ich hoffe wir werden noch viele „Koch-und Silvester- Abende“ miteinander verbringen.

...Florian Wölfle, „das Beste“ was mir passieren konnte, der mir immer den Rücken frei gehalten hat und immer für mich da ist. Danke, für die doch immer schönen 48 Stunden…

…last not least meiner Familie, vor allem meiner Mutter, die mir das Studium ermöglicht hat und mich in jeder Lebenssituation unterstützt hat und natürlich meinem Vater, der immer in Gedanken bei mir ist.