Differentiation of human teratocarcinoma cell line into motor neurons

Differentiation of human teratocarcinoma cell line into motor neurons:

investigation of cellular phenotype in vitro and in transplantation studies.

Thesis

Submitted in partial fulfillment of the requirements for the degree

Doctor of Philosophy -Ph.D.-

Division of Cell Biology, Institute of Physiology University of Veterinary Medicine Hannover

and

Center for Systems Neuroscience Hannover Awarded by University of Veterinary Medicine Hannover

by

Grzegorz Podrygajlo

Trzebnica / Poland

Hannover, Germany, 2009

University of Veterinary Medicine Hannover

Advisory group: Prof. Dr. Gerd Bicker Prof. Dr. Claudia Grothe Institute of Neuroanatomy, Hannover Medical School

` Prof. Dr. Wolfgang Baumgärtner Department of Pathology,

University of Veterinary Medicine Hannover

First evaluation: Prof. Dr. Gerd Bicker Prof. Dr. Claudia Grothe

` Prof. Dr. Wolfgang Baumgärtner

Second evaluation: PD Dr. Wolfgang Härtig

Paul Flechsig Institute for Brain Research, University of Leipzig

Date of final examination: 24.10.2009

The present work was supported by grants from the Federal Ministry for Education and Research (BMBF 0313732 B and BMBF 0313925 D).

Grzegorz Podrygajlo was supported by Marie Curie Scholarship to ZSN- Hannover (MEST-CT-2005 No. 021014).

Podrygajlo G. Tegenge M. A. Gierse A. Paquet-Durand F. Tan S. Bicker G. and Stern M. Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture. Cell Tissue Res 336: 439-452 (2009).

CHAPTER 1: GENERAL INTRODUCTION ... 1

1. INTRODUCTION... 1

2. NEURONAL DIFFERENTIATION OF NT2 CELLS ... 2

3. MORPHOLOGY AND PHENOTYPES OF NT2 NEURONS ... 4

4. ELECTROPHYSIOLOGY OF NT2 NEURONS... 4

5. INDUCTION OF MOTONEURONS IN VIVO AND IN VITRO ... 5

6. NT2 NEURONS IN TRANSPLANTATION STUDIES ... 8

7. AIMS OF THE STUDY ... 9

8. REFERENCES: ... 10

CHAPTER 2: CELLULAR PHENOTYPES OF HUMAN MODEL NEURONS (NT2) AFTER DIFFERENTIATION IN AGGREGATE CULTURE ... 16

1. ABSTRACT ... 17

2. INTRODUCTION... 17

3. MATERIALS AND METHODS ... 19

1. Cell culture ... 19

2. Immunocytochemistry ... 20

3. Stimulation with nitric oxide donors ... 21

4. Positive controls... 22

5. Microscopy and statistical analysis... 22

4. RESULTS ... 23

1. Neuronal differentiation ... 23

2. Neuronal phenotype... 25

3. Neurotransmitters ... 29

5. DISCUSSION ... 36

1. Neuronal phenotype and synaptic markers ... 36

2. Neurotransmitters ... 37

3. Cholinergic phenotype ... 38

4. NO-cGMP signaling pathway ... 39

5. Concluding remarks ... 39

NEURONS DIFFERENTIATED IN AGGREGATE CULTURE ... 44

1. ABSTRACT ... 45

2. REFERENCES ... 54

CHAPTER 4: INTEGRATION OF HUMAN MODEL NEURONS (NT2) INTO EMBRYONIC CHICK NERVOUS SYSTEM ... 57

1. ABSTRACT ... 58

2. INTRODUCTION... 58

3. RESULTS ... 60

4. DISCUSSION ... 68

5. EXPERIMENTAL PROCEDURES ... 70

1. Cell culture ... 70

2. Co-culture of NT2 neurons and myocytes. ... 71

3. Chick embryos ... 71

4. Cell transplantation... 71

5. Immunocytochemistry ... 72

6. Immunohistochemistry ... 72

7. Microscopy and statistical analysis... 72

6. REFERENCES ... 74

CHAPTER 5: DISCUSSION... 77

REFERENCES ... 82

ABSTRACT ... 86

ZUSAMMFASSUNG ... 88

ACKNOWLEDGEMENTS ... 90

ALS amyotrophic lateral sclerosis

BK(Ca) large-conductance calcium-activated potassium channel BMP bone morphogenetic protein

BrdU 5-bromo-2’-deoxyuridine

CFDA-SE 5-(and-6)-carboxyfluorescein diacetate- succinimidyl ester cGMP cyclic guanosine-monophosphate

ChAT choline acetyl-transferase

CM-Dil chloromethyl-benzamidodialkylcarbocyanine CNS central nervous system

Cy3 cyanine dye 3

DAPI 4',6-diamidino-2'-phenylindol-dihydrochloride DMEM Dulbecco's modified Eagle medium

ECC embryonal carcinoma cells EGC embryonic germ cells

EGTA ethylene glycol tetraacetic acid EPSC excitatory postsynaptic current ESC embryonic stem cells

FBS fetal bovine serum FGF1 fibroblast growth factor 1 GABA gamma-amino butyric acid GAD glutamate decarboxylase GFAP glial fibrillary acidic protein

Glu glutamate

HTS high throughput screening

Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IBMX 3-isobutyl-1-methylxanthine

IPSC inhibitory postsynaptic current LPS lipopolysaccharides

MAP-2 microtubule-associated protein 2 MEA multi electrode array

NMDA N-methyl-D-aspartic acid NO nitric oxid

NOC-15 (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate

NT neural tube

ODQ 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one PBS phosphate-buffered saline

PFA paraformaldehyde RA retinoic acid

SC stem cell

sGC soluble guanylyl-cyclase Shh sonic hedgehog

SMI32 monoclonal antibody anti-neurofilament H non-phosphorylated SNP sodium nitroprusside

TH tyrosine hydroxylase

VAChT vesicular acetylcholine transporter VG1uT1 vesicular glutamate transporter 1

YC-1 3-(50-hydroxymethyl-20-furyl)-1-benzyl indazole

1

Chapter 1: General introduction

1.

I

Disorders of the central nervous system such as ischemic brain injury (Casper et al. 2003; Dirnagl et al.

1999), amyotrophic lateral sclerosis (Hedlund et al. 2007; Boillée et al. 2006), Alzheimer’s disease (Roberson and Mucke 2006; Jellinger 2006) or Parkinson’s disease (Biskup and Moore 2006) are a major concern in the aging human societies. Therefore, among basic and applied science exists a high demand for a reliable model system to analyze the pathogenesis of those diseases.

Until now a major effort was directed towards the development of animal models, which mimic the conditions of human neurodegenerative disorders. Unfortunately, the use of animal models meets many of limitations considering e.g. relatively high costs, requirement of separate animal facilities, specialized technicians, complex surgical techniques, and special licenses. It is extremely time consuming to run high throughput screening (HTS) using animals. Therefore, its application into testing new substances and drugs is rather limited also because of species specific differences between human and animal nervous tissue (Munir et al. 1995; Hoyte et al. 2004a; Hoyte et al. 2004b). In the other hand, the use of human tissue raises ethical concerns. To avoid those limitations, researchers use cell culture models, which with the help of modern methodology can be a proper equivalent of the experimental animals because of simplicity, lower costs and lack of ethical issues. Additionally, production of human neurons is especially desirable due to limited potency of human CNS for self renewal (Pera and Trounson 2004).

Generation, multiplication and differentiation on a large scale of such neurons can serve as a model for screening of novel drugs or for analyzing mechanisms of neurodegeneration. In the future, neurons that are obtained from the in vitro culture might serve as a source for the transplantations in the human CNS (Björklund and Lindvall 2000; Correia et al. 2005; Goldman and Windrem 2006).

Unfortunately, human progenitor cells, which can be induced to become neurons have some major limitations, which are listed as follows: a limited number of usable cell lines, material transfer agreements, patents, lack of the clear criteria for use from regulatory agencies, stability of phenotype, potential tumorgenity, contamination by infectious agents and finally ethical considerations (Donovan and Gearhart 2001; Allegrucci and Young 2007) (Fig. 1).

In presented PhD thesis I focus on a well characterized teratocarcinoma cell line (NT2), which can be

2

induced to differentiate into fully functional, postmitotic neurons as was shown over the last two decades in a number of investigations.

2. N

NT2

Neuronal differentiation is a complex process, which involves many steps, from proliferation of the precursor cells through migration of the neurons to their target regions, neurite outgrowth, and finally maturation within synaptic circuits (Spitzer 2006).

NT2 cells (Ntera2/cloneD1), which have been derived from a human testicular cancer (Andrews 1984), are induced to differentiate into neurons by application of micromolar doses of retinoic acid (RA). This biological compound is a derivative of vitamin A and plays an important role in the development of vertebrate central nervous system (Maden 2002). A method of neuronal differentiation of NT2 cells into postmitotic neurons has been described for the first time by Andrews (1984), and later on improved by Pleasure et al. (1992). This “classical” protocol involves 6 weeks of treatment with RA, followed by 2 replates (2 days), a treatment with mitotic inhibitors (7-10 days) and 1-2 selective trypsinization steps to obtain pure fractions of neuronal cells. Thus, the whole process requires about 2 months of cell culturing, which is the major disadvantage of this differentiation method. Another attempt to the differentiation of NT2 cells was a cell aggregation procedure that shortens the culturin g time twice (Cheung et al. 1999). This resulted in a low percentage of neuronal cells in the aggregates, which were in addition hard to purify (Leypoldt et al. 2001). To overcome the two major disadvantages of the described methods, our lab combined them into one procedure, in which precursor cells are multiplied and differentiated in free floating spheres, then expanded and seeded on adherent surface (Paquet-Durand and Bicker 2003) like in the procedure described by Pleasure et al. (1992) (Fig 1).

Immunocytochemical staining for neuronal markers showed that this protocol greatly enhanced nerve cell differentiation and allowed to obtain postmitotic NT-2 neurons in less than half of the time needed for the conventional differentiation protocol (Paquet-Durand et al. 2003). Eventually, the cell aggregate differentiation protocol has been used successfully by others (Paquet-Durand and Bicker 2004; Prehaud et al. 2005) and modified in order to increase number of neurons (Jain et al. 2007).

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Fig. 1 Three distinct methods to generate neurons from stem cells.

Human neurons can be obtained from the three sources: embryonic stem cells (ESC), embryonic germ cells (EGC) and embryonal carcinoma cells (ECC). ESC are derived from inner cell mass of the preimplanted human embryo. EGC are derived from primodal germ cells isolated from embryonic gonad. ECC are derived from testicular tumors in adult humans. Human NT2 neurons are obtained from ECC through the free-floating aggregate method (marked by thick arrows). This procedure is based on the proliferation of the precursor NT2 cells in a Petri dish in normal medium, differentiation in medium with RA, expansion of attached cells in a flask with the RA medium, a purifying step with mitotic inhibitors and finally selective trypsinization to obtain pure, postmitotic neurons.

Modified from Donovan and Gearhart 2001

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3. M

NT2

Matured NT2 neurons have a neuronal morphology characterized by small rounded cell bodies and thin processes. In cell culture NT2 neurons migrated and formed clusters that extended neurites in all directions with bundle axons connected to the surrounding clusters of the cells. NT2 neurons co- cultured with rat astrocytes did not form aggregates but remained dispersed as single neurons.

Additionally, in such conditions the assemblies of functional synapses was observed both by electron microscopic and patch clamp analysis (Hartley et al. 1999). Moreover NT2 neurons express a variety number of neurotransmitter phenotypes in vitro including GABA-ergic, glutamatergic, catecholaminergic, cholinergic, dopaminergic and serotonergic (Zeller and Strauss 1995; Yoshioka et al. 1997; Guillemain et al. 2000; Iacovitti et al. 2001).

4. E

NT2

There are only a rather limited number of studies that characterize cultured NT2 neurons in electrophysiological recordings. In the absence of astrocytes, NT2 neurons show spontaneous firin g activity, normal action potentials and signal propagation speed typical for non-myelinated neurons when grown on multi-electrode arrays (MEA). However, NT-2 neurons did not show a synchronized firing activity (“bursts”) typical for embryonic rat cortical neurons, which might be due to insufficient synaptic coupling under these conditions (Görtz et al. 2004). When seeded on the rat astrocytes monolayer NT2 neurons established functional synapses, whose properties (kinetics, ion selec tivity, pharmacology, and ultrastructure) were similar to those of mammalian neurons in primary cultures (Hartley et al. 1999). NT2 neurons were also responding to stimulation with glutamate and GAB A (Neelands et al. 1999; Hartley et al. 1999). Further electrophysiological experiments characterized in more details the properties of GABA receptors (Gao and Greenfield 2005; Wegner et al. 2007). There is electrophysiological evidence for functional NMDA-type receptors in NT2 neurons derived both from the standard differentiation protocol (Sandhu et al. 2003; Garcia de Arriba et al. 2006) and from floating spheres differentiated neurons utilizing fluorometric calcium-imaging (Paquet-Durand and Bicker 2004; Paquet-Durand et al. 2006).

Large-conductance calcium-activated potassium (BK(Ca)) channels were studied in inside-out patches.

This was the first characterization of a potassium channel in human NT2 neurons showing that it is

5

similar to the BK(Ca) channel of other preparations (Chapman et al. 2007).

5. I

The classical differentiation procedure of Pleasure et al. (1992) generates heterogeneous neurona l phenotypes (Guillemain et al. 2000). Since neuronal specification in the vertebrate neural tube is one of the best known processes of embryonic pattern formation, I examined in my thesis whether NT2 precursor cells can be directed towards a fate resembling motoneurons. In the neural tube distinct neuronal subtypes are generated from precursor cells in a precise, spatial manner accord ing to their localization through the anterior-posterior and dorsal-ventral axes. The whole process is under comple x control of multiple extrinsic and intrinsic factors (Kandel et al. 2000; Dessaud et al. 2008).

Motor neurons located at different positions in the spinal cord project their axons in a highly stereotyped manner to innervate muscles in the periphery. This high degree of spatial order establishes a topographic neural map. The organization of the spinal cord motor projections is a consequence of the generation of subtypes of motor neurons during development (Pfaff and Kintner 1998; Shirasaki and Pfaff 2002). Several key regulatory molecules play an important role in the differentiation of motoneurons. Progenitor cells of the neural tube acquire a more caudal identity by exposure to RA which is released by surrounding tissues, mainly by the adjacent somites (Maden 2006). The notochord and floor plate secrete the diffusible protein Sonic hedgehog (Shh) which in turn has a ventralizin g action on neural tube tissue. Shh, acts in a concentration-dependent manner. High concentration (close to the floor plate) induces motor neurons and lower concentration induces several classes of ventra l interneurons (Wichterle et al. 2002; Wilson and Maden 2005). BMPs, other important signalin g molecules that pattern the spinal cord in the dorsal- ventral axis, are produced in the roof plate (Yang et al. 2006; Alvarez-Medina et al. 2008). They are responsible for generating dorsal cell types in a concentration-dependent fashion, similar to Shh at the ventral floor plate (Lee and Jessell 1999; Helms and Johnson 2003). Generation of motoneurons in vitro can be stimulated by applying high concentration of RA (Maden 2002) to the developing neural progenitor cells. In addition, others various factors like human Shh-N protein (Li et al. 2005), purmorphamine as an activator (through stimulation of Smoothened) of the Shh pathway (Briscoe 2006; Li et al. 2008), or noggin as an inhibitor of the BMP pathway (Novitch et al. 2003) might lead to generation of motor neurons. Signal transduction mediated by Shh is characterized a series of repressive interaction on membrane proteins (Fig. 2). Shh

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acts through a 12-pass transmembrane receptor, which is called Patched (PTCH) by inhibition of its activity. Blocked PTCH can no longer suppress Smoothened (Smo). Released Smo activates intracellularly the Gli class of zinc finger transcription factors. This results in Gli accumulation in the nucleus and induction of Shh target genes transcription, which leads to generation of motoneurons (Dessaud et al. 2008).

Among many phenotypes expressed by NT2 neurons, the cholinergic appears to be the major one. It is displayed at the same proportions in conventionally differentiated NT2 neurons (Guillemain et al.

2000) and in our rapid floating-aggregate differentiation procedure reaching 35% of the total number of the cells (Podrygajlo et al. 2009). Moreover NT2 neurons transplanted into rat striatum displayed 33%

immunoreactivity to ChAT (Saporta et al. 2000). All of these data support a potential use of NT2 cells as a model system for studying motoneuronal generation in vitro.

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Fig. 2 Schematic overview on the key events during specification of motoneurons on the level of the developing neural tube and its ventral cells.

Specification of motoneurons in the developing neural tube in vertebrates, involving action of RA and Shh. RA is secreted from the surrounding tissues, whereas Shh origin is the notochord, which stimulates floor plate cells for further secretion of this factor. Generation of motoneurons might be stimulated by adding recombinant Shh-N protein, the small molecule agonist purmorphamine, and/or noggin (inhibitor of BMPs pathway). At the cellular level, Shh binds to Patched (PTCH) receptor and relieves the Patched-dependent inhibition of the protein Smoothened (Smo). Smo protein is also a critical point for the action of purmorphamine. Once the repression is relieved, smoothened activates the members of Gli transcription factors that enter the nucleus and induce expression of Shh-dependent genes.

Modified from Kandel et al. 2000

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6. NT2

Cell transplantation therapy is an alternative approach for the CNS disorders, in which the neurologica l defect is caused by a loss of defined population of the neurons. The major source of cells, used in the transplantation studies, is fetal embryonic tissue. However, the widespread application of such tissue is limited by logistical barriers and ethical considerations. Thus, cell lines from human tissue, which could be multiply and differentiated in vitro into neurons, are a promising source of cells used for transplantation.

NT2 neurons can be considered as a potential source of transplantable neurons. Until now, successfu l transplantation of NT2 neurons into rodent brains was accomplished in many studies (Trojanowski et al. 1993; Kleppner et al. 1995; Hartley et al. 1999; Miyazono et al. 1996; Lee et al. 2000; Rivas- Arancibia et al. 2000; Saporta et al. 2000; Zigova et al. 2000; Willing et al. 2002; Fricker-Gates et al.

2004) showing that neurons survive, integrate and differentiate in the intact environment of the host nervous system. Transplanted neurons extend neurites, express neuronal markers, survive for up to 15 months and show no signs of phenotype reversion (Hartley et al. 1999b). Additionally loca l environment of host tissue seems to play a key role in the specification of neurotransmitter phenotypes in the NT2 cells (Saporta et al. 2000). Moreover, the undifferentiated NT2 precursor cells did not show tumor formation after transplantation into mice brain (Ferrari et al. 2000) or caudoputamen of nude mice (Miyazono et al. 1996). Human NT2N cells express markers of cholinergic phenotype (Guillemain et al. 2000; Saporta et al. 2000), therefore it was logical to proceed with the engraftments to restore motor function in animal models of ALS. Transplants of NT2 neurons into the lumbar ventra l horn of the spinal cord were used to restore the motor neurons function (Garbuzova-Davis et al. 2001;

Garbuzova-Davis et al. 2002). Transplantation of NT2 neurons improved a motor function measured as a progression in various behavioral tests. However, future studies (Garbuzova-Davis et al. 2006) showed that a positive effect of engrafted cells was based mainly on the secretion of trophic factors.

The promising findings from the animal transplantation experiments have generated great expectations of using of NT2 neurons for clinical trials in patients (Kondziolka et al. 2000; Nelson et al. 2002).

Though, issues such as monitoring of cell survival and progression in the therapy need still to be resolved (Kondziolka and Wechsler 2008).

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7. A

The major aim of this study was to investigate whether the human teratocarcinoma cell line can be differentiated into motor neurons. Up to now, I utilized neuronal differentiation protocols according to the floating cell aggregate method.

In the first part I characterized cellular phenotypes of the human model neurons. Immunocytochemistry showed that NT2 neurons express cholinergic and synaptic markers. To support these findings I co - cultured the NT2 neurons with mouse myotubes. The neurons establish close contact with the myotubes and form neuromuscular junction-like structures that bind -bungarotoxin. Moreover, electrophysiological recordings with the patch clamp technique show spontaneous postsynaptic currents and transmitter responses in cultured NT2 neurons in vitro.

Finally, I grafted human NT2 cells into the embryonic chick nervous system. NT2 cells at different stages of neural development were injected into chick embryo spinal cord and brain. In these transplantation studies I examined how grafted precursor cells and neurons responded to the environment of the embryonic neural tube, whether they extended neurites, and how they populated the different parts of the central nervous tissue.

The findings of my thesis are presented in three chapters. The arrangement of the introductory, results, discussion, methods and references sections is arranged according to the style of the journal where the manuscript has been submitted.

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8. R

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16

Chapter 2: Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture

Grzegorz Podrygajlo & Million A. Tegenge & Andrea Gierse & Francois Paquet-Durand & Saime Tan

& Gerd Bicker & Michael Stern

G. Podrygajlo : M. A. Tegenge : A. Gierse : S. Tan : G. Bicker : M. Stern (*) Div. of Cell Biology, Institute of Physiology,

University of Veterinary Medicine Hannover, Bischofsholer Damm 15,

30173 Hannover, Germany

e-mail: Michael.stern@tiho-hannover.de

* Corresponding author

G. Podrygajlo : M. A. Tegenge : G. Bicker Center for Systems Neuroscience (ZSN), Hannover, Germany

F. Paquet-Durand

Centre for Ophthalmology, Institute for Ophthalmic Research, Division of Experimental Ophthalmology,

University of Tübingen, Röntgenweg 11,

72076 Tübingen, Germany

Cell and Tissue Research (2009) 336(3);439-52.

17

1. A

The well-characterized human teratocarcinoma line Ntera2 (NT2) can be differentiated into mature neurons. We have significantly shortened the timeconsuming process for generating postmitotic neurons to approximately 4 weeks by introducing a differentiation protocol for free-floating cell aggregates and a subsequent purification step. Here, we characterize the neurochemical phenotypes of the neurons derived from this cell aggregate method. During differentiation, the NT2 cells lose immunoreactivity for vimentin and nestin filaments, which are characteristic for the immature state of neuronal precursors. Instead, they acquire typical neuronal markers such as β-tubulin type III, microtubule-associated protein 2, and phosphorylated tau, but no astrocyte markers such as glia l fibrillary acidic protein. They grow neural processes that express punctate immunoreactivity for synapsin and synaptotagmin suggesting the formation of presynaptic structures.

Despite their common clonal origin, neurons cultured for 2–4 weeks in vitro comprise a heterogeneous population expressing several neurotransmitter phenotypes. Approximately 40% of the neurons display glutamatergic markers. A minority of neurons is immunoreactive for serotonin, gamma-amino-butyric acid, and its synthesizing enzyme glutamic acid decarboxylase. We have found no evidence for a dopaminergic phenotype. Subgroups of NT2 neurons respond to the application of nitric oxide donors with the synthesis of cGMP. A major subset shows immunoreactivity to the cholinergic markers choline acetyl-transferase, vesicular acetylcholine transporter, and the nonphosphorylated form of neurofilament H, all indicative of motor neurons. The NT2 system may thus be well suited for research related to motor neuron diseases.

Keywords

Ntera2 . Neurotransmitter . Choline acetyltransferase .Vesicular acetylcholine transporter . Glutamate decarboxylase . Nitric oxide . Human

2. I

Disorders of the central nervous system (CNS) such as ischemic brain injury (Dirnagl et al. 1999), Parkinson’s disease (Biskup and Moore 2006), Alzheimer’s disease (Roberson and Mucke 2006), or amyotrophic lateral sclerosis (Boillee et al. 2006) are an ever-increasing health concern. Much effort is

18

being invested into both basic and applied research on the underlying mechanisms and development of new therapies for CNS disorders. There is, hence, an ever-increasing demand for reliable model systems to satisfy these research needs. A large number of animal models have been established that mimic the development of the human brain or the conditions of human CNS disorders (Hirsch 2007;

Lassmann 2008). However, the use of laboratory animals is time consuming and expensive and creates ethical concerns. Moreover, species differences may impair the direct applicability of results obtained from animal experiments to the human CNS (Dragunov 2008). For instance, drugs proven to be neuroprotective in one species may be ineffective or even have adverse effects on the CNS of another species (Munir et al 1995; Hoyte et al. 2004). Similar problems arise in the field of neurotoxicologica l risk assessment, where recent legislation has prescribed increased testing of a large number of chemica l compounds to predict possible hazards to human brain development (Radio and Mundy 2008).

Since the use of original human tissues is limited because of availability and ethical concerns, cell- based in vitro models are the most suitable alternatives. The use of human nerve cells, generated and multiplied on a large scale in culture, is especially desirable since the bias created by species differences is of lesser concern and their availability is in principle unlimited. Cultured human neurons would, for instance, allow for the high-throughput screening of chemical compounds, both for possible therapeutical use and for risk assessment of developmental neurotoxins (Hill et al. 2008). They might also provide experimental material for investigating neuronal degeneration mechanisms in vitro.

Eventually, cultured human neurons might serve as a source for neural transplantation and repair in CNS disorders (Saporta et al. 2002; Correia et al. 2005; Goldman and Windrem 2006).

Human neurons can be generated from human embryonic stem cells (Glaser and Brüstle 2005), but their availability is limited both for technical and ethical reasons, in particular with respect to high- throughput screening. A promising alternative is the well-characterized teratocarcinoma cell line Ntera2 (NT2) derived from a human testicular cancer (Andrews 1984). NT2 cells (Ntera2/clone D1) can be induced to differentiate into fully functional, postmitotic neurons and other cell types of the neuronal lineage displaying a variety of neurotransmitter phenotypes (Guillemain et al. 2000). These cells have been used in a large number of biomedical investigations in the past two decades (for a review, see Paquet-Durand and Bicker 2007), including successful transplantation into human stroke

patients (e.g., Nelson et al. 2002).

One drawback of the NT2 system has been the timeconsuming process (up to 2 months) needed to generate purified postmitotic neurons. We have developed a new method utilizing freely flowin g aggregates allowing us to shorten significantly the differentiation process up to 1 month (Paquet- Durand et al. 2003). This method is based on the observation that a high concentration of retinoic acid

19

(RA), together with the influence of cell-cell adhesion in neurosphere aggregates, facilitates neurogenesis (Pardo and Honegger 2000) in comparison with classicaldifferentiation in surface - attached monolayer culture (Pleasure et al. 1992). The aim of this work has been to describe, by immunocytochemical analysis, the phenotypes of the population of neurons derived from the improved differentiation process.

3. M

1. Cell culture

All chemicals were obtained from Sigma (Taufkirchen, Germany), unless stated otherwise. The human NT2 cell line was obtained from the American Type Culture Collection (Va., USA). NT2 precursor cells were maintained and cultivated in “normal medium” comprising DMEM/F12 culture medium (Gibco-Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS, Gibco - Invitrogen) and 1% penicillin/streptomycin (Gibco-Invitrogen) under an atmosphere of 5% CO2 at 37°C (Andrews 1984). Generation of NT2 neurons was performed by using the differentiation protoco l in freefloating aggregates (Paquet-Durand et al. 2003). Briefly, NT2 precursor cells were seeded in 96- mm bacteriologicalgrade Petri dishes (Greiner, Hamburg, Germany) at a density of 4×106–5×106 cells per dish. On the first day, 10 ml culture medium was added to each Petri dish. On the next days, medium with 10 μM RA was added and changed every 2–3 days by transferring the cell suspension to centrifuge tubes and centrifuging at 200g for 7 min. After 7–8 days, the cells from one Petri dish were transferred and seeded into one T75 cell culture flask (Falcon, Franklin Lakes, N.J., USA) and cultured for another 7–8 days in RA medium at 4×107 cells per flask. For the induction of the cholinergic phenotype in Petri dishes and T75 culture flasks, cells were treated additionally with recombinant human Sonic hedgehog (Shh) amino-terminal peptide (75 nM, 1314-SH), recombinant human noggin (10 nM, 3344Ng; both from R&D Systems, Wiesbaden-Nordenstadt, Germany), and purmorphamine ( 2 μM, 540220; Calbiochem, San Diego, Calif., USA; for details, see Results). Cells were trypsinized (trypsin-EDTA; Gibco-Invitrogen), transferred to T175 cell culture flasks, and cultured for 2 days in normal medium. The cells were subsequently trypsinized once again, transferred to T75 flasks, and supplied with culture medium with mitotic inhibitors (1 μM 1–6-D-arabinofuransylcytosine, 10 μM 2′- deoxy-5-fluorouridine, 10 μM 1-β-D-ribofuranosyluracil). After 7–10 days, neurons were selectively trypsinized and collected for future experiments or storage. Differentiated NT2 neurons were plated on

20

poly-D-lysine and matrigel (Becton-Dickinson, Bedford, Mass., USA)-coated 96-well plates (Falcon) or cover-slips at a density of 10,000–50,000 cells per well and cultured for 1–5 weeks.

In order to induce a dopaminergic phenotype, differentiated NT2 neurons were incubated for up to 1 week with 1 mM lithium chloride and a mixture of fibroblast growth factor 1 (FGF1, 1 ng/ml), 12-O- tetradecanoylphorbol 13- acetate (200 nM), dopamine (20 μM), 3-isobutyl-1-methylxanthine (IBMX, 250 μM), and forskolin (50 μM). As a positive control for dopamine immunostaining, the cells were preincubated for 15 min in 1 mM dopamine prior to fixation. For the detection of cell proliferation, cells were incubated with 50 μM BrdU (5-bromo-2’-deoxyuridine) for 3 h or 3 days prior to fixation.

2. Immunocytochemistry

NT2 neurons were washed with phosphate-buffered saline (PBS) and fixed for 30 min at room temperature with 4% paraformaldehyde (PFA). Additionally, for the detection of dopamine, 1%

glutaraldehyde, and for gamma-amino-butyric acid (GABA), 0.1% glutaraldehyde was added to the fixative. The detection of dopamine required a reduction step with 1% sodium borohydride and 1%

sodium metabisulfite after fixation. Antigen retrieval for VAChT staining was performed by cookin g samples in 10 mM citrate buffer (pH 6.0) for 10 min. Detection of BrdU required a 20-min incubation in 2 N HCl after fixation. Cells were washed (3×5 min) in PBS containing 0.2% Triton X-100 (PBS-T).

Blocking solution containing PBS-T and 5% normal horse serum was applied for 1 h. Primary antibodies diluted in blocking solution were applied overnight at 4°C or for 1 h at room temperature (for a list of primary and secondary antisera, see Table 1). After three washes, secondary biotinylated antibodies (Vector, Burlingame, Mass., USA), diluted 1:250 in blocking solution, were added for 1 h at room temperature. After further washes, immunofluorescence was detected by applying streptavidin- Alexa Fluor-488 (Mobitec, Göttingen, Germany) or streptavidin-CY3 (Sigma) for 1 h at room temperature (dilution 1:250). Finally, cells were incubated for 5 min with 2 µM DAPI (4′,6-diamidino- 2′-phenylindol-dihydrochloride) as a nuclear counterstain. For visualisation of actin, a solution of phalloidin-Alexa Fluor-488 (Mobitec) in PBS (1:100) was applied for 15 min after immunostaining for β-tubulin type III as described above.

21 3. Stimulation with nitric oxide donors

For the detection of nitric oxide (NO)-induced cyclic guanosine-monophosphate (cGMP) levels, cultures were preincubated for 20 min at room temperature with 1 mM sodium nitroprusside (SNP) or NOC-15 ((Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate, Calbiochem) as the NO donor, 20 µM YC-1 (3-(50-hydroxymethyl-20-furyl)-1-benzyl indazole) as an enhancer of NO- induced activity of soluble guanylyl-cyclase (sGC), 50 µM ODQ (1H-[1,2,4]-oxadiazolo[4,3- a]quinoxalin-1-one) as an inhibitor of sGC, or 1 mM IBMX (3-isobutyl-1-methylxanthine) as a phosphodiesterase inhibitor. They were washed once with PBS and subsequently treated with the same staining procedures as above with blocking solution containing PBS-T and 5% normal rabbit serum.

The polyclonal sheep anti-cGMP (1:10,000; a kind gift from Dr. Jan de Vente, Maastricht University, Netherlands) was used as the primary antibody to detect the level of cGMP.

22 4. Positive controls

As positive controls for astrocyte markers, primary astrocytes prepared from newborn Sprague -Dawley rats (a kind gift from Dr. Martin Stangel’s laboratory, Hannover Medical School) were fixed and stained as described above.

Mouse brain tissue (a kind gift from the Institute of Physiology, University of Veterinary Medicine, Hannover) was used as a positive control for tyrosine hydroxylase immunostaining. Mouse brains were fixed in PBS containing 4% PFA and placed in PBS with 30% sucrose. After the tissue had been frozen in liquid nitrogen, the brain was sectioned at 20 μm on a Reichert-Jung Frigocut 2800E cryostat microtome at −20°C and collected on poly-D-lysine coated slides. Immunocytochemical staining was performed as described above.

As a second positive control for tyrosine hydroxylase immunostaining, we used a human brain tissue section microarray (Oligene, Berlin, Germany; catalog no. 401 1210). Immunocytochemical stainin g was carried out as described above.

5. Microscopy and statistical analysis

Preparations were viewed with a Zeiss Axioscope equipped with an Axiocam3900 digital camera and Zeiss Axiovision software or with a Zeiss Axiovert 200 equipped with a CoolSnap camera (Photometrics, Tucson, Ariz., USA) and MetaMorph software (Molecular Devices, Sunnyvale, Calif., USA). Micrographs were arranged, converted to grayscale, inverted, and contrast-enhanced in Adobe Photoshop. For quantification, at least five randomly chosen areas of 440×330 µm were counted on images obtained with MetaMorph. Cell numbers are expressed as the percent positively stained cells of the total cell number determined by counting DAPI-labeled nuclei. Numbers are given as mean±SEM.

Statistical evaluation of experimental data was performed by using an unpaired two-tailed Student’s t- test. Error bars indicate SEM, whereas levels of significance are indicated as ***P<0.001.

23

4. R

1. Neuronal differentiation

NT2 precursor cells were initially kept in adherent culture until 90% confluency (Fig. 1a) and then differentiated according to the method of Paquet-Durand et al. (2003). When NT2 cells were seeded in the presence of RA onto non-adhesive bacteriological-grade Petri dishes, they formed free-floatin g spheres that supported cellular proliferation and neural differentiation (Fig. 1b). After being replated to adherent substrate, some of the cells that migrated out of the spheres showed long processes, typical for neuronal morphology (Fig. 1c). During cell culture in medium containing mitotic inhibitors (Fig. 1d), the preferential adhesion of differentiating neurons to each other became apparent. After inhibitor treatment and selective trypsinization, purified neurons were cultured for at least 10 days on matrigel- coated cover-slips prior to immunocytochemical characterization. During this period, the initially dispersed NT2 neurons formed clusters and extended numerous long neurites (Fig. 1e). As reported in other studies (P leasure et al. 1992), cell cultures contained more than 95% po stmitotic neurons, fewer than 5% large (100–300 µm) undifferentiated, but postmitotic, epitheloid cells, and no proliferatin g precursor cells. Exposure to BrdU for up to 3 days failed to label nuclei of both epitheloid cells and neurons, whereas BrdU was incorporated into 45.1±3.92% of precursor cells after 2 h and into 95.5±0.49% after 4 h of exposure (n=5 cultures counted) under the same culture conditions (Fig. 2).

24

Fig. 1 Differentiation of Ntera-2 (NT2) precursors into neurons. a Stock of undifferentiated precursor cells, grown on an adherent substrate. b Day 8: neurospheres after 1 week of differentiation in the presence of 10 µM retinoic acid in a non-adherent Petri dish. c Day 17: mixed culture of NT2 neurons and NT2 precursors after being replated. d Day 22: culture of NT2 neurons in inhibitor medium, with few precursor cells remaining. e Day 41: purified neurons, 14 days after final replating on matrige l (PDL poly-D-lysine). Bars 100 µm (a–d), 50 µm (e).

25

Fig. 2 NT2 precursors proliferate, whereas neurons do not. a After a 2-h exposure to BrdU, 45.1±3.92%

of NT2 precursor cells are labeled for incorporated BrdU (red), whereas some remain unlabeled (arrows). Nuclear counterstaining (DAPI) appears blue. b After a 3 -day exposure to BrdU, both flat epitheloid cells (arrows) and neurons (small nuclei) in a mature culture (23 days after plating on matrigel) remain unlabeled for BrdU. Bars 50 µm (a), 100 µm (b).

2. Neuronal phenotype

To confirm neuronal differentiation in the aggregate culture, we tested the expression of cytoskeleta l markers both for immature precursors and mature neurons. NT2 precursor cells did not express type III β-tubulin (Fig. 3a). They expressed the intermediate filaments nestin and vimentin (Fig. 3b, c), typica l markers for neuronal precursor cells (Chu et al. 2006). NT2 neurons displayed immunoreactivity for type III β-tubulin, microtubule-associated protein 2 (MAP-2), and tau (Fig. 3d, g, h, i). Tau staining

26

was weak in cultures stained 8 days after replating but became much stronger after prolonged outgrowth and maturation of the cell culture (see Fig. 3h for tau staining after 24 days in culture). The few large epitheloid cells that accompanied the NT2 neurons displayed only faint staining for type III β-tubulin and no immunoreactivity to antibodies against MAP-2 and tau (arrowheads in Fig. 3d, g, h).

Instead, these cells retained cytoskeletal proteins characteristic for the precursor stadium, such as the intermediate filaments vimentin and nestin (Fig. 3e, f), which were not expressed by neurons (arrowheads in Fig. 3e, f). They differed from the proliferating precursors, however, in their large size (diameter of 100–300 µm compared with precursors at 20–30 µm) and their lack of cell division. In contrast to other reports on NT2 cultures (Sandhu et al. 2002; Ozdener 2007; Hill et al. 2008), neither neurons nor undifferentiated epitheloid cells were immunoreactive for markers characteristic for astrocytes, such as glial fibrillary acidic protein (GFAP, Fig. 4b) or glutamine synthetase (Fig. 4d). Both of these markers were consistently stained in primary rat astrocytes (Fig. 4a, c). Since astrocytes can be induced to increase GFAP expression by exposure to bacterial lipopolysaccharides (LPS; Brahmachar i et al. 2006), we challenged differentiated NT2 cultures with LPS (1 µg/ml) for 24 h. Again, we found no immunoreactivity to GFAP and glutamine synthetase (data not shown).

27

Fig. 3 Cytoskeletal elements (red) and corresponding DAPI-labeled nuclei (blue) in NT2 precursor cells (a–c) at 24 h after seeding onto plastic and in purified NT2 neurons (d–i) cultured for 10 days on matrigel before fixation (except for h: 24 days). a NT2 precursor cells express only minimal amounts of β-tubulin type III. b Precursors express nestin. c Precursors express vimentin. d All neuronal cells display immunoreactivity for β-tubulin type III, whereas non-neuronal epitheloid cells (arrowheads) do not. e Nestin is expressed only by non-neuronal epitheloid cells, but not by neurons (arrowheads). f Vimentin is expressed only by non-neuronal epitheloid cells, but not by neurons (arrowheads). g MAP - 2 is expressed by neurons, but not by non-neuronal epithelial cells (arrowheads). h Processes (but not perikarya) of neurons express tau, whereas non-neuronal epitheloid cells (arrowhead) do not. i Multiple growth cones (arrowheads) on NT2 neurons, at 24 h in culture, labeled with green-fluorescent phalloidin; the red staining is β-tubulin type III. Bar 100 µm (a, b, d–h), 50 µm (c, i).

28

Fig. 4 Both flat epitheloid cells and NT2 neurons lack astrocyte markers (blue DAPI nuclear counterstaining). a Rat astrocytes stained for GFAP (red) as a positive control. b In a 14-day-old culture, neither NT2 neurons (arrows) nor epitheloid cells (arrowheads) stain for GFAP. c Rat astrocytes stained for glutamine synthetase (positive control). d In a 14-day-old culture, neither NT2 neurons (arrows) nor epitheloid cells (arrowheads) stain for glutamine synthetase. Bar 100 µm.

Shortly after purified neurons were replated onto matrigel, they displayed multiple growing neurites (Fig. 3i). Within 10 days of culture, NT2 neurons aggregated into clusters and formed multiple neurites with numerous contacts (Figs. 1e, 3d, g, h). To test whether some of these contacts contained functiona l connections, we stained for synaptic markers. We found strong staining for synapsin (Fig. 5a), a soluble protein associated with the reserve pool of synaptic vesicles, and for the integral synaptic vesicle protein, synaptotagmin (Fig. 5b). Both markers were found predominantly in the neural processes. The staining appeared punctate (arrowheads in Fig. 5a, b), indicating local areas of high concentrations of

29

the synaptic markers, as would be expected in presynaptic structures.

Fig. 5 NT2 neurons stain for synaptic markers (inverted immunofluorescence staining). Punctate staining (arrowheads) shows expression of synapsin (a) and synaptotagmin I (c) in cultures of NT2 neurons on matrigel after 2 weeks; DAPI nuclear counterstaining (b, d). Bar 25 µm.

3. Neurotransmitters

In 14.52±2.29% of the neurons, strong immunostaining was seen against GABA (Fig. 6a–d); this was above the weak background staining that also appeared in the non-neuronal epitheloid cells (arrow in Fig. 6a). Consistently, only a few cells (11.66±0.84%) were labeled strongly for the GABA- synthesizing enzyme, glutamate decarboxylase (Fig. 6e, f). When stained with an antiserum against glutamate (Fig. 6g, h), strong labeling was detected in cell bodies and neurites of approximately half of

30

the neurons (black arrowheads in Fig. 6g) and moderate labeling in all other cells (white arrowheads in Fig. 6g) including the epitheloid cells, possibly representing the staining of ubiquitous metabolic glutamate. The presence of glutamatergic neurons was confirmed by using an antibody against the vesicular glutamate transporter protein, VGluT1, which strongly labeled 39.7±3.45% of the neurons (Fig. 6i, j). NT2 neuron cultures contained only a small population (1.77±0.69%) strongly immunoreactive for serotonin (Fig. 6k, l).

Fig. 6 Inverted immunofluorescence images of NT2-neurons stained for neurotransmitters (a, c, e, g, i, k) and the corresponding DAPI-labeled nuclei (b, d, f, h, j, l). a, c A subset of NT2-neurons is strongly immunoreactive for GABA. Note the weak background staining in the non-neuronal epitheloid cells (arrow). e A similar proportion of neurons is stained for glutamate decarboxylase. g A subset of NT2 neurons displays strong glutamate-like immunoreactivity (black arrowheads), whereas epitheloid ce lls and many neurons display only weak immunoreactivity (white arrowheads). i A subset of NT2 neurons is labeled with an antibody against vesicular glutamate transporter 1 (black arrowheads), whereas other neurons display only background staining (white arrowheads). k A single serotonin-immunoreactive neuron is stained within a large aggregate of NT2-neurons. Bar 50 µm (a, b, e, f, i-l), 100 µm (c, d, g, h).

31

A large population of neurons was immunopositive for cholinergic markers such as choline acetyl- transferase (ChAT, Fig. 7a), vesicular acetylcholine transporter (VAChT, Fig. 7b), and non- phosphorylated filaments stained with the SMI32 antibody (Fig. 7c), all being characteristic for motor neurons (Tsang et al. 2000). Each of these markers was detected in the soma and processes. Cell counting revealed 34.0±6.7% ChAT-positive neurons and 37.6±1.26% SMI32-positive neurons under control conditions. In human embryonic stem cells, the percentage of cholinergic neurons after differentiation can be increased by the addition of Shh (Li et al. 2005), purmorphamine (an activator of the Shh pathway; Briscoe 2006), and the inhibitor of bone morphogenetic factor (BMP) signaling, noggin (Novitch et al. 2003). However, in our experiments, none of these factors were able to induce a significant change in the percentage of cholinergic neurons. With Shh, 36.01±7.14% ChAT-positive and 36.64±10.25% SMI32-positive cells were counted. A combination of Shh and noggin resulted in 46.03±5.03% ChAT-positive and 36.98±2.49% SMI32-positive cells (not significantly different from control).

32

Fig. 7 Inverted immunofluorescence images of NT2 neurons showing expression of cholinergic markers (arrowheads representative cell bodies or neurites positive for the stained marker). a Choline acetyl-transferase. c Vesicular acetylcholine transporter. e Non-phosphorylated neurofilament H (SMI- 32 antibody). b, d, f Corresponding DAPI nuclear counterstaining. Bars 50 µm.

33

Previous reports showed differentiation into dopaminergic NT2 neurons by exposure either to lithium (Zigova et al. 1999, 2000) or to a mix of growth factors, dopamine, and high intracellular cAMP levels by the application of forskolin and IBMX (Iacovitti et al. 2001). In each case, with or without these treatments, we observed no indicators of a dopaminergic phenotype. We used two different primary antibodies against tyrosine hydroxylase, viz., anti-tyrosine hydroxylase MAB318 (Fig. 8a) and anti- tyrosine hydroxylase AB152 (Fig. 8b), both of which stained appropriate control tissue (mouse brain slice, see inset in Fig. 8a; human tissue microarray, see inset in Fig. 8b). Anti-dopamine immunostaining was absent (Fig. 8c) unless the cells were preincubated for 15 min in 1 mM dopamine prior to fixation (see inset in Fig. 8c). This confirmed the reliability of our staining procedure.

Preincubation with a physiological concentration of dopamine (5 µM), which would have been taken up by presumptive dopaminergic neurons containing the high affinity dopamine reuptake transporter, DAT, also failed to induce dopamine-immunostaining (data not shown).

34

Fig. 8 Inverted immunofluorescence images of NT2 neurons cells stained for tyrosine hydroxylase and dopamine; DAPI was used as a nuclear counterstain (b, d, f). Several primary antibodies were used all of which delivered negative results. a Anti-tyrosine hydroxylase MAB318. c Anti-tyrosine hydroxylase AB152. e Anti-dopamine AHP847. Insets: Positive controls, viz., a mouse brain slice (a), human brain tissue microarray (c), and NT2 neurons pretreated with 1 mM dopamine for 15 min before fixation (e).

Bars 50 µm.

35

NO is an unconventional messenger molecule that has been implicated in the modulation of synaptic transmission, neurogenesis, neuronal differentiation, and a variety of pathophysiological mechanisms (Garthwaite 2008; Bicker 2005). In target cells, NO acts largely through the activation of sGC and the subsequent production of cyclic guanosine monophosphate (cGMP). We tested for the presence of the NO/cGMP signaling pathway by stimulation with NO donors. cGMP immunostaining could be detected in up to 28% of NT2 neurons (Figs. 9, 10). The strongest staining was observed after incubation with a mixture of the NO-donor SNP, the enhancer of sGC activity, YC-1 (Friebe and Koesling 1998), and the inhibitor of phosphodiesterases, IBMX (Fig. 9d). Even without the addition of an NO donor, a small percentage of NT2 neurons were cGMP -immunoreactive when exposed to IBMX alone (Figs. 9a, 10) suggesting the presence of endogenous NO. Addition of the inhibitor of sGC, ODQ (Boulton et al. 1995), abolished NO-induced cGMP-staining (Figs. 9d, 10).

Fig. 9 Stimulation with NO donors causes cGMP synthesis in NT2 neurons. cGMP immunostainin g (red) and nuclear DAPI staining (blue) of NT2 neurons at 30 days in culture. a When exposed to IBMX alone, a small number of NT2 neurons were cGMP-immunoreactive. b When stimulated with 1 mM of the NO-donor SNP, a large number of cells became cGMP-immunoreactive. c In the presence of the inhibitor of sGC, viz., ODQ, SNP failed to induce cGMP-immunoreactivity. d The activator of sGC, viz., YC-1, increased NO-induced cGMP immunoreactivity. Bar 50 µm.

36

Fig. 10 Quantitative analysis of NO-induced cGMP production of NT2 neurons at 30 days in culture on matrigel. The NO donors NOC-15 and SNP (both 1 mM) significantly increased the numbers of cGMP-positive neurons, whereas the inhibitor of sGC, viz., ODQ, significantly reduced the number of SNP-induced cGMP-positive cells. The activator of sGC, YC-1, did not significantly increase the number of cGMP-positive neurons (bars means±SEM of six independent measurements).

5. D

1. Neuronal phenotype and synaptic markers

Morphological analysis and staining for the major cytoskeletal markers have confirmed that our improved differentiation protocol (Paquet-Durand et al. 2003) results in the development of differentiated human neurons within less than 1 month. The cultures contain pure neurons with less than 5% contamination by undifferentiated, but postmitotic, cells. Studies from other groups using the conventional adherent culture protocol have reported the generation of astrocytes in addition to neurons (Sandhu et al. 2002; Ozdener 2007). We have seen, in our cultures, large flattened cells reminiscent of the cytoplasmic type of astrocytes. However, these cells do not display the typical astrocytic markers GFAP and glutamine synthetase and cannot be induced to do so by stimulation with LPS. We conclude,