Integration of human model neurons (NT2) into embryonic chick nervous system

Authors

  • Grzegorz Podrygajlo,

    1. Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, Hannover, Germany
    2. Center for Systems Neuroscience (ZSN) Hannover, Hannover, Germany
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  • Christoph Wiegreffe,

    1. Institute of Anatomy and Cell Biology, Department of Molecular Embryology, University of Freiburg, Freiburg, Germany
    2. Faculty of Biology, University of Freiburg, Freiburg, Germany
    Current affiliation:
    1. Institute of Molecular and Cellular Anatomy, Ulm University, Ulm, Germany
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  • Martin Scaal,

    1. Institute of Anatomy and Cell Biology, Department of Molecular Embryology, University of Freiburg, Freiburg, Germany
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  • Gerd Bicker

    Corresponding author
    1. Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, Hannover, Germany
    2. Center for Systems Neuroscience (ZSN) Hannover, Hannover, Germany
    • Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany
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Abstract

Postmitotic neurons were generated from the human NT2 teratocarcinoma cell line in a novel cell aggregate differentiation procedure. Approximately a third of the differentiated neurons expressed cell markers related to cholinergic neurotransmission. To examine whether this human cell model system can be directed toward a motoneuronal fate, postmitotic neurons were co-cultured with mouse myotubes. Outgrowing neuronal processes established close contact with the myotubes and formed neuromuscular junction-like structures that bound α-bungarotoxin. To determine how grafted precursor cells and neurons respond to embryonic nerve tissue, NT2 cells at different stages of neural development were injected into chick embryo neural tube and brain. Grafted NT2 neurons populated both parts of the nervous system, sometimes migrating away from the site of injection. The neural tube appeared to be more permissive for neurite extensions than the brain. Moreover, extending neurites of spinal grafts were approaching the ventral roots, thus resembling motoneuronal projections. Developmental Dynamics 239:496–504, 2010. © 2009 Wiley-Liss, Inc.

INTRODUCTION

The teratocarcinoma line Ntera2 has been widely used as a cell culture model for the differentiation of human neurons (Andrews, 1984; Pleasure et al., 1992). Upon treatment with retinoic acid (RA), NT2 cells (Ntera2/clone D1) can be induced to differentiate into fully functional, postmitotic neurons, which display a variety of neurotransmitter phenotypes (Guillemain et al., 2000; Podrygajlo et al., 2009a). A caveat of the NT2 system, the lengthy differentiation process (up to 2 months) needed to generate and to purify postmitotic neurons, has been successfully overcome. Because a high concentration of RA, together with the influence of cell–cell adhesion in freely floating aggregates, facilitates neurogenesis (Pardo and Honegger, 2000), our lab developed a novel cell aggregate method for NT2 neuron differentiation (Paquet-Durand et al., 2003). This method, in comparison to the classical differentiation in surface attached monolayer culture (Pleasure et al., 1992), significantly shortens the differentiation process to only 1 month. Meanwhile, these rapidly generated postmitotic neurons have been used in the pathophysiological analysis of brain cell injury and neuroprotection (Paquet-Durand and Bicker, 2004; Paquet-Durand et al., 2006; Pistritto et al., 2009).

Numerous transplantation studies have demonstrated the successful grafting of NT2 neurons into rodent brains, including studies of animal models of Parkinson's disease, amyotrophic lateral sclerosis, traumatic brain injury, and stroke (Iacovitti et al., 2001; Garbuzova-Davis et al., 2002; Zhang et al., 2005; Hara et al., 2007). The effectiveness of NT2 neuron grafts in promoting behavioral recovery in animal stroke models together with the absence of tumorigenicity eventually led to a successful clinical trial using transplantation of NT2 neurons into human stroke patients (Kondziolka et al., 2000; Nelson et al., 2002).

There is growing interest in the study of inductive interactions and differentiation processes in the development of human neuronal progenitor cells. Because the differentiation of NT2 neurons in cell culture follows a pattern of differential gene expression similar to that of neural precursors during neurogenesis (Przyborski et al., 2003), the NT2 cell culture system may be a useful approach to study cellular mechanism of early human neural development. So far, transplantation studies into host neural tissue have, however, been limited to adult or neonatal experimental animals. Thus, we considered to establish a vertebrate embryonic animal model as a host for the developing NT2 neurons. The chick embryo is a well-characterized and easily accessible experimental system for developmental biology, because many surgical manipulations can be readily carried out in ovo, and novel molecular techniques for gain and loss-of-function analysis are currently being improved (Stern, 2005). Several studies have shown that mammalian cells and tissues transplanted to avian embryos can respond to local cues and develop into tissues appropriate to their location in the host (i.e., Fontaine-Perus et al., 1997; White and Anderson, 1999; Goldstein et al., 2002). The advantage of using the chick embryo rather than rodents is the ability to follow human neuronal cell development in early embryonic environment, as opposed to the more mature nervous tissue accessible in rodents.

Using our cell aggregate method for neuronal differentiation, we have recently shown that a major subset of the NT2 neurons shows immunoreactivity to choline acetyltransferase, vesicular acetylcholine transporter, and the nonphosphorylated form of neurofilament H (Podrygajlo et al., 2009a), all indicative of a motoneuronal cell phenotype. To test whether NT2 neurons exhibit aspects of motoneuronal morphology in vivo, we grafted NT2 cells into embryonic chick CNS.

In this study, we examined first the in vitro cellular distribution of synaptic markers such as synapsin and α-bungarotoxin in co-cultures of NT2 neurons with myotubes. Then, we tested whether NT2 neurons survive, integrate and extend axons in the embryonic environment of the chick spinal cord. Finally, we compared the integration of NT2 precursor cells and differentiating neurons transplanted into the avian neural tube with grafts into brain tissue.

RESULTS

Coculture of Human NT2 Neurons With Mouse Myotubes

When seeded on a poly-D-lysine/Matrigel coated surface, dispersed neuronal NT2 cell bodies tend to aggregate and form ganglion-like cell clusters that are interconnected by long fiber bundles (Paquet-Durand et al., 2003; Podrygajlo et al., 2009a). Here, we cultured the NT2 neurons together with differentiated mouse myotubes (MT) and found striking differences in culture morphology. The dispersed neuronal cell bodies did not form aggregates and extended process that often spread in parallel to the spindle-shaped myotubes. Occasionally, we observed that the Tau-1–immunoreactive axonal processes (Fig. 1A,C) expressed a high intensity of synapsin staining (Fig. 1A) on the surface of myotubes (Fig. 1B), which is indicative of the formation of synaptic contacts. In some axonal fibers that connected to the mouse myotube, we detected immunostaining for choline acetyltransferase (ChAT; Fig. 1D) in the terminal branches. The majority of NT2 neurons displayed long neurites which stained with the SMI32 antibody against nonphosphorylated neurofilament H (Fig. 1E,F), indicating motoneuron characteristic (Tsang et al., 2000). As a specific marker for nicotinic cholinergic transmission, the axonal terminal arborizations on the myotube were aligned with α-bungarotoxin labeling (Fig. 1E,F).

Figure 1.

Co-cultures of NT2 neurons with mouse myotubes (MT). A: Pattern of immunofluorescent neuronal processes, which tend to align with the surface of myotubes (arrows). Intense immunofluorescence (yellow) is derived from co-labeling against the axonal marker Tau 1 (red) and synapsin (green). Arrowheads indicate DAPI (blue; 4′,6-diamidino-2′-phenylindol-dihydrochloride) stained nuclei of myotubes. For better visibility of myotube shape, the background fluorescence was enhanced by digital image processing. B: Magnification of the boxed area in A showing a high intensity of synapsin staining (green) on the surface of a myotube (arrowheads) together with labeling for Tau-1 (red). C: Co-labeling for Tau-1 (red) and ChAT (green; choline acetyltransferase) on axons (arrowheads) in contact with a myotube (arrow). For better visibility the outline of the myotube is stippled. D: Magnification of the boxed area in C, resolving ChAT immunofluorescence (green, arrow) in the terminal axonal process stained for Tau-1 (red, arrowhead). E: Neuronal processes stained with SMI32 antibody (green, arrowheads) are extending along groups of MT. Terminal arborizations of neurites (box) align with staining for α-bungarotoxin (red). F: Magnification of the boxed area in E, showing the α-bungarotoxin (red, arrow) and SMI32 (green) staining. Nuclei counterstained with DAPI (blue). For reasons of clarity, nuclear staining was not included into enlargements. Scale bars = 100 μm in A,C,E, 50 μm in B,D, 25 μm in F.

General Observations on Transplantation of NT2 Cells Into Chick Embryo

To explore how the NT2 neurons respond to an embryonic neural tissue environment, we performed cell transplantations into the brain ventricle (stage 24–26) and neural tube (stage 14–18) of chick embryos. In initial experiments, cell transplantation into neural tube was performed also at stage 24.

Before grafting, the NT2 neurons and precursor cells were subjected to diverse differentiation conditions. Cells were differentiated in aggregate culture for 5 days with the addition of retinoic acid (NT2+RA) or until final postmitotic stages (NT2 neurons) (Podrygajlo et al., 2009a). To facilitate conversion toward a motoneuronal phenotype, one group of cells was pretreated for 5 days with retinoic acid and sonic hedgehog protein (NT2+RA+Shh; Wichterle et al., 2002; Li et al., 2005). Undifferentiated NT2 precursor cells (NT2) were also used as a fourth group. To visualize the injection site in the operated neural tube NT2 cell colonies were prestained with fluorescent dyes (NT2 neurons, NT2+RA and NT2+RA+Shh with CFDA-SE; NT2 precursors with CM-DiI or CFDA-SE) before transplantation (Fig. 2 A,B). Immediately after microsurgery, the operation site was checked under fluorescence illumination showing that initially all cell types remained as a cluster together. In total, we performed 248 cell grafts of which the majority was targeted to the neural tube at wing bud level (corresponding to somite levels 17–19). In 72 cases of successful implantations, we sectioned regions of the spinal cord and brain on a cryomicrotome.

Figure 2.

Schematic representation of the injection site and immunofluorescence of the grafted cells. A: Schematic diagram of chick embryo at stage 14 with marked somite 17–19. The enlarged box shows the unilateral injection of the fluorescent labeled (CFDA-SE) cells into the operated neural tube. B: Schematic view of the chick embryo at stage 24. The gray plane indicates the orientation of coronal sections. Fluorescent labeled (CFDA-SE) NT2 cells were injected into brain ventricle. C: A low magnification view on the site of transplantation of NT2N cells in the neural tube. Neural tube (NT), dorsal root ganglia (DRG), and notochord (N) are indicated with dashed lines. Magnification of box in C showing neural β-III-tubulin staining in cell bodies and extending processes (arrows). Unilaterally injection of the cells was performed at chick embryo stage 24 at the level of wing bud. Embryo with transplanted cells was incubated for 5 days. D: Cross-section of stage 14 embryo showing integration of NT2+RA (retinoic acid) cells in the neural epithelium after 1 day after injection. Visible immunoreactivity in DRG (arrow) indicate cell migration. Magnification of box in D shows integration of NT2+RA within the neural epithelium of developing neural tube (arrow). Red fluorescence indicates immunoreactivity for β-III-tubulin. Nuclei counterstained with DAPI (blue; 4′,6-diamidino-2′-phenylindol-dihydrochloride). Scale bars = 200 μm in C; 100 μm in D; 50 μm in inserts.

The examination of cross-sections through the spinal cord at the site of transplantation showed no visible surgical damage and revealed the intact structures of neural tube (NT), dorsal root ganglia (DRG), and notochord (N; Fig. 2C). The grafts of human cells were probed with an antibody against β-III-tubulin that did not cross-react with chick neural tissue. From a comparison of large (human) and small (chick) diameter nuclei, we estimated the percentage of β-III-tubulin–negative cells injected into the neural tube. For all transplantation experiments, we counted 18.2% (±8.1) β-III-tubulin–negative cells.

Fully differentiated NT2 neurons that were grafted into the neural tube (Fig. 2C, insert) extended β-III-tubulin–stained processes some of which covered considerable distances larger than 150 μm. Moreover, NT2 precursor cells pretreated for 5 days with RA start to express β-III-tubulin. To resolve the cellular distribution after the operation, we injected cells into the neural tube and incubated the embryo for only 1 day. Figure 2D shows the presence of immunoreactive NT2+RA cells in the neural epithelium and the absence in the central canal. β-III-tubulin–stained NT2 cells can be also located in DRGs suggesting a rapid migration from the injection site.

Transplantation of NT2 Cells Into the Neural Tube

NT2 neurons grafted at stage 18 extended ventrally directed neurites in the neural tube (Fig. 3A). In some cases, NT2 neurons could be found in aggregates aside the neural tube. However, localization within the DRG together with processes outgrowth is also evident (Fig. 3B).

Figure 3.

Cross-sections of NT2 cell grafts into developing neural tube. A: NT2N cells localized in the lateral part of the neural tube sending elongated processes (arrowheads) toward the ventral part along the midline. This morphology was observed in 66.6% preparations. Injection of the cells was done at chick embryo stage 18 at the level of wing bud. Embryo with cell graft was incubated for 5 days. B: Migration and incorporation of the injected NT2N cells into dorsal root ganglia (DRG; arrowheads). Intensely β-III-tubulin–positive small aggregate of the cells bodies is located in the vicinity of the neural tube (arrow). Injection of the cells was done at chick embryo stage 18 at the level of wing bud. Embryo with cell graft was incubated for 5 days. C: Massive incorporation of NT2+RA (retinoic acid) cells into ventral part of neural tube accompanied by extensive neurites outgrowth (arrowheads). In 33.3% of the preparations, processes approaching the ventral root (asterisk) are clearly visible (arrowheads). Cells were injected at stage 14 at the level of the wing bud. Embryo with cell graft was incubated for 5 days. D: Massive invasion of DRG by NT2+RA cells stained for β-III-tubulin (arrowheads). Injection of the cells was done at chick embryo stage 14 at the level of wing bud. Embryo with cell graft was incubated for 5 days. E: NT2 cells treated with RA and Shh localized in the lateral part of neural tube (arrow). Extending processes stained for β-III-tubulin (arrowheads) were visible in 41.6% of preparations. Some cells have migrated and incorporated into the DRG. Injection of the cells was done at stage 16 at the level of wing bud. Embryo with cell graft was incubated for 3 days. F: Single example of cylinder-like structure formed by NT2 precursors labeled with the CM-Dil dye (red) before the transplantation. Injection of the cells was done at stage 14 at the level of wing bud. Embryo with cell graft was incubated for 3 days. Red fluorescence in the figures indicates immunoreactivity for β-III-tubulin (apart F). Nuclei counterstained with DAPI (blue). Scale bars = 100 μm in B,C,E,F, 50 μm in A,D.

Grafts of NT2+RA cells integrated also easily into the neural tube tissue. An example of a large incorporation of NT2+RA cells into ventral parts of the neural tube with extensive neurite outgrowth is shown in Figure 3C. Some of the β-III-tubulin–positive neurites extended toward the ventral root (Fig. 3C). A view of the DRG with massive invasion of β-III-tubulin–positive NT2+RA cells is shown in Figure 3D. NT2 cells pretreated with RA and Shh (NT2+RA+Shh) were mainly detected in the lateral part of neural tube at the wing bud level. It is of note that their β-III-tubulin–positive processes had a tendency to orient toward the ventral region of neural tube (Fig. 3E).

Transplanted NT2 precursors which were not differentiated into neurons were identified by CM-Dil dye labeling. Typically, the injected NT2 precursor cells lacked staining for β-III-tubulin (Fig. 3F). They rarely formed tubular-like structures, a potential sign for malignant tissue formation (Reubinoff et al., 2000; Busch et al., 2009). Figure 3F depicts the only example which we found of such a tubular structure.

Transplantation of NT2 Cells Into the Brain Ventricle

Initial trials established that it was difficult to graft cells directly into brain tissue in young embryos of stage 14–18. Therefore we injected the NT2 cells into the brain ventricles of older embryos at stage 24. The neuroanatomical analysis revealed that labeled cell groups often remained attached to the ventricular surface. Alternatively, migratory cells were found fully integrated into the brain tissue, extending β-III-tubulin–positive neurites (Fig. 4A). The cell bodies of NT2 neurons could easily be discerned from chicken tissue because the nuclei of the human cells (ca. 20 μm) were noticeably bigger (Fig. 4A) than those of the host (ca. 10 μm). NT2 neurons were mainly found close to the scar at the ventricular surface of the brain. They extended extensive processes stained with β-III-tubulin along the wound canal made by the injection needle (Fig. 4B). NT2+RA cells integrated less efficiently compared with the NT2 precursor cells, forming at the same time aggregates inside the brain ventricles in which most of the cells were stained for β-III-tubulin (Fig. 4C). CFDA-SE–labeled NT2 precursor cells migrated out of the injected ventricles and incorporated into the host brain tissue showing immunopositive staining for β-III-tubulin (Fig. 4D). The remaining cells stayed attached to the ventricular surface sending short processes into scarred brain tissue (Fig. 4D).

Figure 4.

Incorporation of NT2, NT2+RA (retinoic acid), and NT2N cells into 5-day-old chick embryo brain. A: NT2 neurons extending neurites in the host brain stained for β-III-tubulin (red, arrows). Nuclei of human cells are noticeably bigger than those of host (arrowheads). Injection of the cells into brain ventricles was done at chick embryo stage 24. Transplanted cells were incubated with host tissue for 3 days. B: NT2 neurons attached to the scar at the ventricular surface of the brain. Neurons are elaborating extensive processes stained for β-III-tubulin (red, arrows) along the wound caused by the injecting needle (asterisk). Injection of the cells into brain ventricles was done at chick embryo stage 26. Transplanted cells were incubated with host tissue for 3 days. C: NT2+RA cells integrated poorly, creating aggregates within the brain ventricle with many cells showing β-III-tubulin immunoreactivity (red, asterisk). Some of integrated cells are β-III-tubulin positive (red, arrow) whereas other are not (green, arrowhead). Injection of the cells into brain ventricles was done at chick embryo stage 24. Transplanted cells were incubated with host tissue for 3 days. D: NT2 cells migrate away from the ventricular surface and incorporate with host brain tissue showing staining for β-III-tubulin (red, arrow) and CFDA-SE (green, arrowhead). The rest of the cells remain attached to the ventricular surface sending short processes into scarred brain tissue (asterisk). Injection of the cells into brain ventricles was done at chick embryo stage 24. Transplanted cells were incubated with host tissue for 3 days. Nuclei counterstained with DAPI (blue; 4′,6-diamidino-2′-phenylindol-dihydrochloride). Scale bars = 200 μm in A,B, 100 μm in C,D.

Evaluation of the Neurite Outgrowths in the Brain and Spinal Cord

When we sectioned the neural tube along the sagittal plane, we noticed that the grafted neurons hardly extended processes in an anterior–posterior direction. To provide a quantitative estimate of neurite outgrowth from β-III-tubulin–stained neurons, we evaluated the size of the longest neurite in multiples of the corresponding soma diameter in cross-sections of spinal cord and coronal sections of the brain. We have included in our calculation only those neurons, in which the traced neurite does not extend beyond the same plane of a section. Thus, our measurements will occasionally underestimate outgrowth in case where long neurites leave the section. Figure 5 shows the different distributions of neurite length, obtained after integration of cell grafts into the neural tube and brain. The percentage of neurons is plotted versus its neurite length for the three different pretreatment groups: NT2+RA, NT2+RA+Shh, and differentiated NT2 neurons. In the spinal cord, the major percentage of β-III-tubulin–stained cells extended neurites in the range of two times the soma diameter. For all three pretreatment groups, neurites of 5 times the soma diameter or longer were also found. (Fig. 5A).

Figure 5.

A,B: Comparison of neurite outgrowth for cells grafts in sections of spinal cord (A) and brain (B). The size of the neurites is expressed as multiple of the corresponding soma diameter. The histograms list the percentage β-III-tubulin–stained neurons extending a certain neurite length. For each of the three different pretreatment groups (NT2+RA, NT2+RA+Shh, and NT2N), the number of successful cell transplantations (n) is indicated.

In samples from brain tissue, we observed almost no cells with neurites longer than 3 times the soma diameter. The majority of β-III-tubulin–positive cells incorporated into the brain showed neurites in the range of a single soma diameter. (Fig. 5B) These results suggest that embryonic chick spinal cord tissue is more permissive to NT2 neurite extension than brain tissue.

DISCUSSION

Using the cell aggregate differentiation protocol (Paquet-Durand et al., 2003), we found in a previous in vitro study that approximately 35% of the postmitotic neurons express immunoreactivity for ChAT, VAChT and for the nonphosphorylated form of neurofilament H, all of which are markers of a motoneuronal phenotype (Podrygajlo et al. 2009a). To obtain further evidence for motoneuron-like cell properties, we co-cultured NT2 neurons with mouse myotubes (MT). Human NT2 neurons were able to make close contact with myotubes, sending in their direction axons with a strong, punctuate synapsin staining (Fig. 1A). Moreover, the seeded neurons remained more dispersed than in pure culture conditions, suggesting adhesive interactions between them and the myotubes. In co-culture, neurons appeared to produce acetylcholine (positive staining to ChAT, Fig. 1C,D) and nonphosphorylated neurofilaments H (Fig. 1E,F). In addition, the ChAT-stained structures on the myotube display the morphological characteristic of neuromuscular junctions (Fig. 1D). These structures were also associated with α-bungarotoxin staining (Fig. 1F; Li et al., 2005), indicating the presence of nicotinic ACh-receptors. These cytological features together with immunocytochemical staining suggest the formation of synapses between NT2 neurons and myotubes.

So far, functional synapses between NT2 neurons that were co-cultured with astrocytes have been detected in electrophysiological recordings (Hartley et al., 1999). There is also evidence from electron microscopy for synaptic contacts among NT2 neurons in the absence of glial cells (Guillemain et al., 2000). To show the functionality of the synapses formed by NT2 neurons in the absence of glia, we performed a patch-clamping study in vitro (Podrygajlo et al., 2009b).

Terminally differentiated NT2 neurons have been used in several transplantation studies, demonstrating successful engraftment into rodent brain and specific animal models for Parkinson's disease, amyotrophic lateral sclerosis, traumatic brain injury and stroke (Iacovitti et al., 2001; Garbuzova-Davis et al., 2002; Zhang et al., 2005; Hara et al., 2007). The transplantation of various NT2 cells (NT2, NT2+RA, NT2+RA+Shh, and NT2 neurons) into developing chicken embryos clearly showed the ability of those cells to survive and integrate in the host embryonic tissue. Except for the NT2 precursors, most of these cells had neuronal morphology and displayed axon-like processes (Fig. 3A,C,E).

Due to limitation of the surgical accuracy, we often found injected cell types distributed in all parts of neural tube. Although the injection was made unilaterally, the contralateral side of the neural tube may have been accidentally opened by the tungsten needle, thus creating a wound for easy access of grafted cells. Immunostaining for β-III-tubulin demonstrated that the successfully transplanted cells that received RA treatment localized inside the neural tube and elaborated neurites. In the spinal cord but also in brain tissue, transplanted cells have also been found to migrate away from the injection site. For example, some of the NT2+RA cells incorporated into the host DRG (Fig. 3C).

A similar observation has been made with human embryonic stem cells which migrated after implantation from the somites to DRGs of the chick embryo (Goldstein et al., 2002). Differentiation of some NT2 cells toward neural crest cell phenotype which might contribute to the formation of DRG remains a distinct other possibility (White and Anderson, 1999). Figure 3C shows neurites from NT2+RA differentiated cells reaching the ventral root. This indicates that the embryonic environment of the neural tube facilitates the differentiation toward a motoneuronal morphology.

In grafts of NT2 precursor cells, tubular structures similar to teratomata were found (Busch et al., 2009; Fig. 3F). We found no evidence that the local environment of embryonic chick tissue can direct the undifferentiated NT2 precursor cells into a neuronal fate with neurite outgrowth. This contrasts with human metastatic melanoma cells, which could be reprogrammed to a neural crest cell-like phenotype in an embryonic microenvironment (Schriek et al., 2005; Kulesa et al., 2006). However, as visualized by the β-III-tubulin expression, pretreatment of NT2 cells with RA greatly enhanced the acceptance, integration, and outgrowth of the neuronal processes in the host tissue.

In case of injecting NT2 cells into the chick embryonic brain ventricle, the results obtained were different from those observed for the neural tube. Those differences may at least in part be caused by differences in the delivery procedure. For grafting into the brain, the cells were injected into the ventricular lumen, whereas for neural tube grafts the cells could be directly transplanted into the nervous tissue. Most of the transplanted cells remained inside the ventricle creating aggregates stained for β-III-tubulin (Fig. 4C), whereas some of the cells integrated into the host brain tissue. A remarkable aspect of the NT2 cells injection was the ability to attach to the ventricular surface and to migrate into the brain tissue.

Comparison of the neurites extension process between the two sites of transplantation showed that the neural tube was more permissive to neurite outgrowth than the brain (Fig. 5). To enable a comparison, in the brain we evaluated only neurite outgrowth from somata that were integrated into the tissue. A potential explanation for the differential outgrowth is that the treatment with RA induces a caudal positional identity (Maden, 2002), which is more compatible with neurite extensions in the spinal cord. We found that the distribution of neurite lengths of the two pretreatment groups (NT2+RA and NT2+RA+Shh) were within comparable ranges. Thus, pretreatment of the NT2 cells with Shh before transplantation did not appear to affect the extension of neurites in vivo (Fig. 5). It is possible that the high concentration of RA (10 μM) overrules any effect of Shh application. Moreover a recent study of transcriptional targets indicated that RA down-regulates components of the canonical Shh pathway in NT2 cells (Vestergaard et al., 2008). This is in line with our former in vitro analysis where we did not observe an effect of Shh during on the differentiation of NT2 neurochemical phenotypes (Podrygajlo et al., 2009a).

This study has opened an avenue to analyze the development of human model neurons in the permissive environment of an intact embryonic nervous system. The chick embryo preparation is accessible to experimental manipulations by incubation of the embryo in ovo with drugs. Using human NT2 neurons transplanted into the neural tube, we will now screen for biological factors or small molecule compounds that stimulate axonal outgrowth toward the muscles.

EXPERIMENTAL PROCEDURES

Cell Culture

Unless stated otherwise, all chemicals were purchased from Sigma, Taufkirchen, Germany. The human Ntera2/D1 cell line (NT2) was obtained from ATCC (American Type Culture Collection, Manassas, VA). NT2 precursor cells were maintained and cultivated in DMEM/F12 culture medium (Gibco-Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS, Gibco-Invitrogen) and 1% penicillin/streptomycin (Gibco-Invitrogen) in the atmosphere of 5% CO2 at 37°C (Andrews, 1984). Generation of NT2 neurons was performed by using the differentiation protocol in free-floating aggregates (Paquet-Durand et al., 2003). Briefly, NT2 precursor cells were seeded in 96-mm bacteriological grade Petri dishes (Greiner, Hamburg, Germany) at a density of ∼5 × 106 cells per dish. On day one, 10 ml of culture medium was added to each Petri dish. On the next days, medium containing 10 μM RA was added and changed every 2–3 days. After 7–8 days, the cells from one Petri dish were transferred and seeded in one T75 cell culture flask (Falcon, Franklin Lakes, NJ) and cultured for another 7–8 days in RA medium at the density 4 × 107 cells per flask. Cells were trypsinized (Trypsin-EDTA, Gibco-Invitrogen), transferred to T175 cell culture flasks and cultured for 2 days in normal medium. Then the cells were 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. Differentiated NT2 neurons (NT2N) were transplanted or plated on poly-D-lysine and matrigel (Becton-Dickinson, Bedford, MA) coated cover glasses at a density of 20,000 cells per glass and cultured for 3–4 weeks.

Co-culture of NT2 Neurons and Myocytes

Mouse C2C12 myoblasts were purchased from American Type Culture Collection (ATCC) and were cultured in DMEM/F12 medium supplemented with 10% FBS. For differentiation into myotubes (MT), C2C12 cells were cultured 4–5 days in DMEM/F12 medium supplemented with 2% FBS. Differentiated MT were plated together with NT2 neurons on poly-D-lysine and matrigel coated cover glasses at a density of 10,000– 20,000 cells per glass and cultured for 3–4 weeks in DMEM/F12 medium with 5% FBS and mitotic inhibitors.

Chick Embryos

Fertilized White Leghorn chick (Gallus domesticus) were incubated at 38°C under 80% humidity. The majority of transplantations was performed on embryos at stage 14–18 (neural tube) and 24–26 (brain) according to Hamburger and Hamilton (1951). However, we checked in initial experiment also other stages.

Cell Transplantation

To prepare cells for transplantation experiments, NT2 precursor cells (NT2) were treated for 5 days in the 96-mm bacteriological grade Petri dishes with 10 μM RA (NT2+RA) or 10 μM RA and 0.1 μM recombinant human Sonic hedgehog amino terminal peptide (1314-SH; R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany; NT2+RA+Shh). Before transplantation, cells were labeled by incubating at 5% COmath image and 37°C for 20 min with 20 μM 5-(and-6) carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE; Invitrogen) or 2 μg/ml chloromethyl-benzamidodialkylcarbocyanine (CM-Dil; Invitrogen) in phosphate buffered saline (PBS). After washing 3 times with PBS, cells were centrifuged at 6,400 rpm for 2 min and kept on ice. Eggs were windowed and the shell treated with wax, which allowed a drop of Locke's solution (Locke and Rosenheim, 1907; Rugh, 1962) to be placed onto the window. Together with injection of Indian Ink below the embryo and proper use of light, this method greatly enhanced contrast and visualization. The site of transplantation was prepared by gently tearing the amnion with the use of a tungsten needle (for brain transplantation) or dissecting part of the NT on the level of somites 17–19. The latter was done either by use of a tungsten needle or by inducing a suction lesion with a fire polished glass pipette mounted to a mouth-controlled aspirator tube assembly (Sigma). A total of 0.5–2 μl of concentrated cell suspension was injected into neural tube or brain with a fire polished glass pipette. Directly after injection, embryos were checked under a fluorescent lamp for successful labeling. Injected embryos were sealed and incubated for additional 3 or 5 days.

Immunocytochemistry

Cells were washed with PBS and fixed for 30 min at room temperature with 4% paraformaldehyde (PFA). Cells were washed after each step three times for 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 hr. Primary antibodies used in experiment were: monoclonal mouse anti-β-III-tubulin (T8660, Sigma, 1:10,000), polyclonal goat anti-choline acetyltransferase (ChAT, AB144P Millipore International, 1:100), monoclonal mouse anti-TAU-1 (MAB3420, Millipore International, 1:2,000), monoclonal anti-neurofilament H nonphosphorylated (SMI32, Covance, 1:2,000), monoclonal mouse anti-synapsin 1 (106001, Synaptic Systems, 1:500), polyclonal rabbit anti-TAU (AB1512, Chemicon, 1:200), and α-bungarotoxin conjugated with Alexa488 (B13422, Invitrogen, 1:1,000).

All antibodies were diluted in blocking solution and applied overnight at 4°C or for 1 hr at room temperature. Secondary biotinylated antibodies (Vector, Burlingame, MA), diluted 1:250 in blocking solution, were added for 1 hr at room temperature. Immunofluorescence was detected by applying streptavidin-Alexa Fluor 488 (Mobitec) or streptavidin-CY3 (Sigma) for 1 hr 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.

Immunohistochemistry

Immunostaining was carried out according to the standard procedures. Briefly, embryos were fixed overnight in Serra's fixative (Serra, 1946). After dehydration, the embryos were embedded in paraffin and serially sectioned at 10 μm. Before immunostaining antigen retrieval was performed by boiling samples in 10 mM citrate buffer (pH 6.0) for 20 min.

Immunohistochemistry on frozen sections was carried out according to standard procedures. Embryos were fixed in 4% PFA, cryoprotected in 30% sucrose, and frozen in OCT compound (Leica) with liquid nitrogen. Then 20-μm cross-sections for the spinal cord and coronal-sections for the brain were cut on a Reichert-Jung Frigocut 2800E cryostat microtome at −20°C, collected on poly-D-lysine coated slides, air-dried for 1 hr, and stored at −80°C until use. Immunohistochemical staining was done as described above.

Microscopy and Statistical Analysis

Preparations were viewed with a Zeiss Axioscope, equipped with an Axiocam3900 digital camera and Zeiss Axiovision software, Zeiss Axiovert 200, equipped with a CoolSnap camera (Photometrics) and MetaMorph software (Molecular Devices) and Leica TCS-SP5 Spectral Laser Scanning Confocal Microscope with LAS AF software. Photographs were processed (contrast and brightness enhanced) in Adobe Photoshop. Cell counts were made on images obtained with Metamorph from each section that contained stained neurons integrated properly into the target region (neural tube or brain). The percentage of β-III-tubulin–negative cells injected into the chick neural tube was estimated by counting the number of large diameter human nuclei from digital images of immunostained sections. Data were processed using GraphPad Prism 4.0 software. Cell numbers are expressed as a percent of positively stained cells for β-III-tubulin. The neurite outgrowths were represented in histograms as: 1×, 2×, 3×, 4×, 5×, or more than 5× times greater than the soma diameter. Data are expressed as mean ± SEM.

Acknowledgements

We thank S. Tan for assistance with the cell cultures. G.P. was supported by the Marie Curie Actions program awarded to the ZSN Hannover, C.W. was supported by the Deutsche Forschungemeinschaft (GRK1104 awarded to M.S.), and G.B. received a DFG grant.

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