Professor Andreas Wree, Institute of Anatomy, University of Rostock, Gertrudenstr. 9, PO Box 100888, D-18055 Rostock, Germany. Tel. +49 381494 8401; fax: +49 381494 8402, e-mail: email@example.com
The use of neural stem cells as grafts is a potential treatment for Parkinson's disease, but the potential of stem cells to differentiate into dopaminergic neurones requires investigation. The present study examined the in vitro differentiation of the temperature-sensitive immortalized mesencephalic progenitor cell line CSM14.1 under defined conditions. Cells were derived from the mesencephalic region of a 14-day-old rat embryo, retrovirally immortalized with the Large T antigen and cultured at 33 °C in DMEM containing 10% fetal calf serum (FCS). For differentiation, the temperature was elevated at 39 °C and FCS was reduced (1%). Using histology, immunocytochemical detection of the stem cell marker Nestin and the neuronal marker MAP5 and, in addition, Western blotting to determine the presence of neurone-specific enolase and the neurone nuclei antigen we demonstrated a differentiation of these cells into neuronal cells accompanied by a decrease in Nestin production. In Western blots, we detected the orphan nuclear receptor Nurr1 in these cells. This was followed by a time-dependent up-regulation of the enzymes tyrosine hydroxylase and aldehyde dehydrogenase 2 characteristic of mature dopaminergic neurones. Our in vitro model of dopaminergic cell differentiation corroborates recent in vivo observations in the developing rodent brain.
In the present study, we characterized the in vitro differentiation of the temperature-sensitive immortalized neuronal progenitor cell line CSM14.1 (Zhong et al. 1993) thought to be rather immature dopaminergic neuronal progenitors. This clonal cell line was derived from the ventral mesencephalic region of an E14 rat. At a well-defined temperature (39 °C, non-permissive temperature) and serum-deprived in vitro conditions we demonstrate decrease of Nestin, differentiation into a neuronal morphology, up-regulation of neuronal proteins, and the detection of Nurr1 and ALDH2 in these cells under conditions for differentiation. The time courses of the appearance of the respective markers established in vitro during differentiation largely corroborate the sequence of molecular events found in vivo.
Materials and methods
Cell culture and cell differentiation
Conditionally immortalized CSM14.1 cells (Zhong et al. 1993) were cultured and expanded in DMEM supplemented with 10% fetal calf serum (FCS), 100 U mL−1 penicillin, 100 µg mL−1 streptomycin in a humidified incubator (95% air/5% CO2, at 33 °C). The cells were passaged every third day. For in vitro differentiation, CSM14.1 cells were treated as described previously for primary neural stem cells (Winkler et al. 1998). In brief, CSM14.1 cells were passaged and cultured overnight in a humidified incubator (95% air/5% CO2, at 33 °C) in Petri dishes for Western blotting or on poly L-lysine-coated culture slides (Becton-Dickinson, Heidelberg, Germany) for immunocytochemistry. Thereafter, the medium was changed for DMEM supplemented with 1% FCS, 100 U mL−1 penicillin, 100 µg mL−1 streptomycin and the incubation continued at 39 °C (non-permissive temperature; 5% CO2, humidified environment). Cells grew for 3, 7, 10 and 14 days, respectively; the medium was changed every third day. All reagents for tissue culture were obtained from Gibco Life Technologies (Karlsruhe, Germany).
Immunocytochemical and Western blot analysis of the cultured cells were repeated at least three times in independent experiments performed under respective culture conditions with reproducible results.
Immunocytochemistry and histology
Cells grown on culture slides for 2 days at 33 °C in DMEM containing 10% FCS or for 3, 7, 10 and 14 days under the non-permissive conditions (see above) were washed with 0.9% sodium chloride and fixed with 4% formaldehyde (prepared freshly from paraformaldehyde dissolved in phosphate-buffered saline; PBS, pH 7.4) for 60 min at room temperature. Cultured cells were then rinsed three times for 5 min in 0.1 m Tris buffer (pH 7.4) and endogenous peroxidases were blocked by 20 min incubation in 3% H2O2 (dissolved in 0.1 m Tris buffer, pH 7.4). After three further washes in 0.1 m Tris buffer (pH 7.4) the specimens were pre-incubated for 1 h in Tris buffer containing 0.05% Triton-X100 (Sigma, Deisenhofen, Germany), 3% BSA (Sigma) and 1.25% normal horse serum (Dianova, Hamburg, Germany) to quench non-specific binding sites, and subsequently incubated with primary antibodies against microtubule-associated protein 5 (MAP5, mouse monoclonal, 1 : 500; Sigma), neural stem cell protein (Nestin, mouse monoclonal, 1 : 500, Becton-Dickinson), glial fibrillary acidic protein (GFAP, mouse monoclonal, 1 : 400, Sigma), neurone-specific enolase (NSE, rabbit polyclonal, 1 : 500, Chemicon, Hofheim, Germany) and tyrosine hydroxylase (TH, mouse monoclonal, 1 : 500, Sigma) overnight at 4 °C. The cell preparations were then washed three times for 10 min in 0.1 m Tris (pH 7.4), incubated again at 4 °C overnight with biotinylated secondary antibodies (polyclonal, 1 : 80, Dianova), washed three times in 0.1 m Tris buffer (pH 7.4), then incubated for 45 min in ABC-Complex (1 : 80, Dianova) at room temperature. After three further washes in 0.1 m Tris buffer (pH 7.4) the labelled binding sites were visualized with a 3,3′-Diaminobenzidine tetrahydrochloride substrate solution (Sigma). Finally, the cells were washed three times for 5 min in 0.1 m Tris buffer (pH 7.4), dehydrated in graded ethanol concentrations and mounted in DePeX (Serva, Heidelberg, Germany). After fixation, parallel culture slides were also stained for 3 min at room temperature with 0.1% Cresyl Violet acetate (Sigma), washed in water and also mounted in DePeX. Specimens were observed and documented with a Leitz Aristoplan microscope (Wetzlar, Germany).
Preparation of cell cultures for Western blotting
Cells grown on Petri dishes were rinsed with 0.1 m PBS (pH 7.4), trypsinized (Gibco Life Technologies), centrifuged (10 min at 400 g), washed and neutralized with DMEM containing 10% FCS. After a final centrifugation (10 min at 400 g), cells were resuspended in PBS. The viability and the number of cells were determined by trypan blue exclusion (Sigma). The cells were then lysed by several freeze/thaw cycles. Equal total cell protein concentrations were determined using a spectrophotometer (Model DU640, Beckman, Fullerton, CA, USA) and a bicinchoninic acid assay (Pierce Chemical Co, Rockford, IL, USA) according to the manufacturer's instructions. Comparable concentrations of whole protein lysates were boiled for 5 min in SDS (sodium dodecyl sulphate) sample buffer (Laemmli, 1970). Fifty micrograms of whole protein dissolved in 20 µL SDS sample buffer was loaded in each lane.
SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed with ready-to-use criterion mini gels (Biorad, München, Germany) consisting of 4–15% polyacrylamide. After electrophoresis, proteins were transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Freiburg, Germany). Membranes were blocked for 2 h at room temperature in 0.1 m PBS (pH 7.4), 0.1% Tween 20 (PBS-T) and 1% BSA (Sigma). Primary antibodies directed against β-actin, the product of a housekeeping gene (1 : 3000, mouse monoclonal, Sigma), the immortalizing gene product SV40 Large T (SV40LT-ag, 1 : 500, mouse monoclonal, Becton-Dickinson), glial fibrillary acidic protein (GFAP, mouse monoclonal, 1 : 400, Sigma), the neurone-specific enolase (NSE, 1 : 1000, rabbit polyclonal, Chemicon), the neuronal nuclei antigen (NeuN, 1 : 5000, mouse monoclonal, Chemicon), the tyrosine hydroxylase (TH, 1 : 3000, mouse monoclonal, Sigma), the orphan nuclear receptor Nurr1 (Nurr1, 1 : 1000, mouse monoclonal, Becton-Dickinson) and aldehyde dehydrogenase 2 (ALDH2, 1 : 1500, rabbit polyclonal, Kathmann et al. 2000) were performed overnight at 4 °C. Membranes were washed in PBS-T (4 × 15 min) and incubated for 1 h at room temperature with secondary antibodies conjugated with horseradish peroxidase (anti-mouse, 1 : 5000 or anti-rabbit, 1 : 10 000, both Vector Laboratories, Burlingame, CA, USA). After washing the membranes in PBS-T (4 × 15 min), the peroxidase activity was visualized with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).
Morphological changes during neuronal differentiation
Histology of cultured CSM14.1 cells stained with cresyl violet revealed morphological changes in differentiating CSM14.1 cells (Fig. 1). CSM14.1 cells cultured at a permissive temperature of 33 °C (Fig. 1A) had small cell bodies and a fibroblast-like phenotype. Their processes were short, and the cells formed an epithelial-like monolayer after reaching confluence. Cell divisions occurred every 18 h. Under differentiation conditions the CSM14.1 cells began to change their morphology (Fig. 1B,C). After one week at 39 °C and supplemented with 1% FCS only, cells with multipolar somata similar to those of cultured neurones could be found (Fig. 1B). After 14 days culture under differentiation conditions cell bodies and processes of these differentiating cells were considerably larger and began to form a connective network (Fig. 1C).
Undifferentiated CSM14.1 cells were highly immunoreactive for the neural stem cell marker Nestin (Fig. 1D), whereas in cells cultured for 7 and 14 days at 39 °C the amount of Nestin drastically decreased (Fig. 1E,F). In contrast, the neuronal protein MAP5, which was weakly detectable in cells cultured at 33 °C (Fig. 1G), increased during differentiation in the cell processes and somata of CSM14.1 cells (Fig. 1H,I).
Cell counts on cell suspensions and on the histological stainings (Fig. 1A–C) revealed a reduction of viable cells during differentiation. This could be explained by the down-regulation of the immortalizing temperature-sensitive Large T antigen whose gene is only expressed at the permissive temperature of 33 °C (Fig. 2A,B). When CSM14.1 cells were cultured at 39 °C, the immortalizing Large T antigen was not detectable after 3 days (7 d, 10 d and 14 d) (Fig. 2B).
Using Western blotting to compare equal amounts of whole cell protein lysates (Fig. 2A) and further investigation of possible neuronal differentiation, we observed an increase of several neuronal markers in the differentiating CSM14.1 cells over time. An increased amount of the neuronal marker NSE protein was found in these cells (Fig. 2C). It was low at 33 °C, but increased when cultured at 39 °C, and reached a maximum at 7 d. NeuN, another marker for post-mitotic mature neurones, was also found to be increased in CSM14.1 cells over time (Fig. 2D). In Western blots, antibodies against this protein labelled two bands of ∼46 kDa and ∼48 kDa. No obvious changes in the NeuN content could be observed in the fraction of ∼48 kDa over time, whereas the band of ∼46 kDa was not detectable in cells cultured at 33 °C. Cells cultured at 39 °C for 3–14 days showed clearly detectable bands of NeuN in the 46-kDa range with a peak at 7 d similar to the temporal pattern of the synthesis of NSE.
In contrast, the glial marker GFAP could never be detected by immunocytochemistry or Western blotting in the CSM14.1 cells at any time in culture (not shown).
Undifferentiated CSM14.1 cells (i.e. at 33 °C) synthesized Nurr1 (Fig. 3). In contrast, when cultured at 39 °C, the amount of this putative receptor increased after 3 days and then decreased until, after 14 days in culture, it reached levels even below those observed in undifferentiated cells.
In Western blots, TH immunoreactivity was weak for protein extracts of CSM14.1 cells cultured at 33 °C, but strong when cells were exposed to differentiation conditions (Fig. 4). After 3 days at 39 °C, TH content in these cells was markedly increased and thereafter remained constant until 14 d. However, we were not able to demonstrate these changes by immunocytochemical staining of plated cells (not shown).
In preliminary Western blotting studies, the expression of the enzyme ALDH2, a putative marker for mature dopaminergic neurones, in the homogenates of rat hippocampus (containing no dopaminergic perikarya) revealed only a very weak ALDH2 immunoreactivity, whereas it was intense in tissue homogenates from adult rat substantia nigra (containing abundant dopaminergic neurones) and in adult rat liver (according to Kathmann et al. 2000) (Fig. 5A). In further experiments with tissue homogenates from adult rat liver tissue, which served as a positive control, and cell lysates from undifferentiated and differentiating CSM14.1 cells (Fig. 5B), ALDH2 was not detectable in CSM14.1 cells when cultured at 33 °C, but when cultured at 39 °C the CSM14.1 cells showed a time-dependent increase of this enzyme, with the highest quantity at 14 d (Fig. 5B). Unfortunately, cellular diversity or the percentage of Nurr1-ir and ALDH2-ir neurones could not be evaluated because the respective antibodies were not suitable for immunocytochemistry.
The aim of this study was to characterize the potential of the CSM14.1 cells to differentiate under defined culture conditions, and to compare these data with those from in vivo studies concerning the differentiation of dopaminergic neurones. At the permissive temperature of 33 °C these cells proliferated, had a flat neuroepithelial-like morphology and possessed the neural stem cell marker Nestin indicating that, indeed, these cells have the character of neural progenitor (stem) cells when cultured at 33 °C (Lendahl et al. 1990). An elevation of the temperature to 39 °C, which is in the range of that of the rat brain (Gordon, 1990; Martinez-Serrano & Björklund, 1997), and which has been routinely used in previous studies dealing with neural stem cells immortalized with the temperature-sensitive Large T antigen (Frederiksen et al. 1988; Redies et al. 1991; Zhong et al. 1993; Snyder, 1994; Cattaneo & Conti, 1998), together with a deprivation of the serum content of the medium (Winkler et al. 1998) prevented them from expanding. The down-regulation of the Large T antigen was demonstrated in Western blots and observed in a reduction of cell numbers with extended cultivation. CSM14.1 cells then differentiated into a neuronal fate which was shown by morphological changes, a decrease of Nestin protein and, in contrast, an increase of various neuronal marker proteins. CSM14.1 cells were never immunoreactive for GFAP. This indicates that under serum reduced in vitro conditions this cell line derived from a single successfully immortalized cell clone (Zhong et al. 1993) was committed to a neuronal fate as has been shown for other neuroepithelial cells (Buc-Caron, 1995; Cattaneo & Conti, 1998). These phenomena are in line with both the in vivo situation of neuronal differentiation and the in vitro observations published by several groups working with reversibly immortalized stem cell lines under differentiation conditions (Frederiksen et al. 1988; Redies et al. 1991; Snyder, 1994; Cattaneo & Conti, 1998).
Until now, data concerning dopaminergic differentiation of the CSM14.1 cells were rare. CSM14.1 cells were shown to be TH-positive by RNA analysis in vitro (Zhong et al. 1993; Anton et al. 1994, 1995). After grafting into the brain of hemiparkinsonian animals, apomorphine-induced rotations were diminished compared to control animals; nevertheless, TH protein was not detectable in the cells (Anton et al. 1995). This could be due to the content of TH in these cells being too low to be detected when using immunocytochemistry (Anton et al. 1995), but probably high enough to produce some improvement in the grafted animals. No further data, however, were available about the time course of the synthesis of TH or other proteins indicating a dopaminergic differentiation.
The fact that CSM14.1 cells contain Nurr1 and, after the onset of differentiation, at later time points, also the dopaminergic markers TH and ALDH2, is interesting in view of the study of Wagner et al. (1999). These authors transfected mouse cerebellar stem cells with a retrovirus which over expressed the gene of Nurr1. With their protocol used for differentiation, no TH and ALDH2 were observed in those cells. TH and also ALDH2 were exclusively detectable when Wagner et al. (1999) co-cultured the transfected cells with primary type 1 astrocytes derived from the mesencephalon of E16 mouse embryos. Wagner et al. (1999) supposed that yet unknown factors secreted by the astrocytes could have bound to Nurr1. Nevertheless, after grafting into the adult mouse striatum no TH was seen in the transplanted cerebellar stem cells over-expressing the gene of Nurr1 (Wagner et al. 1999). However, Nurr1 was present in the CSM14.1 cells cultured at 33 °C in DMEM containing 10% FCS, even without any co-cultivation of type 1 astrocytes. The mature dopaminergic marker proteins TH and ALDH2 were detectable with subsequent cultivation at 39 °C in medium supplemented only with 1% FCS. This might indicate that mesencephalic stem cells were committed to a dopaminergic fate whereas cerebellar stem cells were not.
We gratefully acknowledge Dr D. E. Bredesen (Neuroscience Department, University of California, San Diego) for providing us with the CSM14.1 cells, Dr J. J. Lipsky (Clinical Pharmacology Unit, Mayo Clinic and Foundation, Rochester) for the ALDH2-antibodies and Dr Christian Thode (Neuroscience and Signal Transduction Laboratory, Department of Life Sciences, The Nottingham Trent University, Nottingham) for his helpful comments on the manuscript.