[1] 21w Regulation of neurogenesis and gliogenesis of retinoic acid-induced P19 embryonal carcinoma cells by P2X2 and P2X7 receptors studied by RNA interference

[1] 234w Embryonic carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis. We have now investigated how down-regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Wild-type P19 embryonal carcinoma cells or cells stably expressing shRNAs targeting P2X2 or P2X7 receptor expression were induced to differentiate into neurons and glial cells in the presence of retinoic acid. Silencing of P2X2 receptor expression along differentiation promoted cell proliferation and an increase in the percentage of cells expressing glial-specific GFAP, while the presence of beta-3 tubulin-positive cells diminished at the same time. Proliferation induction in the presence of stable anti-P2X2 receptor RNAi points at a mechanism where glial proliferation is favored over growth arrest of progenitor cells which would allow neuronal maturation. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells resulted in a decrease in cell proliferation and GFAP expression, suggesting the need of functional P2X7 receptors for the progress of gliogenesis. The results obtained in this study indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down-regulating P2X2 or P2X7 receptor gene expression, developed during this work, present useful tools for studying mechanisms of neural differentiation in other stem cell models.

[1] 477w Neural differentiation requires complex series of cellular programming, intercellular communication events and depends on intrinsic and extrinsic factors that promote or inhibit neurogenesis and gliogenesis. Previous studies suggest that differentiation is induced by the upregulation of transcription factors that control the expression of ion channels (Bertrand et al., 2002;Borodinsky et al., 2004). These ion channels generate particular patterns of spontaneous activity and transient elevations of cytosolic Ca 2+ concentration [Ca 2+ ] i that are modulated by signaling proteins (Borodinsky et al., 2004). It has been well documented that expression of many neurotransmitter receptor genes, such as those coding for GABA receptors (Siegel, 1998), glutamate receptors (Monyer et al., 1994), purinergic receptors (Resende et al., 2007) and dopamine receptors (Chen and Weiss, 1991;Gurevich et al., 1999), undergoes regulation during neuronal differentiation. ATP both acts as a fast excitatory neurotransmitter and induces longterm (trophic) purinergic signaling involved in cell proliferation, differentiation, migration and death in embryonic development, and in disease and cytotoxicity (Abbracchio and Burnstock, 1998;Fields and Burnstock, 2006;Zimmermann, 2006;Burnstock and Ulrich, 2011;Glaser et al., 2012). The nucleotide receptors belong to two major classes, ionotropic P2X receptors assembled from P2X1 to P2X7 subunits and eight metabotropic P2Y receptors. P2X receptors are expressed in neurons, astrocytes, oligodendrocytes and microglia and exert fast ATP-induced neurotransmission (Nörenberg and Illes, 2000;North, 2002). Modulation of P2 receptor expression has been observed as a function of neuronal maturation, with well-established functions for P2Y1 receptors in the induction of calcium waves directing and synchronizing migration of neural progenitor cells for cortex development. Much less is known about the functions of P2X receptors which are already expressed in early brain development. P2X2, P2X3 and P2X7 receptor subunits were detected as early as E14 of embryonic rat brain (reviewed by Burnstock and Ulrich, 2011), a period characterized by high levels of neurogenesis (Trujillo et al., 2009). In vitro studies with neurospheres showed that increased neurogenesis as consequence of short-term mitogen deprivation was accompanied by significantly augmented the expression of P2X2 and P2X6 receptor subunits (Schwindt et al., 2011). Moreover, recent studies of our laboratory revealed increasing P2X2 receptor expression during differentiation. By means of pharmacological tools using cocktails of purinergic receptor agonists and antagonists we showed that P2X2 receptors participated in differentiation of P19 embryonal carcinoma (EC) cells into defined neuronal phenotypes expressing NMDA-glutamate and cholinergic receptors (Resende et al., 2007(Resende et al., , 2008)). P2X7 receptors have been attributed to induction of apoptosis, thereby eliminating unnecessary cells during neural development, and to inhibition of neuronal differentiation. For instance, P2X7 receptors, present on neural progenitor cells, induced cell death independent of caspase activation even at ATP concentrations which did not lead to pore formation (Delarasse et al., 2009). Consequently, retinoic acid (RA)-induction of differentiation of neuroblastoma cells is accompanied by inhibition of apoptosis due to down-regulation of P2X7 receptor expression (Orellano et al., 2010;Wu et al., 2009).

[2] 89w The pluripotent P19 mouse EC cell line provides a flexible model for studying events occurring during early neurogenesis. These cells can be induced to differentiate into neurons and glial cells by the addition of RA and suspension culture for 2 days for formation of embryonic bodies resembling the blastula stage (Jones-Villeneuve et al., 1982;McBurney et al., 1982). Replating of these threedimensional cell aggregates gives then rise to neuronal and glial cells. The proportion of neuronal cells declines after the sixth day, while glial cells continue to proliferate (McBurney, 1993).

[3] 74w Using stable RNA interference we show in this work that P2X2 receptors are involved in the partial inhibition of proliferation and progression of in vitro differentiation of P19 EC cells into neurons, while down-regulation of P2X2 receptor expression results in augmented gliogenesis. Interestingly, P2X7 receptors in former works mostly connected to cell death, promote proliferation and gliogenesis of P19 cells. We provide evidence that down-regulation of P2X7 receptor expression favors differentiation into glial cells.

[1] 83w The RNAi-inducing sequences for silencing P2X2 receptor gene expression were selected by using the program available at http://www.ambion.com and checked for homology with other proteins using the program Basic Local Alignment Search The Tool-BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) from the National Center for Biotechnology Information (NCBI). The selected sequences were transfected into P19 cells using lipofectamine (Invitrogen) and assessed for their efficiencies of P2X2 receptor expression knock-down. Transfection efficiency was almost 100% as verified in control experiments using the block-iT TM fluorescent block-double stranded RNA oligomer.

[2] 168w The selected shRNA sequence (Table 1) was inserted into the NheI site of the bicistronic lentiviral vector p156RRLsinPPTCMV-GFP-PRE/NheI (PLV). The production of lentivirus particles and titration of the viral preparation were performed as described previously by Tiscornia et al. (2006). The virus particles containing the expression cassettes of shRNA-P2X2-9 and a control sequence (control-shRNA) were transduced into undifferentiated P19 cells at MOI (multiplicity of infection) 50 according to Tiscornia et al. (2006). Control shRNA sequences (Table 1) were nonfunctional ones directed to a target (mouse cyclin G) unrelated to P2X2 receptors. However, these sequences can be used to verify that both down regulation of P2X2 receptor expression and subsequent effects on differentiation of P19 cells are specific and not an artifact related to expression of a non-related shRNA sequence or to the overexpression of enhanced green fluorescent protein (EGFP). Furthermore, this control sequence did not promote changes in neural differentiation of P19 cells and did not interfere with mouse cyclin G mRNA and protein levels (data not shown).

[3] 102w The two sequences of RNAi (P2X7-70i and P2X7-71i, Table 1) for silencing of the P2X7 receptor expression and the GFPi sequence (GFPi, Table 1) were obtained from the Mission shRNA library from Sigma-Aldrich which had been already tested for gene silencing. These were cloned into the shRNA lentiviral vector TRC2 (TRC2-pLKO-pure, Sigma-Aldrich). P19 cells stably expressing the above mentioned shRNA sequences were obtained according to the protocol provided by the manufacturer. The cloned cells were named shRNA-P2X7-control or shRNA-control, shRNA-P2X7-70i and shRNA-P2X7-71i. For evaluation of shRNA-P2X2 and shRNA-P2X7 efficiency, we examined mRNA and protein expression levels of P2X2 and P2X7 receptors, respectively.

[4] 73w Among ionotropic purinergic receptors, the P2X2 and P2X7 subtypes have been associated with either promotion or inhibition of neurogenesis, respectively (Resende et al., 2007(Resende et al., , 2008;;Wu et al., 2009). However, these conclusions were mostly obtained from pharmacological studies. Therefore, we have now used stable shRNA expression for systematic down-regulation of the expression of one or the other of these purinergic receptors along differentiation of P19 cells into neuronal and glial phenotypes.

[5] 128w P19 cells transduced with lentiviral particles expressing either control-shRNA or shRNA for down-regulation of P2X2 or P2X7 receptor expression (shRNA-P2X2-9, shRNA-P2X7-70i and shRNA-P2X7-71i) were evaluated for their efficiency in silencing mRNA and protein expression. shRNA-P2X2-9 cells decreased the gene expression of the P2X2 receptor by around 40% when compared to control-shRNA-P2X2 cells (Fig. 1A); reduction of P2X2 receptor expression was also visible in Western-blot assays (Fig. 1B). P2X7 receptor gene expression in P19 cells expressing shRNA targeting P2X7 receptor coding mRNA decreased by 90% when compared with control-shRNA-P2X7 cells (Fig. 1C), and protein expression was below detection limits in Western-blot assays (Fig. 1D). Compared to nontransfected P19 cells, there was no difference between P2X2 or P2X7 mRNA expression in stable, transfected control shRNA-P2X2 or shRNA-P2X7 P19 cells, respectively.

[6] 117w Following 6-8 days of induction of neural differentiation as described in Section 2, we have observed that 65% of the control-shRNA-P2X2 cells population expressed beta-3 tubulin, a neuronal marker protein (Fig. 2A) and 37% of them expressed GFAP, a glial cell marker (Fig. 2B). Moreover, flow cytometry analysis indicated that differentiated cells transformed with shRNA-P2X2-9 diminished beta-3 tubulin expression (from 65% to 33%), while the population of GFAP-positive cells increased from 37% to 69% (Fig. 2A and B). These results were confirmed by real-time RT-PCR analysis showing a decrease of 40% in beta-3 tubulin gene expression and an increase of about 50% in GFAP expression (Fig. 2C and D) when P2X2 receptor expression was down-regulated throughout differentiation.

[7] 109w P2X7 receptor RNAi also interfered with neural marker expression. Flow cytometry analysis revealed beta-3 tubulin (Fig. 3A) and GFAP (Fig. 3B) expression in 69% and 23% of shRNA-P2X7-70i cells differentiated into neurons, while shRNA-P2X7-71i cells were 67% positive immunolabeled for beta-3 tubulin and 30% for GFAP respectively. At the same time 65% and 50% of control cells (control-shRNA-P2X7) were positive for beta-3 tubulin and GFAP immunostaining, respectively. Slight increases in beta-3 tubulin expression and marked decreases in GFAP expression by shRNA-P2X7 cells were also observed by real-time RT-PCR analysis (Fig. 3C and D). In summary, blocked P2X7 receptor expression resulted in largely reduced gliogenesis on day 6 of differentiation.

[8] 96w In the present study, we have studied changes of proliferation of shRNA-P2X2 and shRNA-P2X7 cells on the sixth day of neural differentiation by using a BrdU incorporation assay. The effects exerted by P2X2 receptor expression down-regulation, resulting in inhibited neurogenesis and increased gliogenesis, could involve a reversion of the cell cycle. In fact, shRNA-P2X2-9 cells showed a higher proportion of proliferating cells (53 ± 3%) compared to control cells (39 ± 9%) on day 6 of differentiation (Fig. 4A). Subsequent increased proliferation provides a mechanism for the higher number of glial cells observed under these conditions.

[9] 145w P2X7 receptors are important for cytotoxic processes. However, on the other hand, this receptor may also have implications in cell proliferation (Baricordi et al., 1999;Jacques-Silva et al., 2004), since some tumors express high concentrations of the P2X7 receptor (Adinolfi et al., 2002;Slater et al., 2004;Tamajusuku et al., 2010). We have verified that the percentage of shRNA-P2X7-70i and shRNA-P2X7-71i cells labeled with BrdU was decreased to 33 ± 7% and 29 ± 7% respectively, when compared to control-shRNA-P2X7 cells (50 ± 1%) (Fig. 4B). This result is in agreement with the observed decrease in the percentage of glial cells (Fig. 3B) when P2X7 receptor expression was suppressed during differentiation. It can be concluded that the P2X7 receptor in P19 cells may have a slight inhibitory effect on the progress of differentiation, but has prominent functions in promoting proliferation and thereby augmenting the number of glial cells.

[1] 190w Nucleotides are neurotransmitters, but they also act as trophic agents in differentiation and regeneration processes including neuritogenesis, neuroprotection and neurogenic differentiation (Abbracchio et al., 1995;D'Ambrosi et al., 2001;Neary et al., 1994Neary et al., , 1996;;Shukla et al., 2005;Burnstock and Ulrich, 2011). In view of developmental functions of purines and related nucleotides, previous work of our group focused on the characterization of purinergic receptor expression and activity during proliferation and differentiation of P19 cells into neurons. Expression of P2X2, P2X6, P2Y2 and P2Y6 receptor subtypes increased with simultaneous expression reduction of P2X3, P2X4, P2Y1 and P2Y4 receptors (Resende et al., 2007). P2Y1, P2Y2 and P2X2 receptors were identified as key players for the progress of neurogenesis and neuronal phenotype determination by using pharmacological assays (Resende et al., 2007(Resende et al., ,2008)). Different from the above-cited works, no cytosine arabinoside was employed in our present study for inhibition of glial cell proliferation. In our study, P19 cells expressing shRNA for silencing P2X2 or P2X7 receptor expression were differentiated into neuronal and glial phenotypes. Thus, through the silencing of these subtypes of purinergic receptors we sought to understand their roles in neural differentiation.

[2] 188w The sixth day of neural differentiation was chosen for the analysis of silencing the expression of P2X2 and P2X7 purinergic receptors, since this study aims to investigate the effect of inhibition of these receptors in the formation of neurons and glia. We have verified that inhibition of P2X2 or P2X7 receptors caused changes in the profiles of beta-3-tubulin and GFAP expression. Flow cytometry analysis revealed that shRNA-P2X2-9 cells showed a decreasing in the number of beta-3 tubulin-positive cells, while the number of GFAP-positive cells increased compared to the control group (control shRNA-P2X2). This experiment is in agreement with the results obtained from quantitative PCR showing increasing expression levels of ␤-3 tubulin and decreasing GFAP levels and following inhibition of P2X2 expression. The increase of GFAP gene expression should be the result of increased proliferation of progenitor and/or glial cells. We also found that part of the cell population reveals immunostaining for both GFAP-and ␤-3-tubulin expression, being in agreement that immature neurons may be positive for GFAP and neuronal marker proteins, and that GFAP-positive cells may be yet capable to neuronal differentiation (Piper et al., 2000;Song et al., 2002).

[3] 110w For verification of the hypothesis, that increases of GFAP are connected with augmented proliferation rates, we performed BrdU, which quantifies the incorporation of this deoxy-uridine analogue into newly synthesized DNA by proliferating cells. The effect of exposure shRNA-P2X2-9 cells to BrdU for 14 h caused a significant increase in the rate of labelling on the sixth day of differentiation when compared to proliferation rates of control-shRNA cells. Inhibition of P2X2 receptor expression promoted cell proliferation thereby providing a higher percentage of glial cells. Thus, our data show that P2X2 receptors could participate in the regulation of the progress of neural cell differentiation and phenotype determination in this in vitro model.

[4] 168w P2X7 receptors are known to be responsible for apoptosis in the prolonged presence of agonists; on the other hand, these receptors can also promote proliferation (Adinolfi et al., 2005). In view of that P19 cells interfered for P2X7 receptor expression were incubated for 14 h with BrdU. shRNA-P2X7-70i and shRNA-P2X7-71i cells showed decreased incorporation rates of BrdU suggesting a participation of P2X7 receptors in the cell proliferation control. In agreement, we have observed that the expression of GFAP was decreased when compared to the control group, possibly due to inhibition of glial cell proliferation. Moreover, chronic treatment of control wild-type P19 cells with 10 M 2 (3 )-O-(4-benzoylbenzoyl) adenosine 5 -triphosphate (Bz-ATP), mainly activating P2X7 receptors with less activation of further P2X receptor subtypes (Lambrecht, 2000) resulted in a slight increase (ca. 10%) in GFAP expression of differentiating wildtype P19 cells (data not shown). However, we did not observe any changes in ␤-3 tubulin gene expression of shRNA-P2X7-70i and shRNA-P2X7-71i cells when compared to cells expressing control shRNAs.

[5] 149w Some studies have demonstrated the role of P2X7 receptors in promoting cell proliferation, including human neuroblastoma (Raffaghello et al., 2006) and microglial cells (Bianco et al., 2006;Monif et al., 2009) as well as in inducing neurite elongation (Díaz-Hernández et al., 2008;Ortega et al., 2009;Sperlágh et al., 2006). Moreover, basal activation of the P2X7 receptor increases the mitochondrial calcium concentration in mitochondria, which increases metabolic rates and stimulates the synthesis of ATP, favoring cell growth. However, the prolonged activation of the receptor generates a massive influx of calcium into the cytoplasm, which is addressed directly to the mitochondria promoting changes in metabolism and thus leading to cell death (Adinolfi et al., 2005). Therefore, a well-regulated basal P2X7 receptor activity is involved in neural differentiation of P19 cells promoting proliferation and formation of glial cells; however, prolonged activation would promote cell death and thereby also affect the number of glial cells.

[6] 172w Several neurodegenerative conditions, including stroke, multiple sclerosis, Parkinson's disease, Huntington's disease and epilepsy (Skaper et al., 2009;Skaper and Giusti, 2009), exhibit high levels of P2X7 receptor expression in the neuroinflammatory focus to which also activated microglia participates. In this context, it is not clear whether P2X7 receptor actions are only related with induction of cell death. Studies of Monif et al. (2009) indicate that overexpression of P2X7 receptors promotes activation and proliferation of microglial cells in the rat hippocampus, and that this purinergic receptor subtype activates the pro-survival PI3K/Akt pathway in astrocytes (Jacques-Silva et al., 2004). Thus, the P2X7 receptor is an important signaling protein in physiological and pathological conditions of the nervous system (Skaper et al., 2009;Skaper and Giusti, 2009); however, its exact function in neurodevelopmental processes and also possibly in adult neurogenesis and gliogenesis needs yet to be elucidated. It is clear that modulation of P2X7 receptor activity is an important approach for combating neurodegenerative diseases and for successful brain repair by stem cell transplantation or recruitment of endogenous neurogenesis.

[7] 109w In summary, using the technique of RNA interference, we produced cell lines from cloned P19 cells with stable shRNA interference for P2X2 and P2X7 receptors to study their role during neural differentiation. We found that gene silencing of the expression of P2X2 receptors inhibited neurogenesis, but promoted proliferation and an increase in the number of glial cells. On the other hand, silencing of P2X7 receptor gene expression reduced cell proliferation and subsequently the percentage of glial cells, suggesting a role in gliogenesis. Our results confirm the relevance of purinergic signaling in determining the phenotypic fate of neural differentiation, in which P2X2 and P2X7 receptors promote neurogenesis and gliogenesis, respectively.

[1] 200w Total RNA was isolated using TRIzol (Invitrogen) from P19 cells on 6 days following induction to neural differentiation in the presence of RA. RNA integrity was verified by separation of an aliquot of the extracted RNA on a 2% ethidium bromidestained agarose gel. RNAs had previously been treated for 30 min at 37 • C with 1 U RQ1 RNase-Free DNase (Promega, Madison, WI) in the presence of 20 U RNAse OUT (Invitrogen). Total RNAs (1 g) were reverse-transcribed to cDNA, and the reaction products were amplified by ABI Step One Plus Instrument (Applied Biosystems, Foster City, CA, USA) using the Sybr-Green amplification detection system. Thermal cycling was performed using the following protocol: 50 • C for 2 min, 95 • C for 10 min and 35 cycles at 95 • C for 15 s and 60 • C for 1 min. Experiments were performed in triplicate for each data point. The comparative 2 -CT method was used for relative quantification of gene expression as described previously (Livak and Schmittgen, 2001) using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression as an internal control. Primers used for RT-real time PCR listed in Table 2 produced a single peak in the melting curve.

[2] 11w Cloned shRNA sequences for silencing of P2X2 and P2X7 receptor expression.

[3] 29w for 1 h at room temperature. DNA staining was done for 20 min at room temperature in a solution containing propidium iodide (PI) (50 g/mL) and RNase (100 g/mL).

[4] 11w Flow cytometry analysis was performed using a Beckman Coulter Fc500 cytometer.

[5] 90w Cell counting one hundred thousand events per sample were acquired with fluorescence measured on a logarithmic scale data analysis was performed with the software Cyflogic V.1.2.1 (http://www.cyflogic.com). The gates of direct incidence (FS-forward scatter) and perpendicular (SS-side scatter) light scattering were used to exclude dead cells. Negative controls were set up in the absence of primary antibodies or with PI in the absence of both, primary and secondary antibodies. An argon laser line was used for fluorescence excitation of Alexa 488. Compensation was defined with reference to single color controls.

[6] 104w Cells extracts obtained in RIPA buffer containing protease inhibitors (sodium pyrophosphate, sodium fluoride, phenylmethanesulfony fluoride, pepstatin A, aprotinin, leupeptin, antipain, benzamidine -all reagents from Sigma-Aldrich) were used for Western-blot assays. Fifty micrograms of total protein were resolved on a SDS-polycrylamide gel and electroblotted onto nitrocellulose membranes. The blots were probed with specific primary antibodies for P2X2 (1:200, Calbiochem, San Diego, CA, USA) and P2X7 receptor (1:500, Calbiochem) and ␤-actin (Sigma; 1:2000) expression. Immunostaining was performed with secondary antibodies conjugated to horseradish peroxidase (1:2000 HRP, Jackon Laboratories, West-Grove, USA). The reaction was developed with Western Blot luminal reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

[7] 61w Data are reported as mean values ± standard deviation (S.D.) from data obtained in at least three independent experiments, with data from each independent RT-real time PCR experiment being determined at least in triplicate. Statistical differences among groups having received different treatments were detected by Student's ttest. Data of three independent experiments were analyzed by ANOVA with Tukey as post test.

[1] 237w P19 mouse EC cells (ATTC No. CRL-1822) were cultured and differentiated into neurons and glial cells as described previously (Tárnok and Ulrich, 2001). Briefly, P19 cell cultures were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Cultilab, Campinas, Brazil), 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM l-glutamine and 2 mM sodium pyruvate. For induction of neural differentiation, 5 × 10 5 P19 cells/ml in defined medium, containing DMEM medium supplemented with 2 mM glutamine, 2 mM sodium pyruvate, 2.4 mg/ml sodium bicarbonate, 5 g/ml insulin, 30 g/mL human apo-transferrin, 20 M ethanolamine, 30 nM sodium selenite, 100 U/ml penicillin, 100 g/ml streptomycin, and 10 mM HEPES, pH 7.2, were treated with 1 M "all-trans" retinoic acid and plated into bacterial dishes previously coated with 0.5% agarose, to avoid adhesion of the cell culture to plastic surfaces (Martins et al., 2005). After 2 days of culture in suspension in the presence of RA, P19 cells formed embryonic bodies stages (EBs). EBs were collected from suspension cultures replated in adherent culture flasks in DMEM medium with 10% FBS and cultured for 48 h. Serum-containing medium was replaced with defined medium on day 4, followed by two more days of culture until neuronal maturation was completed as determined by neuron-specific protein expression (neurofilament-200 and beta-3 tubulin) (Martins et al., 2005). Glial cells were detected by immunostaining against GFAP.

[2] 247w 5-Bromo-2 -deoxyuridine (BrdU) incorporation by P19 cells on day 5 of differentiation was assessed as a measure for the percentage of cells in proliferation. Cell cultures were exposed to 0.2 M BrdU (Roche Applied Science, Indianapolis, USA), 14 h before analysis. The adherent cells grown in culture flasks (Nunclon 75 cm 3 , Switzerland) were trypsinized, followed by the addition of fetal calf serum (FCS) to neutralize the enzymatic action, and centrifuged for 5 min at 200 × g. Pellets were washed twice with phosphate-buffered saline (PBS). Then cells were resuspended in one part of cold PBS (5 × 10 4 cells/ml), followed by sequential addition of three parts of cold absolute ethanol, while cells were thoroughly mixed after each ethanol addition. For fixation, cells were maintained for at least 4 h at 4 • C protected from light. Following another washing step, cells were incubated for 20 min at room temperature in the dark under gentle shaking in a solution of 2 M HCl and 0.5% Tween 20 for DNA denaturation. After centrifugation, samples were resuspended in 0.1 M sodium tetraborate (pH 8.5) for neutralization of HCl. This procedure was followed by another centrifugation and two washing steps with PBS. The samples were resuspended and incubated for 1 h at room temperature with a 1:200 anti-BrdU antibody (AXYLL, Westbury, NY, USA) dilution. Following two further washing steps immunostaining was verified following incubation with the anti-rat Alexa Fluor 488-coupled secondary antibody (1:500) (Molecular Probes, Eugene, OR, USA)

[3] 124w For flow cytometry experiments, differentiated P19 cells were collected by centrifugation for 10 min at 200 × g and dissociated into a single-cell suspension using trypsin. The experimental procedure of experiments and analysis were essentially the same as described by Schwindt et al. (2011). Briefly, cells were incubated with 1:500 dilutions of primary antibodies for neural markers (beta-3 tubulin, Sigma. GFAP, Dako, Carpinteria, CA, USA). Alexa-Fluor 555 and Alexa-Fluor 488 (1:500) (Molecular Probes) secondary antibodies were used for detection of beta-3 tubulin and GFAP expression, respectively. Data were recorded by using a flow cytometer (Beckman Coulter, Fc500, Fullerton, CA, USA). An argon laser was used for fluorescence excitation of Alexa 555 and Alexa 488. Data were analyzed using the Cyflogic software available at http://www.cyflogic.com.