WO2008100168A1 - Method for the functional identification of new neurons, neural progenitors, astrocytes and immature cells from stem cell cultures and uses thereof - Google Patents

Abstract

The present invention relates to a method for the functional identification of new neurons, neural progenitors, astrocytes and immature cells from stem cell cultures and pharmacological characterization of different cell types differentiating from stem cell cultures comprising the steps of : a) stimulating said stem cells cultures with compounds able to increase the intracellular calcium concentrations specifically in neurons, b) stimulating said stem cells cultures with compounds able to increase the intracellular calcium concentration specifically in immature and neural progenitor cells, c) monitoring intracellular calcium concentrations by the use of a probe, and d) and using ratios of fluorescence values following stimulations to the direct evaluation of the level of differentiation of cells. The invention refers also to the use of the method of the invention in laboratorial or pharmacological studies, for example on undifferentiated nestin positive cells, GFAP positive nest in negative astrocytes, doublecortin positive neuroblasts, MAP-2 positive neurons, in different stages of differentiation.

Description

DESCRIPTION

"METHOD FOR THE FUNCTIONAL IDENTIFICATION OF NEW NEURONS,

NEURAL PROGENITORS, ASTROCYTES AND IMMATURE CELLS FROM

STEM CELL CULTURES AND USES THEREOF"

FIELD OF THE INVENTION

The present invention describes a method for the functional identification of neural populations based in single cell calcium imaging procedures. This method allow the rapid and simultaneous functional/morphological identification of the cell diversity in the populations, allowing subsequent pharmacological intervention in the identified cells. Moreover, this method is suitable for the screening of factors able to promote cell differentiation.

BACKGROUND OF THE INVENTION

Reynolds and Weiss (1992) [1] identified a resident population of stem cells in the SVZ of the mouse brain, endowed with proliferative and self-renewal capacities, and able to generate neurons and glial cells. Since then, stem cells have been identified in the SVZ of other mammalian species, including primates [2, 3] . SVZ- derived neuroblasts migrate along the rostral migratory stream towards the olfactory bulb (OB) , where they functionally differentiate into interneurons, involved in odour discrimination [4, 5, 6, 7, 8] .

Characterization of SVZ neurons and neuronal precursors is mostly performed by immunocytochemistry . SVZ cultures are mixed cultures of immature cells, neurons, astrocytes, oligodendrocytes, neuronal and glial progenitors, in different stages of differentiation [1, 9, 10] . Immature SVZ cells express the intermediate filament protein nestin [9, 11] . Early neuronal commitment is detected through the expression of the neuron-associated class III tubulin isotype β [12] . Migrating immature SVZ neuroblasts express doublecortin (DCX) , a microtubule- associated protein [13, 14], and the polysialylated neural cell adhesion molecule (PSA-NCAM) [15, 16] . As maturation proceeds, SVZ-derived postmitotic neurons express MAP-2, neurofilament (NF) , the enzyme neuron-specific enolase

(NSE) [17] and NeuN [18] . However, immunocytochemistry is time consuming, do not give information about function, and is performed on fixated, i.e. dead cells, hampering their subsequent use for therapeutic purposes.

Measurement of intracellular free calcium variations is useful to rapidly characterize functional cells. Indeed, membrane depolarisation of excitable cells, such as neurons, following exposure to high KCl concentrations, leads to the opening of voltage sensitive calcium channels (VSCC) and massive influx of calcium into the cytoplasm [19] . Immature/stem cells, such as embryonic stem cells, carcinoma and astrocytoma cells express functional histamine receptors [20, 21, 22, 23, 24, 25]. Although the specific role of histamine in neurogenesis and neuronal development is poorly understood, the specific expression of histamine receptors may be used as a marker for undifferentiated neural progenitors. Indeed, stimulation of immature/stem cells with histamine transiently increase [Ca²⁺Ji [20, 22, 26], including immature precursor cells from the postnatal and adult SVZ [25] .

The patent US 7,217,565 describes a method for the identification and isolation of cell populations from stem cells and progenitor cell cultures based in fluorescence activated cell sorting or high gradient magnetic selection, but using monoclonal antibodies conjugated with fluorochromes or conjugated to magnetic particles .

BRIEF PRESCRIPTION OF THE DRAWINGS

Figure 1. Experimental protocol to functionally evaluate neuronal differentiation in SVZ cell cultures. A: SVZ cultures were perfused continuously in Krebs solution during 15 minutes, and stimulated for 2 minutes from 5¹ to 7' with 50 mM KCl, and from 10' to 12 ' with 100 μM histamine. B: Changes in the 340/380 nm ratio of fluorescence. Images were taken from the same field and obtained at different time points. Left image is representative of non-stimulated cells (basal) , whereas right image shows cells upon stimulation with KCl. Scale of fluorescence intensity is indicated at the right : blue and red colours are indicative of low and high ratios, respectively. Observed fields contained around 100 cells. C: Accordingly to our working hypothesis, from all the cells analysed, specific profiles of response for neurons and immature cells were found.

Figure 2. Different profiles of [Ca²⁺Ji variations in SVZ- derived neurons, neuroblasts and astrocytes following stimulation with 50 mM KCl and 100 μM histamine. Representative fluorescent photomicrographs of MAP-2 positive neurons (A; red) , DCX positive neuroblasts (B; green) and GFAP positive astrocytes (C; green) in SVZ cultures and representative associated [Ca²⁺] ± variation profiles. Hoescht 33342 staining (blue) was used to visualize cell nuclei. Scale bar 20 μm. D: Hist/KCl values distribution according to the cell type. A total of 10 MAP- 2 neurons, 8 GFAP astrocytes and 10 DCX neuroblasts were analysed. Data are mean ± SEM. ***P<0.001 using ANOVA with Bonferroni ' s correction for multiple comparisons.

Figure 3. Representative fluorescent photomicrographs of nestin (A ,B, C; red) and GFAP positive cells (B, C; green) in SVZ cultures and the respective associated [Ca²⁺] ± variation profiles of nestin positive cells (A) co- expressing or not (C and B, respectively) GFAP, following stimulation with 50 mM KCl and 100 μM histamine. D: Fluorescent confocal photomicrograph of nestin (red) and GFAP (green) immunodetection in SVZ cell culture. Hoescht 33342 staining (blue) was used to visualize cell nuclei. Scale bar 20 μm.

Figure 4. [Ca²⁺Ji increase in immature SVZ cells following histamine stimulation is mediated by the histamine 1 receptor activation. A, left: RT-PCR detection of Histamine 1 (HlR) and 2 (H2R) receptors in mouse SVZ cells [+: positive controls were done in total murine spleen mRNA] .

Right: fluorescent confocal photomicrograph of nestin (red) and HlR (green) immunodetection. Hoescht 33342 staining

(blue) was used to visualize cell nuclei. Scale bar 20 μm.

Arrows depict double labeling. B: SVZ cultures were perfused continuously in Krebs solution during 20 minutes and stimulated for 2 minutes from 5' to 7 ' with 50 mM KCl, from 10' to 12' with 100 μM histamine and from 15' to 17' with concomitantly 100 μM histamine and either 1 μM mepyramine or 50 μM cimetidine. C: Representative profiles of response of immature SVZ cells following perfusion according to the previously presented protocol. D: Effect of mepyramine and cimetidine on the percentage of immature SVZ cells responding to histamine stimulation (n=3). Data are mean ± SEM. ***P<0.001 using ANOVA with Bonferroni ' s correction for comparison with the response to histamine alone .

Figure 5. Functional and phenotypic evaluation of neuronal differentiation in SVZ cells. A: Bargram depicts percentages of neuronal -like responding cells in SVZ control cultures (no drugs exposure) and in cultures exposed to 20 ng/ml SCF or 10 ng/ml LIF for 7 days. Positive and negative controls were performed following the application of the same protocol of perfusion in hippocampal enriched neuronal cultures (Hipp cells) and cortex glial cultures (Glial cells) (n=2-13) . Data are mean ± SEM. *P<0.05 using ANOVA with Bonferroni ' s correction for comparison with SVZ control cultures. B: Representative fluorescent photomicrographs of NeuN positive neurons (red nucleus) and Hoescht staining (blue nucleus) in SVZ cells. Increased NeuN immunoreactivity was observed in SCF-treated cultures as compared with the control cultures. Bargram depicts the percentage of NeuN-positive neurons, expressed as the percentage of total number of cells, in control cultures and in cultures treated with 20 ng/ml SCF or 10 ng/ml LIF for 7 days (n=6-7) . Data are mean ± SEM. ***P<0.001 using ANOVA with Bonferroni ' s correction for comparison with SVZ control cultures.

SUMMARY OF THE INVENTION

In the present invention, we describe a rapid method to functionally characterize neuronal differentiation, in SVZ cultures, based on the specific

[Ca²⁺] i variations, according to cell type, following KCl depolarization and stimulation with histamine. DETAILED DESCRIPTION OF THE INVENTION

Subventricular zone cell cultures contain mixed populations of immature cells, neurons, astrocytes and progenitors in different stages of development. In the present invention, we examined whether cell types of the SVZ could be functionally discriminated on the basis of intracellular free calcium level ( [Ca²⁺] i) variations following KCl and histamine stimulation. For this purpose, [Ca²⁺] i were measured in SVZ cell cultures from neonatal Pl-3 C57BL/6 donor mice, in single cells, after stimulation with 100 μM histamine or 50 mM KCl. MAP-2 -positive neurons and doublecortin positive neuroblasts were distinguished on the basis of their selective ratio of response to KCl and/or histamine stimulation. Moreover, we could distinguish immature cells on the basis of the selective response to histamine via the histamine 1 receptor activation.

Exposure of SVZ cultures to the pro-neurogenic factor SCF induced an increase of the number of cells responding to KCl and a decrease in the number of cells responding to histamine, consistently with neuronal differentiation. The selective response to KCl/histamine in single cell calcium imaging analysis offers a rapid and efficient way for the functional discrimination of neuronal differentiation in SVZ cell cultures, opening new perspectives for the search of new potential pro-neurogenic factors . Objects of the Invention

The first object of the invention is a method based on single cell imaging for the functional identification of new neurons, neural progenitors, astrocytes and immature cells from stem cell cultures and pharmacological characterization of different cell types differentiating from stem cell cultures comprising the steps of: a) stimulating said stem cells cultures with compounds able to increase the intracellular calcium concentrations specifically in neurons, b) stimulating said stem cells cultures with compounds able to increase the intracellular calcium concentration specifically in immature and neural progenitor cells, c) monitoring intracellular calcium concentrations by the use of a probe, and d) and using ratios of fluorescence values following stimulations to the direct evaluation of the level of differentiation of cells.

In a preferred embodiment, the stem cell cultures are obtained from neural tissue.

In another preferred embodiment, the stem cell cultures are obtained from the subventricular zone of mammals.

Preferably, the probe is a calcium-sensitive fluorescent probe, more preferably Fura 2 -AM.

Usually, the evaluation of the level of differentiation of cells is based on the cell type specific increase in intracellular calcium concentrations following cell stimulations specific for neurons and cell stimulations specific for immature cells and neural progenitors, the previous monitored by means of the calcium probe Fura-2AM.

Preferably, the cell stimulations specific for neurons include exposure of cells to a solution containing high extracellular KCl concentrations, and said cell stimulations specific for immature cells and neural progenitors include exposure of cells to a solution containing histamine.

The cells responding to high extracellular KCl concentrations by increasing intracellular calcium levels and few or not responding to histamine, and therefore with low histamine/KCl ratio of response, include, generally, MAP-2 positive and doublecortin positive cells.

In a preferred embodiment, the cells responding to histamine and not to high extracellular KCl concentrations include nestin positive cells.

Normally, the GFAP positive, nestin negative cells do not respond neither to said cell stimulations specific for neurons nor to said cell stimulations specific for immature cells and neural progenitors. The second object of the invention is use of the method of the invention in laboratorial or pharmacological studies .

In a preferred embodiment, the method is used in pharmacological studies on undifferentiated nestin positive cells, GFAP positive nestin negative astrocytes, doublecortin positive neuroblasts, MAP-2 positive neurons, in different stages of differentiation.

In another preferred embodiment, the method is used for the screening of factors inducing cell differentiation from stem cell cultures.

In another preferred embodiment, the method is used for the culture of neural stem cells in the presence of candidate factors inducing cell differentiation and subsequent evaluation of increase in the percentage of differentiated cells.

In another preferred embodiment, the method is used for the culture of neural stem cells in the presence of putative proneurogenic factors and subsequent evaluation of the increase in percentage of new neurons.

Materials and Methods

All experiments were performed in accordance with NIH and European (86/609/EEC) guidelines for the care and use of laboratory animals. SVZ cultures

SVZ cells were obtained from 1 to 3 -day old C57B1/6 donor mice. Brains were removed following decapitation and placed in HBSS solution (Gibco, Rockville, MD, USA) . Fragments of SVZ were dissected out of 450 μm- thick coronal brain sections, digested in 0.025% trypsin and 0.265 mM EDTA (Gibco) and dissociated by gentle trituration with a PlOOO pipette. The cell suspension was diluted in serum- free culture medium (SFM) composed of Dulbecco's modified eagle medium (D-MEM/F12 + GlutaMAXTM-I , Gibco) supplemented with 100 U ml penicillin 100 μg ml streptomycin (Gibco) , 1% B27 (Gibco) , 10 ng ml epidermal growth factor (EGF; Gibco) and 10 ng ml basic fibroblast growth factor (FGF-2 ; Gibco) . Single cells were then plated on uncoated Petri dishes at a density of 3000 cells cm2. The neurospheres were allowed to develop in a 95% air-5% CO2 humidified atmosphere at 37 ⁰C.

6 to 8 days after plating, the neurospheres were collected with a Pasteur pipette and seeded onto poly-D- lysine coated glass coverslips placed into 12-well cell culture plates for calcium imaging experiments, or 24 -well cell culture plates for immunocytochemistry, and covered with ImI or 500 μl, respectively, of SFM devoid of growth factors .

For the experiments correlating single cell calcium imaging (SCCI) with immunodetection, SVZ neurospheres were seeded onto poly-D-lysine coated microgrid coverslips (Eppendorf CELLocate ^® coverslip, Hamburg, Germany) .

The neurospheres were allowed to develop during 7 days at 37 ⁰C in the absence or the presence of 10 ng/ml LIF or 20 ng/ml SCF (both from Chemicon International, Temecula, USA) .

In all experiments, each experimental condition was assayed in three different wells. Except where otherwise specified, the experiments were replicated three times .

Single Cell Calcium Imaging

To determine the differentiation pattern of SVZ cells, we analysed the variations of intracellular calcium levels following stimulation with 50 rtiM KCl and 100 μM histamine (Sigma) , in accordance to the sequence shown in Figure 1 A. KCl-depolarization causes the increase of the intracellular calcium levels in neurons [19] whereas stimulation with histamine leads to the increase of intracellular calcium levels in stem/progenitor cells [25] . SVZ cultures were loaded for 40 min, at 37 °C, with 5 μM Fura-2 AM (Molecular Probes, Eugene, OR, USA), 0.1% fatty acid free BSA and 0.02% pluronic acid F-127, in Krebs (132 mM NaCl, 1 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4) . After a 10 min post-loading period at room temperature the glass coverslip was mounted on RC-20 chamber in a PH3 platform (Warner Instruments) on the stage of an inverted fluorescence microscope Axiovert 200 (Carl Zeiss) . Cells were continuously perfused with Krebs solution and stimulated by applying 100 μM histamine or high potassium Krebs solution (containing 50 mM KCl, isosmotic substitution with NaCl) . Solutions were added to the cells by a fast-pressurized (95% air, 5% CO2 atmosphere) system (AutoMate Scientific, Inc) The intracellular calcium concentration ([Ca2+]i) was evaluated by quantifying the ratio of the fluorescence emitted at 510 nm following alternate excitation (750 msec) at 340nm and 380nm, using a Lambda DG4 apparatus (Sutter Instruments Company), and a 510 nm bandpass filter (Carl Zeiss ref . 21) before fluorescence acquisition with a 40x objective and a Coll SNAP digital camera (Roper Scientific) . Acquired values were processed using the MetaFluor software (Universal Imaging Corporation) .

Histamine/KCl values for Fura-2 ratio were calculated to determine the extent of neuronal maturation in cultures. The results obtained in SVZ cultures were compared with those obtained in cortical glial cell cultures or in cultured hippocampal neurons [27] .

To determine which histamine receptor was involved in the effect of histamine, we analysed the variations of [Ca2+] i following perfusion of the cells with 100 μM histamine concomitantly with either lμM mepyramine or with 50 μM cimetidine (both from Tocris, Ellisville, MO, USA) , specific antagonists for the histamine receptors 1 (HlR) and 2 (H2R) , respectively [28] in agreement with the sequence shown on Figure 1 B.

Immunocytochemistry

After fixation, for 1 h, in 4% paraformaldehyde, cells were permeabilized and non-specific binding sites were blocked, for lh30, with 0.25% Triton X-100 (Sigma) and 6% bovine serum albumin (BSA, Sigma) dissolved in PBS. Cells were then subsequently incubated with the following primary antibodies: mouse monoclonal anti-nestin (1:200; Chemicon International), mouse monoclonal anti-MAP-2 antibody (1:200; Sigma), rabbit monoclonal anti-GFAP antibody (1:1000; Sigma), mouse monoclonal anti-NeuN antibody (1:100, Chemicon International), rabbit polyclonal anti-doublecortin antibody (1:200, Cell Signaling, Danvers ; MA, USA), overnight at 4 °C. Thereafter, the coverslips were rinsed in PBS and incubated, for 1 h at room temperature, with the secondary goat anti -rabbit Alexa Fluor 488 antibody (1:200, Molecular Probes) or goat anti- mouse Alexa Fluor 594 antibody (1:200, Molecular Probes), respectively. After rinsing with PBS, cell preparations were incubated with Hoescht 33342 (2μg/ml, Molecular Probes) in PBS containing 0.25 % BSA, 5 min at room temperature, for nuclear staining. Finally, the preparations were mounted using Dakocytomation fluorescent medium (Dakocytomation Inc., California, USA). Fluorescent images were recorded using a digital camera coupled to, an Axioskop microscope (Carl Zeiss, Gόttingen, Germany) .

Isolation of total RNA from mouse SVZ cells

Total RNA was isolated from SVZ cells using TRI REAGENT (Sigma) according to manufacturer's instructions. Cells were gently homogenized in guanidium thiocyanate and phenol. Chloroform was added allowing a clear isolation of RNA in the resultant aqueous phase. The RNA was precipitated with isopropanol and the pellet washed with 75% ethanol . The pellet was eluted in diethylpyrocarbonate (DEPC)- treated water.

The total amount of RNA was quantified by optical density (OD) measurements at 260 nm, and the purity was evaluated by measuring the ratio of OD at 260 and 280 nm

(RNA/DNA calculator GeneQuant II, Pharmacia Biotech Amersham Biosciences AB, Uppsala, Sweden) . Additionally, RNA quality was assessed by gel electrophoresis.

RNA extracted from splenocytes was used as a positive control for the detection of histamine receptors 1 and 2 expression.

RT-PCR analysis

Histamine receptors 1 and 2 mRNA expression was determined by reverse transcription-PCR (RT-PCR). First, cDNA was obtained from the transcription of 2 μg RNA using avian microblastosis virus (AMV) reverse transcriptase and Oligo-p (dT) 15 primers (Roche Molecular Biochemicals, Indianapolis, IN, USA) . PCR was performed in a 50 μL reaction system (Roche Molecular Biochemicals) containing 5 μL template cDNA, 1 μL deoxynucleotide mix, 5 μL 10x PCR reaction buffer, 0.2 μL upstream and 0.2 μL downstream primer, a variable volume of water and 0.25 μLTaq DNA polymerase (35 cycles: at 95°C for 30 s, at 56°C/58°C (histamine receptor l/histamine receptor 2) for 30 s and at 72 °C for 30 s) (BIORON GmbH, Ludwigshafen, Germany)).

Primers used in PCR reactions were as follows: Histamine receptor 1: forward primer 5'-GGG CTC AAA GGC CAA TGA C-3¹ and reverse primer 5'-TCC GCC GGC AAG TAC TCA-3 ^■ . Histamine receptor 2: forward primer 5'-CTG GCT GTC AGC TTG AAT CG-3 ' and reverse primer 5'-GCT GCC AGG GAC ACA ATG A- 3'. β-actin: forward primer 5'-GAC TAC CTC ATG AAG ATC CT- 3' and reverse primer 5'-ATC TTG ATC ATG GTG CTG-3 ' (MWG- Biotech AG, Ebersberg, Germany) [29] .

PCR products of each sample were subjected to electrophoresis in a 1.5% agarose gel and stained with ethidium bromide. Photographs were taken in a Versa-Doc

Imaging System (Model 3000, Bio-Rad Laboratories, Hercules,

CA, USA) . Data analysis and statistics

Percentage of NeuN immunoreactive cells were calculated from cell counts in 5 independent fields in each coverslip with a 4Ox objective (about 200 cells per field) .

Because no significant difference were found between experiments, the corresponding data were pooled and expressed as mean ± SEM. Statistical significance of differences was examined by one-way ANOVA followed by the Bonferroni post-test for multiple comparisons, or unpaired t-test. Statistical significance level was set for P-values

< 0.05.

Results

Variations in [Ca²⁺] j following stimulation with KCl or histamine

We characterized the profile of [Ca²⁺] i variations in single SVZ cells upon KCl and histamine stimulation. 6 to 8 day old neurospheres were plated onto poly-D- lysine coated coverslips, and allowed to develop during 7 days in SFM medium devoid of growth factors. The cells were then loaded with the Fura-2 AM calcium probe, perfused continuously for 15 min with Krebs solution and shortly (2 min) stimulated with 50 mM KCl or with 100 μM histamine as shown in Figure 1 A. Changes in 340/380 nm ratios of fluorescence were monitored constantly during all the experiment . Figure 1 B provides an example of [Ca²⁺] ± variations in SVZ cells upon KCl stimulation. Interestingly, some cells display a rise in the [Ca²⁺Ji following KCl stimulation but not following histamine perfusion, consistent with a neuronal-like profile. Moreover, an immature-like profile, characterized by an absence of response to KCl but a rise in [Ca²⁺] i following histamine stimulation was also found (Figure 1 C) .

To associate profiles of [Ca²⁺] i variations with the respective cell phenotype, we monitored [Ca²⁺] i responses using single cell calcium imaging and then performed subsequent immunocytochemical characterization on

SVZ cells cultivated on microgrid coverslips, allowing cell localization. We show that the neuronal-like profile of [Ca²⁺] i variations is displayed by MAP-2 expressing neurons

(Figure 2 A) . Another population of cells responding to

KCl, however weakly than MAP-2 positive neurons, and with minor response to histamine, expresses the immature neuronal marker DCX (Figure 2 B) . Cells responding neither to KCl nor to histamine are GFAP-positive astrocytes

(Figure 2 C) .

We then calculated the ratio of responses due to histamine or KCl exposure (Hist/KCl) in a total of 10 MAP-2 positive neurons, 10 DCX-positive neuroblasts and 8 GFAP expressing astrocytes. The Hist/KCl values were significantly different between groups, as shown in Figure 2 D, indicating that following KCl and histamine stimulation, SVZ-derived neurons and neuroblasts can be discriminated according to the Hist/KCl ratio. Additionally, ratios displayed by both mature and immature neurons were significantly lower than the ones found in GFAP positive cells, since these cells do not respond to stimulation with histamine or with KCl, and hence display a normalized Hist/KCl ratio close to one. Thus, neuronal cells can be undoubtedly discriminated from glial cells on the basis of their Hist/KCl ratio.

Similar single cell calcium imaging experiments were performed, in SVZ cultures subsequently stained for the marker of immature cells, nestin. As shown in Figure 3 A, cells responding to histamine and not to KCl express nestin, suggesting that [Ca²⁺Ji response following histamine stimulation is restricted to immature/undifferentiated cells, in SVZ cultures. However, nestin is expressed by a wide range of SVZ cell types [30] . Accordingly, nestin positive cells expressing GFAP, a marker for mature astrocytes, are found in SVZ cultures (Figure 3 D, cell b) , as well as nestin⁺/GFAP^~ (Figure 3 D, cell a) and nestin^" /GFAP⁺ (Figure 3 D, cell c) cells. Single cell calcium imaging experiments were then conducted in SVZ cultures followed by double immunodetection of nestin and GFAP markers. Immature-like profile of [Ca²⁺Ji was found associated with both nestin⁺/GFAP^" cells as well as nestin⁺/GFAP⁺ cells (Figure 3 B and C, respectively) .

All the cells analysed responding to histamine displayed immunoreactivity to nestin (n=12), but not all the nestin-immunoreactive cells were found to respond to histamine stimulation (n=10) . This is in accordance with the well established fact that nestin is not a selective marker of immature cells since it is also expressed by SVZ- derived differentiated cells [30] . These results further demonstrate that increase in [Ca²⁺] i following histamine stimulation is restricted to SVZ immature/undifferentiated cells. Furthermore, mature neurons can be discriminated from immature cells according to their low and high Hist/KCl ratio, respectively.

Increase in [Ca²⁺] i in SVZ immature cells is mediated by histamine Hl receptor activation

To further investigate which histamine receptor was involved in histamine- induced [Ca²⁺Ji responses, we performed RT-PCR to detect histamine receptors HlR and H2R in SVZ cells. mRNA for both receptors were found in SVZ cells (Figure 4 A) .

To evaluate the involvement of each receptor in the increase of [Ca²⁺] i following histamine stimulation, we performed single cell calcium imaging studies in the presence of specific antagonists for HlR and H2R. Cells were continuously perfused with Krebs solution, for 20 min, and stimulated. with KCl, histamine or with histamine plus 1 μM mepyramine, an HlR antagonist, or with 100 μM histamine plus 50 μM cimetidine, an H2R antagonist (Figure 4 B) . Representative profiles of response of immature SVZ cells are shown in Figure 4 C. Moreover, we also calculated the ratios of response following exposure to histamine and histamine plus antagonist. Accordingly, we evaluated the percentage of histamine-sensitive SVZ cells that still responded to histamine in the presence of the antagonists

(ratio histamine/histamine + antagonist close to 1) .

Following stimulation with mepyramine, a significant decrease of 82% (n=3) in the number of immature cell responding to histamine was observed, whereas stimulation with cimetidine did not block [Ca²⁺Ji responses induced by histamine (n=3) (Figure 4 D) .

Moreover, by performing double immunocytochemistry against HlR and nestin, we showed that immature SVZ cells express the histamine 1 receptor (Figure 4 A; right image) .

Variations of [Ca²⁺Jj in neurons, glia and precursor cells following stimulation with KCl or histamine: evaluation of neuronal differentiation

To validate the method described in the present invention, we performed single cell calcium imaging experiments on phenotypically identified cells, i.e. on neuronal hippocampal primary cultures and on cortical glia cell cultures. In control cultures, 20% of cells display a neuronal-like profile, i.e. with a Hist/KCl ratio below 0.8 (n=13) (Figure 5 A) . In hippocampal cultures, 40% (n=3) of the cells display a low Hist/KCl ratio (below 0.8) (Figure 5 A) , as expected for a primary culture enriched in a neuronal phenotype . Unlikely, in cortical glial cell cultures, we did not observe any cells showing a low Hist/KCl ratio (n=2) (Figure 5 A) .

We then used this method to evaluate neuronal differentiation in SVZ cultures pre-treated with the pro- neurogenic factor SCF [31] or with the gliogenic factor LIF

[32, 33, 34, 35] . For this purpose, we treated the cultures with 20 ng/ml SCF or 10 ng/ml LIF, during 7 days. A 25%

(n=8) significant increase in the proportion of SVZ cells presenting a neuronal -like profile was observed in SVZ cultures treated with SCF (Figure 5 A) . In contrast, following treatment with LIF, the relative number of neurons decreased, although not significantly (n=6) (Figure 5 A) . Consistently, treatment with SCF increased the number of NeuN positive neurons to 25% (n=7) as compared to 9% in the control (n=6) (Figure 5 B) . Treatment with LIF did not modify neuronal differentiation (n=6) .

Discussion

In the present invention, we describe a rapid method to functionally evaluate neuronal differentiation in SVZ cell cultures. This method is based on the profiles of

[Ca²⁺Ii variations, according to cell type, following KCl depolarisation and stimulation with histamine. Hence, KCl depolarisation was used as a feature of neuronal differentiation, since VSCC are absent or expressed at very low levels in neural precursors, whereas during neuronal differentiation and in differentiated neurons there is expression of high levels of these channels [36, 37, 38] . In agreement, we observed that following KCl perfusion, there is an increase in [Ca²⁺] _± in neuronal cells due to the opening of VSCC, whereas in immature cells or glial cells the [Ca²⁺Ji remained unchanged. On the other hand, histamine perfusion increases [Ca²⁺Ji iⁿ SVZ immature cells [25] but not in neurons, nor in glial cells.

We monitored [Ca²⁺] i rises using single cell calcium imaging and correlated these data with subsequent immunocytochemical characterization of the same imaged cells. [Ca²⁺Ji increase following KCl perfusion but not following histamine stimulation was associated with MAP-2 positive neurons, whereas cells responding to KCl and with minor response to histamine are DCX-positive immature neuronal precursors. Cells where [Ca²⁺] i was not significantly affected by either KCl or histamine were identified as GFAP-positive astrocytes. The analysis of the ratio of responses obtained following histamine and KCl perfusion (Hist/KCl) allow us to conclude that cells presenting a Hist/KCl ratio value below 0.80 are mature neurons, phenotypically identifiable by MAP-2 expression. Cells responding only to histamine and thus displaying a high Hist/KCl ratio (higher than 1) were identified as immature nestin positive cells. GFAP expression was found in cells neither responding to KCl nor to histamine, consistently with astrocytic phenotype, as well as in a subpopulation of cells responding to histamine and co- expressing nestin.

Based on the Hist/KCl ratio, we calculated the percentage of cells showing neuronal-like profile, in cortical glial cell cultures, in neuronal -enriched hippocampal cultures and in SVZ cultures. No cells presenting a neuronal-like response were found in cortical glial cell cultures, whereas 40% were found in postnatal neuronal hippocampal cultures. These data strongly indicate that the method now described reliably allows the identification of neurons and neuronal differentiation in SVZ cultures. In non-treated SVZ (control) cultures, about 20% of cells show a neuronal-like response. Pre-treatment of SVZ cells with SCF, a trophic factor reported to stimulate neurogenesis in an in vitro model of cerebral ischemia, as well as in basal conditions in vivo [31] , increased the percentage of cells with a neuronal-like response, as compared to control cultures. In contrast, pre-treatment of SVZ cultures with LIF, a multifunctional cytokine reported to promote self-renewal of neural stem cells or alternatively the differentiation of neural/progenitor cells into GFAP immunoreactive cells

[32, 33, 34, 35, 39], decreased, although not significantly, the percentage of cells with a neuronal-like response, as compared to control cultures. Differentiated neurons expressing NeuN were quantified in each condition and the obtained data corroborated the pro-neurogenic potential of SCF, in postnatal SVZ culture. Thus, the procedure that we describe is fully adapted for the screening of putative pro-neurogenic factors. Moreover, this method characterises, although indirectly, glial and immature cells. Indeed, cells responding neither to KCl nor to histamine are GFAP astrocytes, whereas cells only responding to histamine express nestin. A subpopulation of nestin positive cells, responding only to histamine, was found to be GFAP positive consistently with the wide expression of nestin among cells of the SVZ, including a subpopulation of astrocytes [30] .

Using RT-PCR and histamine receptor-selective inhibitors we show that the increase of the [Ca²⁺] _± in SVZ cells following stimulation with histamine is mediated by the activation of HlR. Indeed, it was already shown by others that embryonic stem and carcinoma cells express functional histamine 1 receptors, suggesting a role for this receptor as a marker for undifferentiated neural progenitors [20, 21, 24, 25].

Our data demonstrate that the monitoring of the increase in the [Ca²⁺Ii following KCl depolarization and stimulation with histamine is a powerful and reliable tool to functionally evaluate neuronal differentiation in SVZ cell cultures. SVZ culture is a mixed culture of neurons, astrocytes, oligodendrocytes, neuronal and glial progenitors in different stages of differentiation, and stem/immature cells [1, 9, 10] . We firstly cultivated single SVZ cells with EGF and FGF-2 during 7 days to allow the formation of neurospheres, i.e. to increase cell proliferation. Plating onto poly-D-lysine as well as withdrawal of growth factors are necessary steps to drive the differentiation of progenitor cells into either glial or neuronal fate [40] . Free floating neurospheres adhere to the poly-D-lysine substrate and cell differentiation occurs in the border of the neurospheres, where migrating cells emerge, forming a dense carpet of cells. All the measurements of [Ca²⁺] ± variations and immunocytolabelling were performed in these cells. In spite of the fact that cells in the selected area might be more differentiated, it is also true that these cells are not only phenotypically diverse (glia versus neurons) but are also different concerning their developmental stages. Indeed, in SVZ cultures, neurons are present in different developmental stages, so it is possible to find markers for mature neurons such as NeuN and MAP-2, as well as markers for immature cells, such as DCX and nestin.

Evaluation of neuronal differentiation by immunocytochemistry involves time consuming procedures performed in dead cells. Single cell calcium imaging studies provide a rapid method to evaluate neuronal function. Functionality can be analyzed by electrophysiology, looking at the ability of neurons to generate currents following stimulation. However, patch- clamp approaches are time consuming and allow recordings from one cell each time, making impossible estimation of neuronal differentiation in the overall culture. This is in clear contrast with the possibility of imaging between 100- 120 cells, at the same time, in 15 min, using single cell calcium imaging.

Another report described the single isolation of immature cells, neuronal and glial progenitors from the embryonic rat telencephalon [41] . In this study, the authors used fluorescent-activated cell sorting and the combination of calcium responses to basic FGF or EFG and subsequent immunocytochemistry . However, instead of the strong response of SVZ immature cells to histamine detected using our method, Barker and collaborators isolated neural stem cells by negative flow cytometric selection for 5 surface epitopes. Moreover, using calcium imaging in embryonic stem cells, the cellular response to basic FGF via FGFRl receptor activation, was not specific for neural precursors since some neuronal and glial progenitors also respond with an increase of [Ca²⁺] i to basic FGF [41] . Our method, using specific increase of [Ca²⁺] i following histamine perfusion, prove to be faster and does not involved expensive FACS methods.

In conclusion, monitoring the increase of [Ca²⁺] i following the application of KCl or histamine, allowed us to design a rapid method to evaluate neuronal differentiation in living SVZ cultures. This method is based on the different patterns of increase in the [Ca²⁺Ji, in SVZ cells, following KCl or histamine stimulation, allowing the characterization of an immature but functional, population of neurons. Moreover, this method allows the rapid screening of putative pro-neurogenic factors that might be relevant for cell -based therapy, as it allows the identification of functional immature neurons; cells that are more plastic than fully- differentiated neurons and so are more adequate for efficient neuronal replacement following grafting in the diseased brain.

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Claims

1. A method based on single cell imaging for the functional identification of new neurons, neural progenitors, astrocytes and immature cells from stem cell cultures and pharmacological characterization of different cell types differentiating from stem cell cultures comprising the steps of: a) stimulating said stem cells cultures with compounds able to increase the intracellular calcium concentrations specifically in neurons, b) stimulating said stem cells cultures with compounds able to increase the intracellular calcium concentration specifically in immature and neural progenitor cells, c) monitoring intracellular calcium concentrations by the use of a probe, and d) and using ratios of fluorescence values following stimulations to the direct evaluation of the level of differentiation of cells.

2. The method of claim 1, wherein said stem cell cultures are obtained from neural tissue.

3. The method of claim 2, wherein said stem cell cultures are obtained from the subventricular zone of mammals.

4. The method of claim 1, wherein said probe is a calcium-sensitive fluorescent probe.

5. The method of claim 4, wherein said calcium- sensitive fluorescent probe is Fura 2 -AM.

6. The method in claim 1, wherein said direct evaluation of the level of differentiation of cells is based on the cell type specific increase in intracellular calcium concentrations following cell stimulations specific for neurons and cell stimulations specific for immature cells and neural progenitors, the previous monitored by- means of the calcium probe Fura-2AM.

7. The method of claim 6, wherein said cell stimulations specific for neurons include exposure of cells to a solution containing high extracellular KCl concentrations, and said cell stimulations specific for immature cells and neural progenitors include exposure of cells to a solution containing histamine.

8. The method of claim 7, wherein cells responding to high extracellular KCl concentrations by- increasing intracellular calcium levels and few or not responding to histamine, and therefore with low histamine/KCl ratio of response, include MAP-2 positive and doublecortin positive cells.

9. The method of claim 7, wherein cells responding to histamine and not to high extracellular KCl concentrations include nestin positive cells.

10. The method of claim 1 or 7 , wherein GFAP positive, nestin negative cells do not respond neither to said cell stimulations specific for neurons nor to said cell stimulations specific for immature cells and neural progenitors .

11. Use of the method of any one of claims 1 to 10 in pharmacological studies on undifferentiated nestin positive cells, GFAP positive nestin negative astrocytes, doublecortin positive neuroblasts, MAP-2 positive neurons, in different stages of differentiation.

12. Use of method of any one of claims 1 to 10 for the screening of factors inducing cell differentiation from stem cell cultures.

13. Use of the method of claims 1 to 10 for the culture of neural stem cells in the presence of candidate factors inducing cell differentiation and subsequent evaluation of increase in the percentage of differentiated cells .

14. Use of the method of claims 1 to 10 for the culture of neural stem cells in the presence of putative proneurogenic factors and subsequent evaluation of the increase in percentage of new neurons .