Negative Regulation of Neural Stem/Progenitor Cell Proliferation by the Pten Tumor Suppressor Gene in Vivo Matthias Groszer, Rebecca Erickson, Deirdre Scripture-Adams, Ralf Lesche, Andreas Trumpp, Jerome A. Zack, Harley I. Kornblum, Xin Liu, Hong Wu

Mice. The morning when the vaginal plug was detected was defined as embryonic day 0.5 (E0.5) and the day of birth as P0. All animals were maintained in specific pathogen-free conditions according to the regulations of the Department of Laboratory Animal Medicine at University of California, Los Angeles.

Histology and Immunohistochemistry of Tissue Sections. Haematoxylin-Eosin staining was performed on formalin-fixed, paraffin-embedded 4m brain serial sections according to standard procedures. Immunohistochemistry was performed on frozen sections using antibodies GluR1 (upstate biotechnology, 0.5g/ml) and TuJ1 (1:500; Berkely Antibodies) as described previously (1).

5-Bromo-2'-Deoxyuridine (BrdU) Labeling. Pregnant females were injected intraperitonally with BrdU (100g/g body weight) at E14.5. Embryos were removed 1h after injection and fixed in 4% phosphate-buffered paraformaldehyde solution. Staining with anti-BrdU antibody (Becton-Dickenson) was performed according to manufacturer's suggestions. To quantify BrdU positive cells [modified from (2)], digital photographs of the BrdU labeled embryonic cortex were analyzed using pro-image plus 4.1 software (Media Cybernetics). In comparable sections, the cortex was subdivided into 100 M segments. The total number of labeled cells/segment of cortex was calculated. n=9.

TUNEL Assay. TUNEL assay (Boehringer-Mannheim) was performed according to manufacturer's suggestions. Briefly, sections were incubated for 10 minutes in 0.1% Triton-X and 0.1% sodium citrate, washed in PBS and incubated in terminal deoxynucleotidyl transferase [TdT] and labeled nucleotide mixture for 1h at 37°C in a humidified chamber. Sections were washed and alkaline phosphatase (AP) conjugated Fab fragment was applied for 30 minutes at 37°C. BM purple AP substrate solution (Boehringer-Mannheim) was used for the color reaction. Quantification of TUNEL-positive cells was performed similar to that of BrdU labeling.

Western Blotting. Brains, spinal cord and neurospheres were sonicated, lysed in buffer containing 1 X PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100g/ml PMSF, 1mg/ml Aprotinin, 500g/ml leupeptide, 1mM sodium orthovanadate and centrifuged two times at 10,000g for 15 minutes at 4°C. Supernatants were separated, and protein concentrations were determined by Bradford assay using BSA as a standard.30g of proteins from brain and spinal cord, or 10g from cultured neurospheres were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% milk protein and incubated with primary antibodies overnight at 4°C. All antibodies (PTEN, Akt, phospho-specific Akt, S6 kinase, and phospho-specific S6 kinase) were purchased from New England Biolabs.

Neurosphere Cultures. Neurosphere cultures were prepared as described previously (3, 4). E14.5 or P0 cortices were dissected and then dissociated by light trituration with a fire-polished glass pipette. For moderate density cultures, cells were re-suspended at 40,000 cells/ml (40K) in DMEM/F12 medium (Fisher), supplemented with B-27 (Gibco) and 20ng/ml basic fibroblast growth factor (bFGF, R&D). For clonal/low density cultures, the cells were plated at 1,000 cells/ml (1K) in media conditioned for three days by high-density-grown neurospheres. bFGF was added daily at a concentration of 20ng/ml. Secondary neurosphere cultures were grown after dissociating the 40K primary cultures into single cells, passage through a 25m nylon mesh, and then plated at 1K with conditioned media.

Immunocytochemistry of Neurospheres. Immunocytochemistry of neurosphere cultures was performed as described previously (1). Differentiation was induced by plating the neurospheres onto coverslips pre-coated with poly-L-lysine (Sigma) in Neurobasal medium. After five days, the plated neurospheres were fixed in 4% paraformaldehyde and immunostained with TuJ1 (Berkely Antibodies) for neurons, GFAP (DAKO) for astrocytes, and O4 (Chemicon) for oligodendrocytes.

Neurosphere Counts and Diameters. Neurospheres were plated onto poly-L-lysine-coated coverslips, and diameters of 50-100 randomly chosen spheres were measured within 20 minutes after plating, using the Microcomputer Imaging Device Program (MCID). A minimum cutoff of 50m was used in defining a neurosphere.

BrdU Labeling of Neurospheres. Single cell suspensions were plated at 40K, and 2M BrdU (Sigma) was added. After 24 hours, neurospheres were transferred onto poly-L-lysine-coated coverslips. The spheres were fixed and immunostained with BrdU (Boehringer Mannheim). Stained cells were counted.

Glial differentiation. The assay was essentially done as described (5). Briefly, E16.5 cortices were dissected in ice-cold dissection buffer (15mM HEPES, NaHCO3, 25mM Glucose in HBSS-CMF), the meninges were removed and the cortices incubated in a buffer containing trypsin 0.025% (Gibco) 1:1 in dissection buffer with 25g/ml DNAseI (Sigma) for 10 minutes at 37°C, then dissociated with fire polished Pasteur pipettes. Cells were plated on acid treated, poly-L-lysine (0.1mg/ml) and laminin (25g/ml, Beckton-Dickenson) coated glass coverslips (Carolina Biologicals) at 5000 cells/mm² in Basal Eagle's medium (Gibco) supplemented with 5% FCS (Hyclone). Cultures were treated with or without 50ng/ml Leukemia-Inhibitory-Factor (LIF). 3 days later, cells were stained with anti-GFAP (DAKO) antibody and a secondary FITC labeled -rabbit antibody (1:200, Jackson Immuno). Ten random viewfield pictures at 200 X were taken with a CCD camera system (Hamamatsu) and GFAP positive cells were counted, blind for genotype, and calculated as a percentage of total cells present in the cultures (n=4).

Neuronal Differentiation. For neuronal differentiation, E14.5 cortices were dissociated as described above. Cells were plated at a density of 110 cells/mm² on coated glass coverslips (see above) in Neurobasal medium (Gibco) with B-27 serum supplement (Gibco), conditions shown to yield nearly pure neuronal populations (6). After 8 days of culture, cells with neurite length more than 3 times of the cell body diameter were counted from 10 randomly chosen viewfields and presented as a percentage of neurons in total number of cells plated. To verify the counting, we also stained the cells with the neuronal differentiation marker TuJ1 (Berkeley Antibodies) and counterstained with nuclear marker DAPI (Vector) (n=2).

Cell-Cycle Time Analysis with Carbofluorescein-Succinimidyl-Ester (CFSE). E14.5 cortices were dissociated as described above. After recovery in serum-containing medium for 4 hours, cells were washed several times in serum-free medium then stained with 5 M CFSE (Molecular Probes) in PBS at 37°C for 15 minutes in the dark. Cells were washed 3X in 5ml of HBSS-CMF. Immediately after staining, one half of the cells were fixed in 1% Paraformaldehyde and analyzed in a Becton Dickenson FacsCaliber flow cytometer. The remaining cells were cultured under neurosphere culture condition (see above). After 6 days, neurospheres were dissociated and fluorescent intensities were measured as above in a genotype blinded fashion. As a control, 10M Aphidicolin (Sigma), a S-phase cell cycle blocker, was added to one of the samples. (n=2).

Statistic analysis. Data were analyzed using the paired t test and presented as the mean ± SD.

References
1. H. I. Kornblum et al., J. Comp. Neurol. 380, 243 (1997).
2. T. Takahashi et al., J. Neurosci. 19, 10357 (1999).
3. V. Tropepe, C. G. Craig, C. M. Morshead, D. van der Kooy, J. Neurosci. 17, 7850 (1997).
4. D. H. Geschwind et al., Neuron 29, 325 (2001).
5. A. Bonni et al., Science 278, 477 (1997).
6. G. J. Brewer et al., J. Neurosci. Res. 35, 567 (1993).

Supplemental Figure 1. Pten deletion leads to severe disturbance of the lamination of the mutant cortex (upper panels) and hippocampus (lower panels) at P0. H&E staining; Bar=100m.

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Supplemental Figure 2. Increased cell proliferation and decreased cell death in the ventricular zone of E14.5 mutant telencephalons. There was a significantly higher number of BrdU-labeled cells in mutant cortex compared to control littermates (upper panels). By counting 9 representative 100m segments of the cortex, we found 52 BrdU-labeled cells in the control samples (con) compared to 73 BrdU-labeled cells in mutants (Mut). Decrease of naturally occurring cell death in mutant brain revealed by TUNEL staining was also significant.

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Supplemental Figure 3. PTEN deficiency resulted in increased neural stem/progenitor cell proliferation and self-renewal. (a). Number of spheres derived from primary neurosphere cultures at initial plating density of 40,000 cell/ml (1st-40K). Mutants: filled bars; controls: open bars. Data represent the mean ± SD, a typical result of 4-6 independent experiments; P<0.012. (b) Clonal analysis of GFP labeled neurospheres. Cells were infected with lentiviral vectors containing green fluorescent protein (GFP). Two days post infection, these small spheres were dissociated and the percentage of GFP-positive cells was quantified. The GFP-positive cells were then mixed with GFP-negative cells at a ratio of 1:10, and cultured at a final density of 1,000 or 15,000 cells per ml. A. GFP positive sphere; B. GFP negative sphere; C Mixed sphere in higher density culture, arrow points to GFP⁺ cells in the mixed sphere. In the 1K/ml culture density, all spheres were either GFP-positive or GFP-negative (lower right). (c). Western blot analysis shows complete Pten deletion in E14.5 brain (Brain) and neurosphere cultures (NS) derived from E14.5 cortex. As a result of Pten deletion, the downstream signaling molecule Akt is hyperphosphorylated in brains and neurospheres. (d) Increased neurosphere diameters in the mutant culture. (e) The overall cell size from mutant cultures was larger than controls, as revealed by higher forward scatter values in flow cytometry.

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