All animal related experiments were conducted in accordance with the guidelines of the Experimental Laboratory Animal Committee of Sun Yat-sen University Health Science Center (Guangzhou, China) and under the approval of the principles of the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. NSCs were prepared from the SVZ of the brain of neonatal Sprague Dawley (SD) rats. Neonatal SD pups (age, 1 day) were sacrificed and their brains were removed. The meninges and blood vessels were removed with fine forceps; a thin tissue layer surrounding the lateral ventricles was isolated from coronal slices of the brain and cut into small sections in ice-cold D-Hanks medium (Genom Co., Hangzhou, China) containing 100 U/ml penicillin and 100 mg/ml streptomycin. Following two washes in D-Hanks medium, the tissues were dissociated mechanically with a Pasteur pipette (Biolegix, Philadelphia, PA, USA) into coarse single-cell suspensions, which were further centrifuged and resuspended prior to being passed through a cell strainer (40-μm, 352340, BD Falcon™; BD Biosciences, Franklin Lakes, NJ, USA) to remove the debris. When the homogenate was centrifuged at 80 × g for 5 min, the cells were resuspended in supplemented culture medium that consisted of DMEM: nutrient mixture F12 (DMEM/F12) (1:1) (Gibco-BRL, Carlsbad, CA, USA) supplemented with 1% B27 (Invitrogen Life Technologies, Carlsbad, CA, USA), 1% N2 supplement (17502-048; Invitrogen Life Technologies), 20 ng/ml epidermal growth factor (EGF) (Invitrogen Life Technologies) and 20 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen Life Technologies). Cells were incubated at 37ºC in a 5% CO₂ atmosphere and half the volume of the culture medium was replaced every 2 days. After 7 days, NSCs aggregated into neurospheres. For passaging, neurospheres were resuspended and replated into non-coated culture flasks at a density of 2×10⁵ cells/ml following mechanical dissociation.

To induce the differentiation of NSCs, neurospheres were seeded onto poly-L-lysine-coated glass coverslips (NEST Biotechnology Co., Ltd., Shanghai, China) following three passages and then cultured in differentiation medium containing DMEM/F12 (1:1), 1% N2, and 10% fetal bovine serum (FBS) (Gibco-BRL). Under this environment, the sedimentary neurospheres attached to the poly-L-lysine-coated surface and began to differentiate into neurons, astrocytes and oligodendrocytes. The cells were maintained at 37ºC in a 5% CO₂ atmosphere and the medium was replaced every 2 days. The differentiation of NSCs was observed using a phase-contrast microscope (DMI4000B; Leica, Wetzlar, Germany), cells were photographed and analyzed for morphology at day 7.

The brains of neonatal SD rats (age, 1 and 7 days) were used to investigate the expression and localization of PGRN during development. The rats were anesthetized with a peritoneal injection of 10% chloral hydrate (Sigma-Aldrich, St. Louis, MO, USA) and perfused transcardially with cold phosphate saline buffer (PBS) followed by ice-cold 4% paraformaldehyde in 0.1 M PBS. The brains were removed and postfixed in 4% paraformaldehyde solution at 4ºC overnight, after which the brains were dehydrated serially in 10, 20 and 30% sucrose in PBS overnight at 4ºC. When the the brain sediment reached the bottom of the tubes, the brains were embedded in Optimal Cutting Temperature compound (Sakura Finetek Co., Ltd., Tokyo, Japan). Serial coronal sections were cut using a freezing microtome at 10 μm and mounted onto poly-L-lysine-coated glass slides. PGRN was detected using immunohistochemistry.

To investigate the expression of PGRN in NSCs, the neurospheres were collected by centrifugation and then washed three times with PBS and fixed in 4% paraformaldehyde solution for 30 min at room temperature. Following three washes with PBS, the neurospheres were blocked using 10% normal goat serum for 1 h at room temperature to reduce non-specific binding. Neurospheres were then incubated with primary antibody against nestin (dilution, 1:1,000; Abcam, Hong Kong, China) for NSCs and PGRN (dilution, 1:500; R&D Systems, Inc., Minneapolis, MN, USA) in PBS containing 0.3% Triton X-100 at 4ºC overnight. Following sufficient washing, cells were incubated with appropriate fluorescence-conjugated secondary antibodies for 1 h at 37ºC. For the identification of PGRN in differentiated NSCs, the cells were grown on poly-L-lysine-coated round glass coverslips, rinsed three times with PBS following 7 days of differentiation, fixed with 4% paraformaldehyde solution for 30 min at room temperature and blocked with 10% normal goat serum for 1 h at room temperature. The cells were then incubated with primary antibody against βIII-tubulin for neurons (dilution, 1:1,000; Abcam), glial fibrillary acidic protein (GFAP) for astrocytes (dilution, 1:1,000; Abcam), Oli for oligodendrocytes (dilution, 1:1,000; Abcam) and PGRN (dilution, 1:500; R&D Systems, Inc.) in PBS containing 0.3% Triton X-100 at 4ºC overnight. The cells were washed with PBS and incubated with the corresponding fluorescence-conjugated secondary antibodies [Alexa Fluor^®488 donkey anti-sheep IgG (H+L), A-11015; Alexa Fluor^®594 goat anti-mouse IgG (H+L), A-11005; and Alexa Fluor^®594 goat anti-rabbit IgG (H+L), A-11012; Invitrogen Life Technologies)] for 1 h at 37ºC. Nuclei were stained for 5 min with the nuclear dye Hoechst 33342 (20 ng/ml; Biotium, Hayward, CA, USA). Images were captured with a fluorescence microscope (BX51WI; Olympus, Tokyo, Japan).

The expression of PGRN was also detected in the brain of neonatal SD rats (age, 1 and 7 days). Frozen sections were heated to 56ºC for 10 min, washed with PBS and incubated with blocking solution with 10% normal goat serum containing 0.3% Triton X-100 for 1 h at room temperature. The sections were then incubated with primary antibodies (anti-PGRN, 1:600; anti-βIII-Tubulin, 1:1,000; anti-GFAP, 1:1,000; and anti-Oli, 1:1,000) overnight at 4ºC. When the sections had been thoroughly washed with PBS, appropriate secondary antibodies Alexa Flour 488 and Alexa Flour 594 (1:1,000 dilution, Invitrogen Life Technologies) were added and incubated for 2 h at 37ºC, and the sections were washed three times with PBS. Nuclei were stained with the nuclear dye Hoechst 33342 (20 ng/ml) for 5 min and rinsed with PBS, and the coverslips were mounted on slides with FluorSave reagent (Beyotime, Jiangsu, China).

Neurospheres were clearly observed at day 3. Numerous neurospheres proliferated in suspension at day 7 following primary culture (Fig. 1A). Immunocytochemistry results showed that neurospheres appeared positive for nestin and PGRN expression (Fig. 2A–D). These results indicated that NSCs were successfully isolated and cultured and expressed PGRN.

After three passages, the neurospheres were transferred in 24-well plates containing poly-L-lysine-coated glass coverslips and were maintained in differential medium, containing DMEM/F12 (1:1), 1% N2 supplement and 10% FBS, for a further 7 days. The neurospheres began to attach to the coverslips and gradually showed the morphological features of neural cells (Fig. 1B). The immunocytochemical staining showed that the NSCs differentiated into βIII-tubulin⁺ neurons, GFAP⁺ astrocytes and Oli⁺ oligodendrocytes (Fig. 1F–H). Moreover, all NSC-derived cells expressed PGRN in vitro (Fig. 3).

The expression of PGRN was also analyzed by immunofluorescent staining in postnatal rat brains at day 1 (P1) and day 7 (P7). PGRN immunoreactivity was observed predominantly in the cortex, corpus callosum and hippocampus. To analyze the cell type-specific expression of PGRN in the neonatal brain, immunofluorescent double labeling was performed with specific markers for neurons (βIII-tubulin), astrocytes (GFAP), microglia (OX-42), NSCs (Nestin) and oligodendrocytes (Oli). The results indicated that the mature neuronal marker, βIII-tubulin (Fig. 4Aa–Ad) and microglia marker, OX-42 (Fig. 4Da–Dd) were double-labeled with PGRN, which indicated that PGRN was predominantly expressed in neurons and microglia during the early stages of rat brain development. By contrast, PGRN immunoreactivity was marginal in astrocytes (Fig. 4Ab–Db), NSCs (Fig. 4Ca–Cd) and oligodendrocytes (Fig. 4Ea–Ed). Thus, these results suggest that PGRN was predominantly expressed in neurons and microglia rather than NSCs, astrocytes and oligodendrocytes in the brain at P1 and P7.