Formylpeptide Receptors Promote the Migration and Differentiation of Rat Neural Stem Cells

Neural stem cells (NSCs) bear characteristics for proliferation, migration and differentiation into three main neural cell type(s): neurons, astrocytes and/or oligodendrocytes. Formylpeptide receptors (Fprs), belonging to the family of G protein-coupled chemoattractant receptors, have been detected on neurons in the central nervous system (CNS). Here, we report that Fpr1 and Fpr2 are expressed on NSCs as detected with immunohistochemistry, RT-PCR and WB assays. In addition, Fpr1 and Fpr2 promoted NSC migration through F-actin polymerization and skewed NSC differentiation to neurons. Our study demonstrates a unique role of Fpr1 and Fpr2 in NSCs and opens a novel window for cell replacement therapies for brain and spinal cord injury.

results obtained with immunocytochemistry. RT-PCR detected a high level expression of Fpr1 and Fpr2 mRNA in NSCs similar to their levels in a positive control cell line K562 (Fig. 1c,d). In addition, WB detected Fpr1 and Fpr2 proteins in NSCs (Fig. 1e,f). Thus, both Fpr1 and Fpr2 are expressed by NSCs.

Fpr1 and Fpr2 enhance NSC migration both in vitro and in vivo.
We then tested the capacity of Fpr1 and Fpr2 to mediate NSC migration by morphological changes, transwell assays in vitro and by cell tracking in brain injury model as well as immunohistochemistry in vivo. Results showed that Fpr specific agonists induced potent NSC migration in vitro and this effect was abrogated by specific Fpr1 or Fpr2 antagonists (Fig. 2). In addition, Fpr1 and Fpr2 synergistically induced NSC migration ( Fig. 2e-h). For in vivo study, a brain injury model was used to test the migration of transplanted NSCs induced by Fpr agonists. The results clearly showed that prelabelled NSCs migrated away from the infusion site to the midline and the contralateral site of fMLF or MMK-1 infusion site through the corpus callosum. The migration was specifically abrogated by Fpr antagonists (Fig. 3 and Supplementary Figure 1). Immunohistochemistry detection also confirmed the directed migration of endogenous NSC by Fpr agonists in vivo (Supplementary Figure 2). Thus, Fprs expressed by NSCs are definitively functional both in vitro and in vivo and play a pivotal role in the migration of NSCs in vivo after CNS injury.
Fpr1 and Fpr2 promote neuronal differentiation of NSCs. We next tested whether Fpr1 and Fpr2 promote NSC differentiation of NSCs. Immunocytochemistry indicated that both Fpr1 and Fpr2 skewed NSC differentiation to immature neurons as revealed by significantly increased immature neuron marker DCX 24 hours after stimulation with Fpr1 and Fpr2 agonists (Fig. 4a-d). Mature neuron marker TUJ-1 was markedly increased on day 3 and lasted at least for 7 days after Fpr agonist treatment (Supplementary Figure 3). The effects of Fpr agonists were blocked by antagonists (Figs 3a-d and 4a-d). The expression of DCX was confirmed by WB (Fig. 4e,f). However, Fpr agonists had no effect on NSC differentiation to astrocytes or oligodendrocytes (Fig. 5). We also observed but failed to found any effects of Fpr agonists on the proliferation of NSCs (Supplementary Figure 4).

Fprs mediate in NSC F-actin polymerization.
To examine mechanisms of Fpr1 and Fpr2 mediated NSC migration, we measured the expression of F-actin upon activation of the cells with Fpr agonists. The expression of F-actin was up-regulated in NSCs stimulated with fMLF and MMK-1. The effect of Fpr agonists was inhibited by Fpr antagonists tBOC and WRW4 (Fig. 6). Since F-actin polymerization is required for ligand-induced cell chemotaxis, Fpr-mediated NSC migration is dependent on F-actin.

Discussion
NSCs bear the capacity for self-renewal and give rise to three main neural cell type(s): neurons, astrocytes and/or oligodendrocytes 1,2 , which involve proliferation, differentiation and migration [24][25][26] . The present study provides the evidence to suggest that NSCs express functional Fprs that promote NSC migration and neuronal differentiation upon stimulation with agonists. This may represent a mechanism for NSCs to home and engraft to the lesions of inflammation and injury in the central nervous system.
Tissue injury, such as cerebral ischemia and/or spinal cord damage, results in inflammation, and leukocyte infiltration 27 . The injured tissue produces inflammatory mediators, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α ), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interferon gamma (IFN-γ ), and interleukin-18 (IL-18) 28,29 .Among the mediators, monocyte chemoattractant protein-1 (MCP-1, CCL2) and stromal-derived factor-1 (SDF-1,CXCL12) regulate the proliferation, migration and neural differentiation of NSCs 9,11,30 . Fprs also regulate the function of neutrophils and/or monocytes through the interaction with their ligands [31][32][33] . Spontaneous migration of NSCs from restricted regions to the lesions is regulated by many molecules. Increasing evidence shows that Fprs are not only expressed by neutrophils and monocytes but are also detected in human brain, spinal cord, anterior horn cells and hypoglossal nucleus neurons 18 . NSCs also interact with some chemokines and cytokines produced by neutrophils and monocytes to enhance their proliferation, migration and final differentiation 6 .
In present study, we showed that both Fpr1 and Fpr2 are expressed on NSCs and both receptors mediate the migration of NSCs in vitro and in vivo, and promote the differentiation of NSCs to neuron. Consistent with previous findings, both Fpr1 and Fpr2 participate in the normal wound healing process for early neutrophil recruitment and subsequent wound closure 34 . Thus, Fprs may play pivotal roles in NSC migration to lesion sites by interaction with specific ligands in the microenvironment. These results also imply that Fpr ligands could be utilized for the treatment of a variety of clinical conditions, including stroke and spinal cord injury. The present study focuses on the Fpr-mediated migration of primary NSCs. Our results using cell tracking in vivo clearly showed that transplanted NSCs migrated in response to Fpr agonists through the corpus callosum. Further studies are needed to identify the subsets of NSCs that utilize Fprs for migration.
Accumulating evidence shows that some signaling pathways coupled to cytokines and chemokines are involved in NSC proliferation, migration and differentiation. The Janus kinase-signal transducer and JAK/STAT alter NSC self-renewal, progenitor cell division and differentiation 28,29 . The c-Jun N-terminal kinase (JNK) pathways mediate neuronal differentiation induced by IFN-γ 35 , and also are required for neural differentiation of embryonic carcinoma cells, embryonic stem cells and PC12 cells [36][37][38][39] . Activated microglia secrete insulin-like growth factor-1(IGF-1) under pathological conditions and the subsequent activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway increases neurogenesis 40 .
Our study suggests that Fprs may serve as candidates for treatment of brain or spinal cord injury.  Primary Neurosphere Culture. We isolated and cultured primary rat NSCs from cortices of fetal E14.5

Animals and Ethics
Sprague-Dawley pups as previously described 41 . Briefly, brains were dissected and incubated in DMEM containing 0.25% trypsin-EDTA and 250U/ml DNase I at 37 °C for 30 min. Then, tissue pieces were washed twice in DMEM with 10% fetal bovine serum to inhibit the digestion of trypsin. The tissue samples were triturated using a fire-polished Pasteur pipette and passed through a 100 μ m Nylon cell strainer to harvest dissociated cell suspensions after washed twice with Dulbecco's Modified Eagle's Medium. Cell suspensions were and cultured in DMEM/ F12 medium supplemented with B27, 20 ng/ml EGF, 20 ng/ml FGF-2 and 1% penicillin-streptomycin at 37 °C   were blocked with 5% (wt/vol) nonfat dry milk in TBST (TBS, 0.1% (vol/vol) Tween 20) for 2 hours at room temperature and subsequently incubated with rabbit polyclonal to Fpr1 (1:2000), rabbit polyclonal to Fpr2 (1:2000), or mouse monoclonal to GAPDH (1:1000) overnight at 4 °C with gentle agitation and peroxidase-conjugated (HRP)-conjugated secondary IgGs (1:5000) for 2 hours at room temperature. All membranes were detected by ChemiDoc ™ XRS + imaging system using the Pierce Fast Western Blot Kits (Thermo Scientific, USA). All immunoblots were carried out in duplicates of three independent cell populations and averaged (n = 3).
ELISA. Quantitative measurement of F-actin and GAPDH were analyzed and determined by the corresponding ELISA kits (Abcam) according to the manufacturer's instructions. The absorbance was measured at 450 nm.

Cell Migration Assay in vitro.
Morphological observation assays were performed with Millipore 96-well cell clusters in accordance with the manufacturer's instructions. Briefly, about 10-20 μ m diameter of 3-5neurosphereswere incubated in 96-well plates in 100 μ L DMEM/F12 containing 2%B27 supplemented with 20 ng/ml EGF, 20 ng/ml FGF-2 and 1% penicillin-streptomycin. The agonist and/or antagonist of Fpr1 and Fpr2 were added to each 96-well plates for 12 h at 37 °C in a humidified incubator with 5% CO2.Cell morphology were observed on 12 h and compared with 0 h under the microscope. Transwell assays were performed with Millipore transwell inserts (8-μ m pore filter, 24-well cell clusters) in accordance with the manufacturer's instructions. The upper chambers were seeded 100 μ L (1 × 105/ml) NSCs in DMEM/F-12 medium containing 2% B27 supplemented with 20 ng/ml EGF, 20 ng/ml FGF-2 and 1% penicillin-streptomycin. The lower chambers were filled with 600 μ L DMEM/F-12 medium with 6 μ M fMLF and 0.5 μ M MMK-1 which served as chemoattractants or 0.2 μ M tBOC and 0.4 μ M WRW4 that served as inhibitors to test Fpr1 and Fpr2 on NSC migration. The NSCs were allowed to migrate from the upper to the lower chambers for 12 h at 37 °C in a humidified incubator with 5% CO2. Non-migratory cells were removed from the top of the membrane with a cotton swab and the cells attached to the lower surface of membrane were fixed in 4% paraformaldehyde at room temperature for 30 min and counterstained with DAPI, and the number was counted under the microscope. A total of 12fields were counted for each transwell filter.

NSC Migration Assay in vivo.
A modified cerebral hemorrhage model was used to corroborate the Fpr effects on NSC migration in vivo. Firstly, transplanted prelebelled NSCs were observed in vivo. Briefly, the animals were anesthetized with 10% Chloral Hydrate. A suspension of 70,000 NSCs cells in 10 μ L prelabeled with CM-Dil (Cell Tracker, MolecularProbes, treated according to the instructions of the manufacturer) 7 was injected into the subcortical white matter on the right (bregma + 0.74 mm, 2.5 mm lateral and 4 mm ventral to skull) used a stereotaxic apparatus. On the same day and the following 10 days, 10 μ L containing 6 μ M fMLF and 0.5 μ M MMK-1 or 0.2 μ M tBOC and 0.4 μ M WRW4 was injected into the contralateral hemisphere at the same site. Animals were sutured and allowed to recover from surgery on a heating pad, then carefully monitored for the duration of the experiment. On day 10, the brain were sliced and the distance of migration (the distance between the NSC injection site to the furthest point in the corpus callosum and the middle line where the transplanted NSCs reached were analyzed. In addition, the migration of endogenous NSCs was also detected with immunohistochemistry. 10 μ LFpr1 and Fpr2 agonists/antagonists with gradient concentrations were injected into basal ganglia near subventricular zone with fine needle in rat brain. Then, we took out the brain on day 1, 3 and 7 post injection and used immunohistochemistry as previously described to observe NSC migration (Supplementary Figure 2). Cell Differentiation Assay. Immunocytochemistry. As previously described, cells were incubated with rabbit polyclonal to rabbit polyclonal to Doublecortin (24 h, 1:200), rabbit polyclonal to glial fibrillary acidic protein (GFAP) (24 h, 1:100), rabbit polyclonal to Olig2 (24 h, 1:100,), rabbit polyclonal toTUJ-1 (3d and 7d, 1:200) (n = 3).
Cell Proliferation Assay. The effects of Fpr1, Fpr2 and their agonist and/or antagonist on the NSC proliferation were measured by a CCK-8 assay kit. Briefly, 8 × 103 cells/well were incubated in 96-well plates in 100 μ L DMEM/ F12 containing B27 supplement without FGF-2 and EGF. The agonist and/or antagonist of Fpr1 and Fpr2 were added every day to each 96-well plates. Cell Counting Kit-8 solution was added after 3 days to each well and incubated for 3 hours. The cell viability in each well was determined by reading the absorbance of the culture medium at a test wavelength of 450 nm and a reference wavelength of 630 nm.
CCK-8 assay. The effects of Fpr1, Fpr2 and their agonist and/or antagonist on the NSC proliferation were measured by a CCK-8 assay kit (Dojindo, Japan). Briefly, 8 × 103 cells/well were incubated in 96-well plates in 100 μ L DMEM/F12 containing B27 supplement without FGF-2 and EGF. The agonist and/or antagonist of Fpr1 and Fpr2 were added every day to each 96-well plates. Cell Counting Kit-8 solution was added on 1, 3, and 7day to each well and incubated for 3 hours. The cell viability in each well was determined by reading the absorbance of the culture medium at a test wavelength of 450 nm and a reference wavelength of 630 nm (Thermo Scientific, USA).
Ki-67 assays were performed with immunocyto chemistry as previously described which incubated with rabbit polyclonal to  or Mouse monoclonal to Nestin (1:250).
Statistical analysis. All data were presented as mean ± SEM and statistical analyses were performed with GraphPad Prism v5.0 (GraphPad Software, Inc. USA).Statistical differences among testing and control groups were analyzed by Student's t-test and one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison post-test. A P value < 0.05 was considered.