Role of pleiotrophin in neural stem cell proliferation

Esther Gramage1, Gonzalo Herradón1, Francisco Molina-Holgado2. 1Department of Food and Pharmaceutical Sciences, Universidad CEU San Pablo, Boadilla del Monte, 28668, Madrid, Spain, 2Departament of Life Science, University of Roehampton, London SW15 4JD, UK

Pleiotrophin (PTN) is a neurotrophic factor important in the normal development of the central nervous system. PTN expression peaks during embryonic development at the same sites where neuronal progenitors are ceasing to proliferate and initiating the differentiation processes to a neuronal phenotype. It has been previously shown that PTN is present in conditioned medium from neural stem cells (NSC). Furthermore, mRNA transcripts of PTN and its receptors receptor protein tyrosine phosphatase (RPTP) beta/zeta, N-syndecan and anaplastic lymphoma kinase (ALK) were detected in neurosphere, suggesting that pleiotrophin signalling systems are present in the NSC and are involved in the modulation of fate of neural stem cells. In the present studies, we aimed to characterize the effect of exogenous PTN on NSC. We have used NSC from C57 mice to test the effects of PTN on cellular proliferation. The cells were cultured as free-floating neurospheres in 75-cm2 flasks at a density of 20 cells/μl (Molina-Holgado et al., 2007; Rubio-Araiz et al., 2008) in Dulbecco's modified Eagle's medium/F12 plus B27, 3 μg/ml heparin and 20 ng/ml FGF and EGF. Primary neurospheres were grown for 7–10 days before secondary cultures were established with mechanical dissociated cells. Thereafter, the spheres were passaged every 5–7 days and experiments were performed after three to seven passages. We treated increasing numbers of NSC per plate (125, 250, 500, 1000, 2000 and 4000 cells) with increasing concentrations of PTN (20, 50 and 100 ng/ml) and tested neurosphere formation. The results obtained in PTN-treated and control (untreated) NSC were analyzed by two-way ANOVA with treatment and number of cells per plate as ANOVA factors, and Bonferroni’s post-hoc tests. ANOVA revealed a significant effect of the number of cells plated (F(5,96) = 104.6, P < 0.0001). Bonferroni’s post-hoc analysis uncovered that 50 ng/ml PTN causes a significant increase in neurosphere formation in 4000 cells-plates (t = 2.928, P < 0.05). The data identify appropriate experimental high cell density conditions to test the effects of PTN on NSC. The data demonstrate that PTN is a cytokine with proliferative effects on NSC and suggest that the presence of PTN and its receptors identified by others in NSC is critically involved in the modulation of the NSC fate, presumably at one stage in the frontier between the previous steps of cessation of proliferation and initiation of differentiation.

Molina-Holgado et al., Eur J Neurosci. 2007,25(3):629-34.

Rubio-Araiz et al., Mol Cell Neurosci. 2008, 38(3):374-80.

EG is supported by a fellowship AP2008-00726 from Ministry of Education (SPAIN) and by a researcher mobility fellowship from CEU – Bank of Santander (SPAIN).