Research reportIdentification of pleiotrophin in conditioned medium secreted from neural stem cells by SELDI-TOF and SELDI-tandem mass spectrometry
Introduction
Neural stem cells (NSCs) are multipotential progenitor cells present in the central nervous system (CNS) that have capacity for self-renewal and generation of all differentiated cells including neurons, astrocytes, and oligodendrocytes and continue to function throughout adulthood [1], [3], [5], [6], [16], [18], [22], [30].
Since recent advances in neurobiology have demonstrated that partial morphological and functional recovery can be achieved in some pathological conditions, such as ischemia [36], there has been enthusiastic interest in NSCs as therapeutic agents for the damaged CNS. In order to obtain the functional recovery by endogenous or transplanted NSCs, we should elucidate the regulatory mechanisms underlying the differentiation and self-renewal of NSCs.
NSCs respond only to fibroblast growth factor (FGF) in early stage [9], [25], [31]. Later appearing NSCs require either FGF or epidermal growth factor (EGF) [7] for proliferation. Platelet-derived growth factor (PDGF) and FGF are important for glial precursor cell proliferation [11], while neuronal precursor cells respond to sonic hedgehog (Shh) [2], FGF [4], and neurotrophin-3 [12]. These results of previous studies suggest that various extracellular signals mediated by growth factors and cytokines have essential roles in normal growth and development as well as the fates of the NSCs in situ that are dependent on their niche [34] containing growth factors and cytokines. To further elucidate the precise regulatory mechanism of NSCs, we attempted to identify specific proteins secreted by neural stem cells into conditioned medium. In the present study, a combination of SELDI-TOF-MS, SELDI-tandem MS and Edman degradation sequencing demonstrated that pleiotrophin (PTN) is secreted from NSCs, and that mRNA transcripts of receptors for PTN such as receptor protein tyrosine phosphatase (RPTP) β/ζ, syndecan-3, and anaplastic lymphoma kinase (ALK) in NSCs exist. Thus, pleiotrophin signaling system is considered to be involved in the biology of NSCs.
Section snippets
Isolation and culture of neural stem cells
Fourteen-day pregnant ICR mouse (Charles River Japan, Yokohama, Japan) was sacrificed by cervical dislocation. The striatum, cortex and hippocampus of embryo brain were removed. Tissues were transferred into conical-bottom tubes, mechanically dissociated to a single-cell suspension and seeded 2×105 cells/ml in flask (Greiner Japan, Tokyo, Japan) with 40-ml culture medium (NPBM; Cambrex, NJ), bFGF 20 ng/ml and EGF 20 ng/ml and incubated for 7 days in 5% CO2 at 37 °C (primary culture).
Primary
Isolation and differentiation of neurospheres
We isolated primary neural stem cells (NSCs) from mouse embryo brain. These cells grew primarily as spherical aggregates like neurospheres (Fig. 1A–C). Single-cell suspensions created from dissociated neurospheres after every passaging were able to form new neurospheres (data not shown).
To investigate whether the NSCs have the same potential to differentiate into all the three main neural cell types, i.e., neurons, astrocytes, and oligodendrocytes, we induced differentiation by plating primary
Discussion
Using a SELDI ProteinChip-based proteomics approach to screen for differentially expressed proteins, we identified pleiotrophin being as elevated in the conditioned medium from neurosphere.
It is well known that growth factors are required for growth, differentiation, and maintenance of viability during CNS development. Pleiotrophin is a secreted heparin-binding growth factor [13], [21], [27] which is highly expressed in the developing nervous system. PTN was originally described as a
Acknowledgements
The authors thank Mrs. Yumiko Oishi and Ms. Sachiko Kawasaki for technical support.
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Both authors contributed equally to this work.