HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/11/1237 03-0927fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by JUNG, C.-G. Articles by NISHINO, H. Search for Related Content PubMed PubMed Citation Articles by JUNG, C.-G. Articles by NISHINO, H.

Pleiotrophin mRNA is highly expressed in neural stem (progenitor) cells of mouse ventral mesencephalon and the product promotes production of dopaminergic neurons from embryonic stem cell-derived nestin-positive cells

CHA-GYUN JUNG^*,1, HIDEKI HIDA^*, KENSUKE NAKAHIRA, KAZUHIRO IKENAKA, HYE-JUNG KIM^* and HITOO NISHINO^*,

^* Department of Neuro-Physiology and Brain Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan;
Department of Physiology, Saitama Medical School, Saitama, Japan;
Laboratory of Neural Information and
Divison of Learning and Memory Research, National Institute for Physiological Sciences, Okazaki National Research Institutes, Okazaki, Japan

¹ Correspondence: Department of Neuro-Physiology and Brain Science, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Ku, Nagoya 467-8601, Japan. E-mail: jung{at}med.nagoya-cu.ac.jp

SPECIFIC AIM

To understand the mechanism of neural differentiation, especially with dopaminergic (DAergic) neurons, the aim of the present study was to create a gene file of neural stem (progenitor) cells derived from mouse (E12.5) ventral mesencephalon using serial analysis of gene expression (SAGE). Among 15,815 tags investigated, pleiotrophin (PTN) mRNA was highly expressed in these cells. Since PTN affects cell migration, repair, neurite outgrowth, and differentiation of neural development, we focused on clarifying the functional role of PTN in DAergic differentiation using mouse embryonic stem (ES) cells.

PRINCIPAL FINDINGS

1. Gene expression in stem (progenitor) cells derived from the ventral mesencephalon of mouse embryos
To understand the mechanism of neural differentiation, a SAGE experiment was performed on neurospheres (P2D2) obtained from the ventral mesencephalon (VM) of E12.5 mouse embryos. Among 15,815 tags analyzed, mRNA of PTN, an 18 kDa heparin binding growth factor was highly expressed in VM-derived neural stem (progenitor) cells.

2. PTN and its receptor genes are expressed in ES-derived nestin positive cells
We investigated the functional role of PTN on ES-derived nestin positive cells generated from mouse ES cells committed to a neuroectodermal lineage, especially in development of DAergic neurons. ES cell-derived nestin positive cells and their differentiation were prepared in the following stages: 1) expansion of undifferentiated ES cells; 2) generation of embryoid bodies (EBs); 3) selection of nestin-positive cells; 4) expansion of nestin-positive cells; and 5) induction of differentiation of nestin-positive cells. RT-PCR analysis showed that mRNAs for both PTN and PTN receptors (syndecan-3 and protein tyrosine phosphatase, PTP-) were first expressed at stage 3. Their expressions were maintained during stage 4 and up-regulated in stage 5.

3. Induction of TH-positive cells by PTN treatment
PTN was applied every second day during stage 4. The culture system induced efficient neural differentiation and resulted in all colonies being microtubule associated protein-2 (MAP-2) and ß-tubulin III (not shown) positive at day 10 of stage 5 (Fig. 1 D). There was no difference in colony size between PTN-treated and non-treated control groups. Immunocytochemistry revealed a network of TH positive cells in both cultures (Fig. 1A, D) . In comparison to the control, the number of TH positive cells in the PTN treated group was increased to 240 ± 27% at day 2 and to 213 ± 10% at day 10 of stage 5 (Fig. 1B, E ). Neurite extension was also promoted by PTN (Fig. 1D ). By semiquantitative RT-PCR analysis, expression of Nurr1 mRNA was significantly up-regulated by PTN at both day 2 and day 10 of stage 5 (Fig. 1C, F ).

View larger version (32K): [in this window] [in a new window]   Figure 1. PTN treatment increases the number of TH-positive cells and promotes neurite extension. PTN (100 ng/mL) was applied every second day during stage 4, then cells were used for immunocytochemistry. The number of TH-positive cells and expression of Nurr1 at day 2 (A–C) and day 10 (D–F) of stage 5 were investigated. A) Immunofluorescence staining for MAP-2 (top) and TH (bottom). D) Immunofluorescence staining for MAP-2 (top) and TH (middle and bottom). B, E) The number of TH-positive cells in 8 randomly selected fields was significantly increased by PTN at day 2, as well as day 10 of stage 5 (n=5, P<0.01). C,F) Semiquantitative RT-PCR showed increased expression of Nurr1 mRNA by PTN at both days.

4. PTN treatment enhances L-dopa synthesis
At day 2 of stage 5, an earlier stage of differentiation, release of L-dopa more than doubled in cultures treated with 100 ng/mL of PTN (Fig. 2 B). This effect of PTN was concentration-dependent (Fig. 2A, B ). A time course study showed that L-dopa release was increased in a time-dependent manner and reached a maximum at day 8 of stage 5, both in control and in PTN-treated cells (Fig. 2B ). At day 8, the effect of a lower concentration of PTN (100 ng/mL) was almost the same as that of a high concentration of Shh (500 ng/mL) (Fig. 2C ).

View larger version (19K): [in this window] [in a new window]   Figure 2. PTN promotes L-dopa release. A) HPLC pattern of L-dopa in culture medium at day 10 of stage 5 in nontreated control (left) and PTN-treated (500 ng/mL, right). B) PTN enhances release of L-dopa in a concentration-dependent manner. PTN was applied every second day during stage 4 and L-dopa released into the medium was measured at days 2, 4, 6, 8, and 10 of stage 5. P < 0.01; n = 3. C) Effect of a lower concentration of PTN was similar to that of a higher concentration of SHH. PTN (100 ng/mL) and SHH (500 ng/mL) were applied every second day during stage 4 and L-dopa was measured at days 8 and 10 of stage 5. P < 0.01; n= 3. Data are shown as mean ± SE.

5. Effect of PTN at different developmental stages
To investigate whether the effect of PTN to produce DAergic neurons was stage specific, PTN (100 ng/mL) and Shh (500 ng/mL) were applied in stage 4 and/or 5. Application of PTN in stage 4 increased the number of TH positive cells to 209 ± 25% of the control. The effect of Shh (213±28% of control) was similar to that of PTN. The number of TH positive cells was increased to 415 ± 31% of the control by co-treatment with PTN/Shh in stage 4. Treatment with PTN in stage 5 was less effective than that in stage 4 (123±9.2% of control). Treatment with Shh in stage 4 and with PTN in stage 5, slightly increased the number of TH positive cells (269±21% of control) compared with Shh alone in stage 5 (213±28%).

CONCLUSIONS AND SIGNIFICANCE

In the present study, we first constructed a gene file of mesencephalic stem (progenitor) cells using SAGE to begin to understand the mechanism of neuronal development, especially in terms of DAergic neurogenesis. We report that among thousands of genes expressed, PTN gene is highly expressed, and that PTN promotes production of DAergic neurons from ES-derived stem cells.

PTN and its receptors were first expressed in nestin positive cells (stage 3). Their expression was not detected in stages 1 and 2 and had increased in stage 5, suggesting that PTN is a molecule involved in neural differentiation of nestin positive cells. Parallel expression of PTN and PTN receptors may indicate an autocrine action since the phenotype of cells in stages 4 and 5 (especially stage 5) is mostly homogeneous and may partly indicate an interaction of neurons and glial cells.

Mesencephalic DAergic progenitors migrate to form reticular formation, substantia nigria pars compata, and the ventral tegmental area. Stem cells first migrate ventrally from the ventricular surface along radial processes of neuroepithelial cells that express tenascin, then laterally along tangentially arranged nerve fibers that express L1 (Fig. 3 B). PTN is detected in the neural plate at E9.5 and restricted to the dorsal ventricular zone from E11.5 to 13.5. Thus PTN may influence cells located at the ventricular zone via paracrine actions or via diffusion through the ventricle (Fig. 3B ). It has been reported that PTP-binds to tenascin, a large glycoprotein molecule in the extracellular matrix. In tenascin gene knockout mice, abnormal behavior is displayed in conjunction with a significant decrease in TH mRNA levels in midbrain. Therefore, during the developing stage it is probable that PTN affects stem (progenitor) cells before and during their migration to induce DAergic neurogenesis. To test this, we designed an in vitro experiment investigating action of PTN on production of DAergic neurons from stem (progenitor) cells.

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic diagram.

Application of a low concentration of PTN during stage 4 more than doubled the number of TH positive cells, and the effect was compatible to that of higher concentration of Shh. We show that PTN is a most effective substance in DAergic neuroproduction. The effect of PTN was most evident with co-treatment with Shh. The number of TH positive cells was also increased by series of treatments with Shh during stage 4 and PTN during stage 5. Consistent with increase in the number of TH positive cells, release of L-dopa was increased.

Information on gene expression of fetal ventral mesencephalic stem (progenitor) cells revealed in the present study should provide basic information necessary for further understanding the molecular mechanism of neuroproduction and DAergic development. Since pleiotrophin was found to be one of the most potent substances enhancing production of DAergic neurons, it is possible to apply pleiotrophin to developing DAergic donor cells and improve transplantation technique for a higher survival of grafted cells and greater functional recovery.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0927fje; doi: 10.1096/fj.03-0927fje

This article has been cited by other articles:

				T. Vazin, J. Chen, C.-T. Lee, R. Amable, and W. J. Freed Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells Stem Cells, June 1, 2008; 26(6): 1517 - 1525. [Abstract] [Full Text] [PDF]

				T.-S. Huang, J.-Y. Hsieh, Y.-H. Wu, C.-H. Jen, Y.-H. Tsuang, S.-H. Chiou, J. Partanen, H. Anderson, T. Jaatinen, Y.-H. Yu, et al. Functional Network Reconstruction Reveals Somatic Stemness Genetic Maps and Dedifferentiation-Like Transcriptome Reprogramming Induced by GATA2 Stem Cells, May 1, 2008; 26(5): 1186 - 1201. [Abstract] [Full Text] [PDF]

				P. U. Magnusson, A. Dimberg, S. Mellberg, A. Lukinius, and L. Claesson-Welsh FGFR-1 regulates angiogenesis through cytokines interleukin-4 and pleiotrophin Blood, December 15, 2007; 110(13): 4214 - 4222. [Abstract] [Full Text] [PDF]

				H. Muramatsu, P. Zou, N. Kurosawa, K. Ichihara-Tanaka, K. Maruyama, K. Inoh, T. Sakai, L. Chen, M. Sato, and T. Muramatsu Female infertility in mice deficient in midkine and pleiotrophin, which form a distinct family of growth factors Genes Cells, December 1, 2006; 11(12): 1405 - 1417. [Abstract] [Full Text] [PDF]

				T. Yasuhara, N. Matsukawa, K. Hara, G. Yu, L. Xu, M. Maki, S. U. Kim, and C. V. Borlongan Transplantation of Human Neural Stem Cells Exerts Neuroprotection in a Rat Model of Parkinson's Disease J. Neurosci., November 29, 2006; 26(48): 12497 - 12511. [Abstract] [Full Text] [PDF]

				I. Hernandez-Gonzalez, I. Gonzalez-Robayna, M. Shimada, C. M. Wayne, S. A. Ochsner, L. White, and J. S. Richards Gene Expression Profiles of Cumulus Cell Oocyte Complexes during Ovulation Reveal Cumulus Cells Express Neuronal and Immune-Related Genes: Does this Expand Their Role in the Ovulation Process? Mol. Endocrinol., June 1, 2006; 20(6): 1300 - 1321. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/11/1237 03-0927fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by JUNG, C.-G. Articles by NISHINO, H. Search for Related Content PubMed PubMed Citation Articles by JUNG, C.-G. Articles by NISHINO, H.