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Our research of stem cell transplantation using mouse embryonic stem (ES) cells and induced pluripotent (iPS) cells was carried out from the aspect of trophic factor, cell differentiation, and better survival of grafted cells. Pleiotrophin, an enhanced trophic factor in the dopamine (DA)-depleted striatum, increased the number of DAergic neurons from ES-derived neural stem cells (ES-NSCs), increased cell survival of cultured DAergic neurons, and affected cell survival of grafted DAergic cells in Parkinson model rats. It was shown that DAergic differentiation from ES-NSCs was mediated by hypoxia inducible factor 1-alpha. Our challenges of the transplantation of ES-NSCs and iPS-derived oligodendrocyte progenitor cells (iPS-OPCs) into periventricular leukomalasia (PVL) model rats are also presented. It was found that grafted ES-NSCs survived better in the corpus callosum without immunosuppressant and most of them differentiated into neurons near the grafted site. It was also revealed that only a few of the grafted iPS-OPCs induced by a stepwise culture method with no use of serum could survive in PVL model rats, indicating that trophic factor (s) and improvement of graft techniques will be needed for better survival of grafted iPS-OPCs.

Perinatal hypoxia-ischemia (HI) frequently causes white-matter injury, leading to severe neurological deficits and mortality, and only limited therapeutic options exist. The white matter of animal models and human patients with HI-induced brain injury contains increased numbers of oligodendrocyte progenitor cells (OPCs). However, the origin and fates of these OPCs and their potential to repair injured white matter remain unclear. Here, using cell-type- and region-specific genetic labeling methods in a mouse HI model, we characterized the Olig2-expressing OPCs. We found that after HI, Olig2+ cells increased in the posterior part of the subventricular zone (pSVZ) and migrated into the injured white matter. However, their oligodendrocytic differentiation efficiency was severely compromised compared with the OPCs in normal tissue, indicating the need for an intervention to promote their differentiation. Erythropoietin (EPO) treatment is a promising candidate, but it has detrimental effects that preclude its clinical use for brain injury. We found that long-term postinjury treatment with a nonerythropoietic derivative of EPO, asialo-erythropoietin, promoted the maturation of pSVZ-derived OPCs and the recovery of neurological function, without affecting hematopoiesis. These results demonstrate the limitation and potential of endogenous OPCs in the pSVZ as a therapeutic target for treating neonatal white-matter injury., (Copyright © 2012 AlphaMed Press.)

The hypoxia-responsive cytokine erythropoietin (EPO) provides neuroprotective effects in the damaged brain during ischemic events and neurodegenerative diseases. The purpose of the present study is to evaluate the EPO/EPO receptor (EPOR) endogenous system between astrocyte and oligodendrocyte precursor cell (OPC) under hypoxia. We report here elevated EPO mRNA levels and protein release in cultured astrocytes following hypoxic stimulation by quantitative RT-PCR and ELISA. Furthermore, the EPOR gene expressions were detected in cultured OPCs as in astrocytes and microglias by quantitative RT-PCR. Cell staining revealed the EPOR expression in OPC. To evaluate the protective effect of endogenous EPO from astrocyte to OPCs, EPO/EPOR signaling was blocked by EPO siRNA or EPOR siRNA gene silencing in in vitro study. The suppression of endogenous EPO production in astrocytes by EPO siRNA decreased the protection to OPCs against hypoxic stress. Furthermore, OPC with EPOR siRNA had less cell survival after hypoxic/reoxygenation injury. This suggested that EPO/EPOR signaling from astrocyte to OPC could prevent OPC damage under hypoxic/reoxygenation condition. Our present finding of an interaction between astrocytes and OPCs may lead to a new therapeutic approach to OPCs for use against cellular stress and injury., (Copyright © 2011 Wiley-Liss, Inc.)

We have previously demonstrated that a G1/S-phase cell cycle blocker, deferoxamine (DFO), increased the number of new neurons from rat neurosphere cultures, which correlated with prolonged expression of cyclin-dependent kinase (cdk) inhibitor p27(kip1) [H. J. Kim et al. (2006)Brain Research, 1092, 1-15]. The present study focuses on neuronal differentiation mechanisms following treatment of neural stem/progenitor cells (NPCs) with a G1/S-phase cell cycle blocker. The addition of DFO (0.5 mm) or aphidicolin (Aph) (1.5 microm) to neurospheres for 8 h, followed by 3 days of differentiation, resulted in an increased number of neurons and neurite outgrowth. DFO induced enhanced expression of transforming growth factor (TGF)-beta1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF-beta1 expression. DFO-induced neurogenesis and neurite outgrowth were attenuated by administration of a cdk5 inhibitor, roscovitine, suggesting that the neurogenic mechanisms differ between DFO and Aph. TGF-beta1 (10 ng/mL) did not increase neurite outgrowth but rather the number of beta-tubulin III-positive cells, which was accompanied by enhanced p27(kip1) mRNA expression. In addition, TGF-beta receptor type II expression was observed in nestin-positive NPCs. Results indicated that DFO-induced TGF-beta1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF-beta1 signaling inhibition, via a TGF-beta receptor type I inhibitor (SB-505124), resulted in decreased DFO-induced neurogenesis, in conjunction with decreased p27(kip1) protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S-phase induces TGF-beta1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27(kip1) activation in NPCs.

Neural stem/progenitor cells (NPCs) reside in the subventricular zone (SVZ) and dentate gyrus in the adult mammalian brain. It has been reported that endogenous NPCs are activated after brain insults such as ischemic stroke. We investigated whether proliferation and migration of endogenous NPCs are increased after a collagenase-induced small intracerebral hemorrhage (ICH) near the internal capsule in rats. Bromodeoxyuridin (BrdU) administration for 14 days after ICH (post-labeling) resulted in an increase in the number of BrdU-positive cells as shown in both ipsilateral and contralateral SVZs. BrdU treatment given for 2 days before ICH to label endogenous NPCs (pre-labeling), caused more BrdU-positive cells to be detected in the ipsilateral dorsal striatum (dSTR) compared to those in the contralateral dSTR 14 days after ICH. BrdU- and doublecortin (Dcx)-positive cells were found in the ipsilateral STR. An increase in the number of Dcx-positive migrating immature neurons was found in the dSTR and peri-hemorrhage area 14 days after ICH, and a cluster of Dcx-positive cells was found in the STR around the lesion 28 days after ICH. Matrix metalloproteinase-2 (MMP-2) was strongly expressed in wide area of the injured brain, particularly around the lesion 14 and 28 days after ICH. Dcx- and MMP-2-positive cells were detected in the ipsilateral STR near the lesion. These data suggest that collagenase-induced ICH enhances the proliferation of endogenous NPCs and the migration of newly born neuroblasts toward the hemorrhage area.

Neural transplantation is a promising approach for treating neurodegenerative disease. Neural stem/progenitor cells (NPCs) are self-renewing and multipotent and thus are good candidates for donor cells when they have been clearly defined to differentiate into neurons. As neuronal differentiation follows cell cycle exit, we investigated whether neuron production from NPCs is increased by treatment with cell cycle blockers. NPCs from E12.5 rat ventral mesencephalon were cultured as neurospheres in DMEM/F12 medium containing N2 supplements and bFGF. Treatment of NPCs with deferoxamine, a G1/S phase blocker, increased the number of beta-tubulin III-positive cells after differentiation, concomitant with increases of MAP2 mRNA and protein, and a decrease of GFAP protein. Further, an increase in beta-tubulin III/BrdU double-positive cells and a decrease in GFAP/BrdU double-positive cells were confirmed. In real-time PCR, the expressions of p21(cip1), p27(kip1) and p57(kip2) mRNAs remained unaltered for 8 h after treatment with deferoxamine but were significantly elevated after 1 day. Deferoxamine specifically enhanced the elevation of p27(kip1) mRNA at 1-2 days and the accumulation of p27(kip1) protein at 3 days, along with the activation of neuroD promoter and the elevation of neuroD mRNA. Transfection of p27(kip1) into NPCs induced activation of neuroD promoter and increase of number of beta-tubulin III-positive cells. These data suggest that pretreatment with deferoxamine increases the number of neurons from NPCs related to prolonged p27(kip1) elevation and activation of the neuroD signaling pathway. In this way, regulation of the cell cycle should be a useful first step in engineering NPCs for neural transplantation.

Neural stem cells are promising candidates for donor cells in neural transplantation. However, the mechanism by which neural stem cells differentiate into neurons is not well understood. In the present study, a serial analysis of gene expression (SAGE) was carried out to generate a gene file of neural stem (progenitor) cells from the mouse ventral mesencephalon. Among the 15,815 tags investigated, the mRNA of the housekeeping genes (elongation factor 1-alpha, ATPase subunit 6, GAPDH, actin), laminin receptor 1, HSP 70, pleiotrophin, and nestin were highly expressed. Because pleiotrophin (PTN) exhibits mitogenic and trophic effects on neural development and exhibits trophic effects on survival of dopaminergic (DAergic) neurons, we investigated the role of PTN in neurogenesis, especially to DAergic neurons. Here, we show that PTN increased the production of tyrosine hydroxylase (TH)-positive neurons from embryonic stem (ES) cell-derived nestin-positive cells. The expression of Nurr1 mRNA was enhanced by PTN. L-dopa in the culture medium was increased by PTN. This effect was as strong as with sonic hedgehog. Data suggest that PTN mRNA is highly expressed in neural stem (progenitor) cells of mouse ventral mesencephalon, and PTN promotes the production of DAergic neurons from ES cell-derived nestin-positive cells.