Mesenchymal stem cells promote proliferation of endogenous neural stem cells and survival of newborn cells in a rat stroke model

Abstract

Mesenchymal stem cells (MSCs) secrete bioactive factors that exert diverse responses in vivo. In the present study, we explored mechanism how MSCs may lead to higher functional recovery in the animal stroke model. Bone marrow-derived MSCs were transplanted into the brain parenchyma 3 days after induction of stroke by occluding middle cerebral artery for 2 h. Stoke induced proliferation of resident neural stem cells in subventricular zone. However, most of new born cells underwent cell death and had a limited impact on functional recovery after stroke. Transplantation of MSCs enhanced proliferation of endogenous neural stem cells while suppressing the cell death of newly generated cells. Thereby, newborn cells migrated toward ischemic territory and differentiated in ischemic boundaries into doublecortin+ neuroblasts at higher rates in animals with MSCs compared to control group. The present study indicates that therapeutic effects of MSCs are at least partly ascribed to dual functions of MSCs by enhancing endogenous neurogenesis and protecting newborn cells from deleterious environment. The results reinforce the prospects of clinical application using MSCs in the treatment of neurological disorders.

Figures

Figure 1

Differentiation of expanded MSCs into mesenchymal lineage cells in vitro. MSCs from the 8th passage were induced to differentiate into mesenchymal lineage cells as described in the 'Materials and Methods'. (A) Phase-contrast picture of MSCs culture. (B) Adipogenesis was visualized by staining of lipid vacuoles with oil-red O. (C) Chondrogenesis was demonstrated with increased proteoglycan rich extracellular matrix by alcian blue staining. (D) Osteogenic differentiation of MSCs was demonstrated by the increased alkaline phosphatase activity. Scale bars, 100 µm.

Figure 2

Transplantation of MSCs ameliorates the behavioral impairments and reduces infarct volume in stroke model of rats. Behavioral performance in the rotarod (A) and adhesive removal (B) tests of PBS (open circles) or MSCs (closed circles) treated animals from 1 to 28 days after ischemia. (C) Brain infarct volumes of eight animals in each group were measured using MRI from 2 to 28 days after stroke. (D) Relative infarct volume of PBS (open bars) or MSCs (closed bars)-treated animals are presented as the mean ± S.D. Statistically significant differences between the MSCs group with PBS group were determined by ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). Tx indicates the time point of transplantation.

Figure 3

Endogenous neurogenesis induced by MSCs. Proliferation of endogenous NSCs and subsequent migration was demonstrated by double staining of BrdU with Dcx at 1 week and 2 weeks after MCAo. (A) Schematic illustration of region of investigated was shown (1, subventricular zone1, SVZ1; 2, subventricular zone2, SVZ2; 3, ischemic cortical boundary, ICB; 4, ischemic striatal boundary, ISB). (C, D) Proliferation of NSCs and subsequent migration of newly generated cells were detected by double staining for BrdU (green) and Dcx (red) in SVZ1 and SVZ2 at 1 week; or ICB and ISB at 2 weeks following transplantation of PBS or MSCs. (B) The numbers of BrdU+, Dcx+ cells in 0.25 mm2 of two regions (SVZ1 and SVZ2) indicated in (A) at 1 week or ICB and ISB at 2 weeks were quantified as described in the Methods. (E) Neuronal differentiation of newborn cells was demonstrated by double immunoreactivity to BrdU and NeuN in ICB at 2 weeks after MCAo. Orthographic images of the inset were acquired by higher magnified z-section. (F) Quantitative analysis of BrdU+ cells co-stained with NeuN in 0.25 mm2 of ICB were carried out. The data are presented as the mean numbers of positive cells ± S.D. (ANOVA, *p < 0.05, **p < 0.01). Arrows indicate the BrdU+ cells co-stained with Dcx (C, D) or NeuN (E); Arrowhead indicates BrdU+ cells that were not colocalized with Dcx or NeuN. Scale bars, 10 µm.

Figure 4

MSCs increase mitotic cell division from NSCs. Proliferation of NSCs resident in SVZ was demonstrated by immunoreactivity of Ki-67 at 1 week after MCAo. (A) Ki-67+ (brown) cells indicate the progenitor cells undergoing mitosis. (B) The numbers of Ki-67+ cells in 0.25 mm2 of SVZ were quantified as described in the Methods. The data are presented as the mean numbers of positive cells ± S.D. (ANOVA, **p < 0.01). Scale bars, 50 µm.

Figure 5

MSCs protect newborn cells from the ischemic environment. Prolonged survival of newborn cells was demonstrated by immunoreactivity to BrdU and TUNEL 2 weeks after MCAo. (A) The TUNEL+ (green) and BrdU+ (red) cells indicate the new born cells undergoing apoptotic cell death. Please note that most BrdU+ cells are TUNEL+ in the control animals. (B) Quantitative analysis of BrdU+ cells co-stained with TUNEL in 0.25 mm2 of ICB and ISB were carried out as described in the Methods. The data are presented as the mean numbers of positive cells ± S.D. (ANOVA, *p < 0.05). Arrows indicate BrdU+ cells colocalized with TUNEL; Arrowheads TUNEL+ cells devoid of BrdU in cortex or striatum. Scale bars, 10 µm.