Recovery of neurological function of ischemic stroke by application of conditioned medium of bone marrow mesenchymal stem cells derived from normal and cerebral ischemia rats

Abstract

Background: Several lines of evidence have demonstrated that bone marrow-derived mesenchymal stem cells (BM-MSC) release bioactive factors and provide neuroprotection for CNS injury. However, it remains elusive whether BM-MSC derived from healthy donors or stroke patients provides equal therapeutic potential. The present work aims to characterize BM-MSC prepared from normal healthy rats (NormBM-MSC) and cerebral ischemia rats (IschBM-MSC), and examine the effects of their conditioned medium (Cm) on ischemic stroke animal model.

Results: Isolated NormBM-MSC or IschBM-MSC formed fibroblastic like morphology and expressed CD29, CD90 and CD44 but failed to express the hematopoietic marker CD34. The number of colony formation of BM-MSC was more abundant in IschBM-MSC than in NormBM-MSC. This is in contrast to the amount of Ficoll-fractionated mononuclear cells from normal donor and ischemic rats. The effect of cm of BM-MSC was further examined in cultures and in middle cerebral artery occlusion (MCAo) animal model. Both NormBM-MSC Cm and IschBM-MSC Cm effectively increased neuronal connection and survival in mixed neuron-glial cultures. In vivo, intravenous infusion of NormBM-MSC Cm and IschBM-MSC Cm after stroke onset remarkably improved functional recovery. Furthermore, NormBM-MSC Cm and IschBM-MSC Cm increased neurogenesis and attenuated microglia/ macrophage infiltration in MCAo rat brains.

Conclusions: Our data suggest equal effectiveness of BM-MSC Cm derived from ischemic animals or from a normal population. Our results thus revealed the potential of BM-MSC Cm on treatment of ischemic stroke.

Figure 1

Characterization of BM-MSC cultured from normal or ischemic rats. (A,B) round-shape colonies of fibroblastic like NormBM-MSC and IschBM-MSC, respectively. (C,D) Proliferative activities of NormBM-MSC and IschBM-MSC were stained with anti-BrdU labeled FITC (green) and Hoechst (blue) incorporation. Magnification: 200× (E) Ficoll-fractionated mononuclear cell numbers from bone marrows of normal or ischemic rats. n = 13 ~ 16; *P < 0.05 (F) Numbers of colony forming unit-fibroblast (CFU-f) from bone marrow cultures at 6 and 14 days in vitro. n = 4 per group; *p < 0.05 IschBM-MSC vs. NormBM-MSC (G) Quantitative cell proliferation of NormBM-MSC and IschBM-MSC.

Figure 2

Flow cytometry analysis of bone marrow mesenchymal stem cells (BM-MSC) from normal and cerebral ischemic rats. (A) analysis of cell surface markers in NormBM-MSC (B) analysis of cell surface markers in ischBM-BMSC (C) comparative analysis of cell surface markers in NormBM-MSC and IschBM-MSC (D-E) FACS analyses of total cell populations in cultured NormBM-MSC (D) and IschBM-MSC (E). Cells (passages 1-3) were lifted, labeled with antibodies against the indicated antigens, and analyzed by flow cytometry. Only representative examples featuring antigen expression profiles are shown. Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 3

Conditioned medium from NormBM-MSC or IschBM-MSC increased cortical neuronal survival by enhancing neuronal connection and decreasing LDH release. (A) Neuronal cells, non-treated control. (B) NormBM-MSC Cm-treated neuronal cells (C) IschBM-MSC Cm-treated neuronal cells (D) βIII tubulin positive signal, as percentage of total area, quantified by Image J, in cortical neuronal cells. (E) LDH release to medium in cortical neuronal cells. Three days after treatment, cortical cell cultures were immunostained with anti-βIII-tubulin, a neuronal marker and medium were collected for LDH assay. Data are presented as means ± SEM from 8–10 independent experiments done in duplicate. *, **P < 0.05, 0.01 Treatment versus control. Magnification 200× (A-C).

Figure 4

Effects of administration of conditioned medium from NormBM-MSC and IschBM-MSC on infarct volume and functional behavior in MCAo rats. (A) The right lateral view of MCAo rat brains showing the stroke outcome from different treatments, (B) The infarct volumes of MCAo rat brains evaluated at 7 days postinjury using TTC staining analysis (C) Quantitative results of TTC staining of MCAo rat brains from different treatment, These results were taken as the mean ± S.D. from 8 ~ 15 rats/group (Control n = 8, NormBM-MSC Cm n = 15, IschBM-MSC n = 9), (D) Grip strength of left forelimbs (g) assessed using grasping power test on stroke-affected side (#p < 0.05 versus pre-MCAo, *p < 0.05 versus Control), (E) Grip strength of right forelimbs (unaffected side). Data were given as the mean ± S.D. from 6 ~ 7 rats/group (7 rats for control, 6 rats for NormBM-MSC Cm and 6 rats for IschBM-MSC Cm).

Figure 5

Promoted sprouting of neuronal progenitor cells in the lateral ventricles of MCAo rats treated with NormBM-MSC Cm and IschBM-MSC Cm. (A) The schematic picture displays the assessed lateral ventricle near hippocampus; Coronal sections of brain tissues from different treatment were immunostained with anti-doublecortin (DCX; green). DCX-positive cells were located along with lateral ventricles in the group given with BM-MSC Cm; magnification 200× (B): The immunoreactive area of DCX-positive cells was determined using Image-Pro Plus software. Representative data were taken as the means ± S.D. of three repetitions.

Figure 6

Attenuated infiltration of microglia/macrophage in the cortical regions of MCAo rats given NormBM-MSC Cm and IschBM-MSC Cm. (A) The illustration indicated the cortical region of the brain tissue was examined. Coronal sections of brain tissue were immunostained with anti-ED1 antibody labeled FITC. Immunofluorescent showed that ED-1 positive cells were abundant in the ischemic cortex on day 7 after MCAo (Control), whereas ED-1 positive cells were fewer in the group given NormBM-MSC Cm or IschBM-MSC Cm. ED-1 positive cells were absent in the unaffected hemisphere after MCAo. (B) The numbers of ED1 immunoreactive cells were determined using Image-Pro Plus software. Representative data were taken as the means ± S.D. of three repetitions.