Transplanted bone marrow stem cells relocate to infarct penumbra and co-express endogenous proliferative and immature neuronal markers in a mouse model of ischemic cerebral stroke

Abstract

Background: Several studies demonstrate that neurogenesis may be induced or activated following vascular insults, which may be important for neuronal regeneration and functional recovery. Understanding the cellular mechanism underlying stroke-associated neurogenesis is of neurobiological as well as neurological/clinical relevance. The present study attempted to explore potential homing and early development of transplanted bone marrow stem cells in mouse forebrain after focal occlusion of the middle cerebral artery, an experimental model of ischemic stroke.

Results: Bone marrow stem cells isolated from donor mice were confirmed by analysis of surface antigen profile, and were pre-labeled with a lipophilic fluorescent dye PKH26, and subsequently transfused into recipient mice with middle cerebral artery coagulation. A large number of PKH26-labeled cells were detected surrounding the infarct site, most of which colocalized with immunolabelings for the proliferating cell nuclear antigen (PCNA) and some also colocalized with the immature neuronal marker doublecortin (DCX) during 1-2 weeks after the bone marrow cells transfusion.

Conclusions: The present study shows that transplanted bone marrow cells largely relocate to the infarct penumbra in ischemic mouse cerebrum. These transplanted bone marrow cells appear to undergo a process of in situ proliferation and develop into putative cortical interneurons during the early phase of experimental vascular injury.

Figure 1

Fluorescence-activated cell sorting analyses the expression of mesenchymal stem cell markers. APC-conjugated anti-mouse CD34 reactivity is detected on 9.67% of the isolated cells (A). PE-conjugated anti-mouse/human CD44 reactivity is present on 53.90% of the cells (B). Percp-conjugated anti-mouse CD45 antibody labels 60.04% of the cells (C), and FITC-conjugated anti-mouse LY-6A/EC Sca-1 antibody reacts to 27.25% of the cells (D).

Figure 2

Localization of transplanted cells and in situ proliferation at the infarct border in experimental model. (A-C) are images of coronal cerebral slices showing the impact of occlusion 6 hours post-occlusion, with the infarct appearing as pale area marked with broken line. Hematoxylin and eosin (H.E.) stain illustrates loss of relatively large cells and infiltration of small cells at the border of the infarct (D). Paired cellular profiles are seen peripheral to the infarct border or the infarct penumbra (E). At low magnification, a large number of transplanted bone marrow cells pre-labeled by PKH26 (red fluorescence) are present in the infarct penumbra 7 days post lesion (F). The labeled cells are small and may occur in cluster (G). No fluorescent cells exist in the cerebral cortex of mice received vehicle infusion (H). In situ cell division reflected by immunoreactivity of proliferating cell nuclear antigen (PCNA) occurs predominantly at the infarct border (I) and penumbra (J), appearing as brown immunoreactive nuclei in hematoxylin counter-stained section.

Figure 3

Identification of PKH26-labeled cells with proliferating cell nuclear antigen and doublecortin after transplantation. Colocalization of PKH26-labeled bone marrow cells with proliferating cell nuclear antigen (PCNA) (A-D) and doublecortin (DCX) (E-G) around the infarct penumbra 7 and 14 days following transplantation. PKH26 and PCNA double-labeled cells are small and occur in pair or small cluster (C, D). PKH26 and DCX double-labeled cells have round or oval somata with visible neuronal processes. PKH26 fluorescence appears weaker in these double-labeled cells relative to those seen at 7 days post cell transplantation. Scale bar (in B) = 200 μm for A, B and 50 μm for C-G.