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Brain to spleen stem cell migration
did on other days, with the highest concentration found in the group with severe stroke (P<0.001) (Figure 4C,D).
OX6-positive cells localized near or within lymphatic vessels in the brain and spleen
OX6-positive cells localized near or within lymphatic vessels in the brain (Figure 5A) and spleen (Figure 5B) and their quantity was higher in animals with mild or severe stroke than in sham-treated animals. Groups with mild or severe stroke had more OX6-positive cells close to or within lymphatic vessels on day 3 than at other time points. OX6 expression near or within lymphatic vessels in the brain and spleen was significantly higher in the groups with mild and severe stroke than in the sham- treated group at all time points (P<0.05; P<0.01; P<0.001) (Figure 5C,D). On day 3, the brains and spleens of the groups with mild and severe stroke, but not sham-treated group brains and spleens, exhibited significantly more OX6-positive cells next to or within lymphatic vessels than they did on other days, with the highest concentra- tion found in the group with severe stroke (P<0.01) (Figure 5C,D).
Human specific phagocytic marker CD68, anti-apopto- sis inhibitor 5 and neuronal marker triple staining in the brain and spleen
Stroke-induced ischemic and apoptotic neurons result in inflammation. To test whether transplanted hBMSC can phagocytose ischemic neuronal cells in the brain and migrate to the periphery, we stained brain and spleen sec- tions with human specific phagocytic marker CD68, anti- apoptosis inhibitor 5, and anti-160kD neurofilament medium antibody-rat specific neuronal marker. The human specific phagocytic marker CD68 was employed to label the transplanted hBMSC demonstrating phagocyt- ic activity, the anti-apoptosis inhibitor 5 was utilized to mark rat neurons undergoing apoptosis, and the anti- 160kD neurofilament medium antibody-rat specific neu- ronal marker was used to label rat neurons. CD68 expres- sion was negligible for these hBMSC in vitro, suggesting that phagocytic expression may be triggered by the ischemic microenvironment of the transplant site. In the brain and in the spleen, apoptotic neuronal cells were found inside phagocytic human cells (Figure 6A,B). The amounts of cells positive for all three staining labels were
AC
B
D
Figure 2. Survival of human bone marrow mesenchymal stromal cells in the brain and spleen. (A) In the brain, transplanted human bone marrow mesenchymal stromal cells (hBMSC) express antigens for human nuclei (HuNu). The hBMSC grafts in the brain were positively stained with HuNu. (B) Representative merged images showing co-localization of HuNu-positive cells and 4,6-diaminodino-2-phenylindole (DAPI) for hBMSC in the spleen. (A and B) Arrow heads indicate HuNu-pos- itive cells. Scale bars = 100 mm. Green: HuNu; blue: DAPI. (C and D) Quantitative analyses of the estimated number of HuNu-positive hBMSC in the brain (C) and in the spleen (D) of stroke and sham-treated animals revealed more HuNu-positive cells survived in the brain and migrated to the spleen on day 3, especially in the groups with severe stroke. Significance bars: **P<0.01; ***P<0.001. (C) a: The group with mild stroke had significantly more HuNu-positive cells in the brain on day 3 than on other days (P<0.01); b: the group with severe stroke had significantly more HuNu-positive cells in the brain on day 3 than on other days (P<0.01). (D) a: The group with mild stroke had significantly more HuNu-positive cells in the spleen on day 3 than on other days (P<0.001); b: the group with severe stroke had sig- nificantly more HuNu-positive cells in the spleen on day 3 than on other days (P<0.001).
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