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K. Xu et al.
significantly higher in brains and spleens of the groups with mild and severe stroke than in sham-treated group brains and spleens (P<0.001) (Figure 6C,D). Animals with mild or severe stroke, but not sham-treated animals, had significantly more triple stain-positive cells in both organs on day 3, relative to other days, especially the group with severe stroke (P<0.05) (Figure 6C,D).
Human bone marrow mesenchymal stromal cells migrate toward bEnd.3, BV2, and bEnd.3+BV2 cells, a process enhanced by tumor necrosis factor-a treatment
hBMSC migrated toward lymphatic endothelial cells (bEnd.3, expressing LYVE-1), microglia (BV2), and a com- bination of bEnd.3+BV2 cells in in vitro cell migration assays (Figure 7A-F). Administering TNF-a escalated hBMSC migration in a dose-dependent manner. At differ- ent time points (0 h - 72 h), the density of hBMSC migrat- ing toward bEnd.3 (Figure 7A,B), BV2 (Figure 7C,D), and bEnd.3+BV2 (Figure 7E,F) increased significantly with pro-
gressively higher concentrations of TNF-a (P<0.05). Additionally, the density of hBMSC migration to bEnd.3, BV2, and bEnd.3+BV2 cells increased over time (Figure 7B,D,F). Cell migration to bEnd.3+BV2 peaked at 48 h with the highest TNF-a dose (Figure 7F), which is consis- tent with the in vivo results showing that the number of hBMSC was highest in the group with severe stroke on day 3 (Figure 2D). BV2 cells had the highest density of hBMSC migration relative to bEnd.3 and bEnd.3+BV2 groups at all time points, demonstrating that hBMSC pref- erentially migrated to BV2 cells (P<0.01) (Figure 7G-J).
Discussion
The brain is traditionally known to play an essential role in governing and coordinating systemic homeostasis. In recent years, it has become increasingly clear that brain health and neurological diseases are intimately associated with other physiological systems.28,29 Growing evidence
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Figure 3. Visualization of human bone marrow mesenchymal stromal cells within lymphatic vessels in the brain and spleen. (A and B) Human bone marrow mes- enchymal stromal cells (hBMSC) were stained with human nuclei (HuNu) and lymphatic endothelial cells were stained with lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) to reveal co-localization of hBMSC and lymphatic endothelial cells in the brain (A) and spleen (B). Arrow heads indicate co-localization of HuNu- positive and LYVE-1-positive cells and show that hBMSC localized within lymphatic vessels in the brain and spleen. Small boxes show 40x magnification. Scale bars = 100 mm. Red: LYVE-1; green: HuNu; blue: DAPI. (A) Images were taken close to the dural sinuses in the brain. (B) Images were taken near to the gate of the spleen, close to the white pulp in the spleen. (C and D) Quantitative analyses of the estimated number of co-localized HuNu-positive and LYVE-1-positive cells in the brain (C) and in the spleen (D) of animals with mild or severe stroke and sham-treated animals. Significance bars: *P<0.05; **P<0.01; ***P<0.001. (C) No significant differences were found in the magnitude of co-localization in the brain between sham-treated groups and those with mild or severe stroke on all days that the assess- ments were made (P>0.05). (D) Groups with mild and severe stroke displayed significantly higher quantities of co-localization in the spleen relative to the sham-treat- ed group on all days the measurements were made (*P<0.05). Staining overlap was most prominent on day 3, especially in the groups with severe stroke. a: The group with severe stroke had significantly more co-localized cells in the spleen on day 3 than on other days (P<0.001).
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haematologica | 2019; 104(5)