Murine wild-type (WT) and TLR4KO MSCs were harvested from bone marrow and grown in?vitro. A total of 0.1 ¡Á 106 cells/well were incubated in hypoxic conditions versus normoxic controls. After 24 h, these groups were examined for cell survival via counting and compared using a t-test with P < 0.05 = statistical significance. AKT and ERK concentrations were measured in lysate using Western blot analysis.
The morphology of WT and TLR4KO MSCs was similar. In line with our previous findings, hypoxia did significantly increase cell death in WT cells (1.79 ¡Á 105 living cells/mL control versus 0.88 ¡Á 105 hypoxia, P < 0.05). Hypoxic injury did not increase cell death in the TLR4KO group (1.68 ¡Á 105 control versus 1.82 ¡Á 105 hypoxia, P < 0.05). Increased AKT activation was observed in all TLR4KO groups. TLR4 did not affect phosphorylated ERK levels.
TLR4-knockout MSCs show improved survival after hypoxic injury because of increased AKT pathway signal. Use of TLR4-knockout MSCs in ischemia/reperfusion studies results in enhanced cardioprotection; improved stem cell survival was likely a contributing factor.