Source Paper
Krisztián Németh, Asada Leelahavanichkul, Peter S T Yuen, Balázs Mayer, Alissa Parmelee et al.
Nature Medicine • 2008
Objective: To evaluate the effect of bone marrow stromal cells (BMSCs) on mortality and organ function in mice with sepsis induced by cecal ligation and puncture (CLP)
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Induce sepsis in mice using the cecal ligation and puncture procedure
Note: This is the primary surgical intervention to create the sepsis model
“inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function”
Administer bone marrow stromal cells to mice before or shortly after CLP-induced sepsis
Note: Timing of administration is flexible (before or shortly after CLP)
“Administering bone marrow stromal cells (BMSCs—also known as mesenchymal stem cells) to mice before or shortly after inducing sepsis”
Deplete macrophages in some mice to test the role of macrophages in BMSC-mediated protection
Note: This is a control/mechanistic experiment to determine if macrophages are necessary for BMSC benefit
“The beneficial effect of BMSCs was eliminated by macrophage depletion”
Pretreat mice with antibodies specific for IL-10 or IL-10 receptor to block these signaling pathways
Note: This is a control/mechanistic experiment to determine if IL-10 signaling is necessary for BMSC benefit
“pretreatment with antibodies specific for interleukin-10 (IL-10) or IL-10 receptor”
Prepare monocytes and/or macrophages from lungs of septic mice for analysis of IL-10 production
Note: Samples collected from both BMSC-treated and untreated septic mice for comparison
“Monocytes and/or macrophages from septic lungs made more IL-10 when prepared from mice treated with BMSCs versus untreated mice”
Culture LPS-stimulated macrophages with or without BMSCs to measure IL-10 production
Note: This is an ex vivo assay to test the direct effect of BMSCs on macrophage IL-10 production
“LPS-stimulated macrophages produced more IL-10 when cultured with BMSCs”
Use BMSCs lacking specific genes (Toll-like receptor 4, myeloid differentiation primary response gene-88, TNF receptor-1a, or cyclooxygenase-2) in culture experiments to determine which genes are necessary for the IL-10 production effect
Note: This mechanistic experiment identifies the molecular pathways required for BMSC-mediated macrophage reprogramming
“this effect was eliminated if the BMSCs lacked the genes encoding Toll-like receptor 4, myeloid differentiation primary response gene-88, tumor necrosis factor (TNF) receptor-1a or cyclooxygenase-2”
Measure tissue peroxidase as a marker of organ injury
Note: Used to assess organ function following sepsis and BMSC treatment
“K.N. formulated the molecular mechanism hypothesis and designed and performed in vitro and ex vivo assays; B.M. performed the measurements for tissue peroxidase”
Perform flow cytometry analysis to characterize cell populations
Note: Used for detailed cellular analysis
“I.J. performed FACS experiments”
Some mice were genetically modified to lack specific genes (Toll-like receptor 4, myeloid differentiation primary response gene-88, TNF receptor-1a, or cyclooxygenase-2)