Abstract 19496: Development of a Novel Model to Mimic Ischemic Microenvironments
Targeting the harsh ischemic microenvironment cell-based therapies have been limited due to altered viability, homing, retention, and functionality of injected cells. To mimic the complexity of ischemia, hypoxia, hypercapnia, excess potassium, metabolic waste accumulation, acidosis, nutrient depletion, and oxidative/inflammatory stress need to be included. Since current models do not reflect this complexity, we aimed to develop a novel model that closely mimics tissue ischemia.
Bone-marrow mononuclear cells (BMNC), largely been used for cell-based therapy, were isolated from 8-weeks-old, male FVB mice. Cells were subjected to standard (ctrl) or novel ischemic conditions (IS; 1% O2, 20% CO2, nutrient depletion, metabolic waste accumulation). In addition, BMNCs were co-cultured with muscle slices from ischemic hind-limbs to account for oxidative/inflammatory stress. As determined by microscopy (HE, TUNEL/sarcomeric actin) tissue slices were stable for >7 days. In serial gas analyses (n=3 for all) the culture milieu showed reduced pO2 (ctrl 179±9 vs. IS 99±6 mmHg, p<0.0001), increased pCO2 (ctrl 35±1 vs. IS 99±7 mmHg, p<0.0001), an acidic pH (ctrl 7.36±0.01 vs. IS 6.99±0.03, p<0.0001), lactate (ctrl 1.6±0.6 vs. IS 3.3±0.9, p<0.05), and K+ accumulation (ctrl 4.8±0.1214 vs. 6.9±0.3, p<0.05). BMNCs remained viable under these conditions (FACS, Life/Death assay, day 7: IS 63.4± 2.7%; n=2). RT-PCR revealed an induction of ischemia-responsive genes (normalized expression vs. ctrl: VEGF 5.7±1.6, HO1 1.6±0.5, iNOS 289.7±176.8 fold, n=2). Consequently, 717 genes were up- and 250 down-regulated ≥2-fold in response to this novel model after 24hrs of ischemia (Whole Mouse Genome Oligo Microarray, Agilent). Ongoing functional clustering and probability-driven prediction analyses will identify interesting genes for functional investigation.
In summary, we developed a novel model that closely mimics the complexity of the ischemic microenvironment. This model allows better understanding of adaptive mechanisms and unmet needs of therapeutic cells within the ischemic target zone. Further insights will enable prior modification and/or the development of supportive biomaterials to enhance the therapeutic potency of stem/progenitor cells.
Author Disclosures: A. Menon: None. L. Napp: None. P. Galuppo: None. J. Bauersachs: None. J. Tongers: None.
- © 2014 by American Heart Association, Inc.