Browsing by Author "Hunsberger, E. Bailey"

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    Human Adipose Stromal Cells Increase Survival and Mesenteric Perfusion Following Intestinal Ischemia and Reperfusion Injury
    (Wolters Kluwer, 2016-07) Jensen, Amanda. R.; Doster, Dominique L.; Hunsberger, E. Bailey; Manning, Morenci M.; Stokes, Samantha M.; Barwinska, Daria; March, Keith L.; Yoder, Mervin C.; Markel, Troy A.; Surgery, School of Medicine
    OBJECTIVE: Intestinal ischemia can quickly escalate to bowel necrosis and perforation. Transplantation of stem cells presents a novel treatment modality for this problem. We hypothesized that: human adipose-derived stromal cells (hASCs) would increase survival and mesenteric perfusion to a greater degree compared with differentiated cellular controls following ischemic intestinal injury, and improved outcomes with hASC therapy would be associated with preservation of intestinal histological and tight junction architecture, and lower levels of systemic inflammation following intestinal injury. METHODS: hASCs and keratinocytes (differentiated cellular control) were cultured on polystyrene flasks at 37°C in 5% CO2 in air. Adult male C57Bl6J mice were anesthetized and a midline laparotomy performed. The intestines were eviscerated, the small bowel mesenteric root identified, and intestinal ischemia was established by temporarily occluding the superior mesenteric artery for 60 min with a noncrushing vascular clamp. Following ischemia, the clamp was removed, and the intestines were returned to the abdominal cavity. Before abdominal closure, 2 million hASCs or keratinocytes in 250 μL of phosphate-buffered saline (carrier for cells and control solution) were infused into the peritoneum. Animals were allowed to recover for 12 or 24 h (perfusion, histology, cytokine, and immunofluoresence studies), or 7 days (survival studies). Intestinal perfusion was assessed by laser Doppler imaging. Intestinal tissue segments were stained with hematoxylin and eosin, as well as antibodies for the tight junction protein claudin-1. Separate aliquots of intestine, liver, and lung tissue were homogenized and assessed for inflammatory cytokines via multiplex beaded assay. RESULTS: Animals administered hASCs following intestinal ischemia and reperfusion (I/R) injury had significantly greater 7-day survival and better postischemic recovery of mesenteric perfusion compared with vehicle or keratinocyte therapy. hASCs also abated intestinal mucosal destruction, facilitated preservation of intestinal tight junctions, and decreased the systemic inflammatory response to injury. CONCLUSIONS: Human adipose-derived stromal cells improved survival and mesenteric perfusion and attenuated the mucosal damage associated with intestinal I/R injury. hASCs should be considered as a plausible cell source for novel cellular treatment plans following intestinal ischemia.
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    Human Mesenchymal Stromal Cells Decrease Mortality Following Intestinal Ischemia and Reperfusion Injury
    (Elsevier, 2015-11) Markel, Troy A.; Crafts, Trevor D.; Jensen, Amanda R.; Hunsberger, E. Bailey; Department of Surgery, IU School of Medicine
    Background Cellular therapy is a novel treatment option for intestinal ischemia. Bone marrow–derived mesenchymal stromal cells (BMSCs) have previously been shown to abate the damage caused by intestinal ischemia/reperfusion (I/R) injury. We therefore hypothesized that (1) human BMSCs (hBMSCs) would produce more beneficial growth factors and lower levels of proinflammatory mediators compared to differentiated cells, (2) direct application of hBMSCs to ischemic intestine would decrease mortality after injury, and (3) decreased mortality would be associated with an altered intestinal and hepatic inflammatory response. Methods Adult hBMSCs and keratinocytes were cultured on polystyrene flasks. For in vitro experiments, cells were exposed to tumor necrosis factor, lipopolysaccharides, or 2% oxygen for 24 h. Supernatants were then analyzed for growth factors and chemokines by multiplex assay. For in vivo experiments, 8- to 12-wk-old male C57Bl6J mice were anesthetized and underwent a midline laparotomy. Experimental groups were exposed to temporary superior mesenteric artery occlusion for 60 min. Immediately after ischemia, 2 × 106 hBMSCs or keratinocytes in phosphate-buffered saline were placed into the peritoneal cavity. Animals were then closed and allowed to recover for 6 h (molecular/histologic analysis) or 7 d (survival analysis). After 6-h reperfusion, animals were euthanized. Intestines and livers were harvested and analyzed for inflammatory chemokines, growth factors, and histologic changes. Results hBMSCs expressed higher levels of human interleukin (IL) 6, IL-8, vascular endothelial growth factor (VEGF), and epidermal growth factor and lower levels of IL-1, IL-3, IL-7, and granulocyte-monocyte colony-stimulating factor after stimulation. In vivo, I/R resulted in significant mortality (70% mortality), whereas application of hBMSCs after ischemia decreased mortality to 10% in a dose-dependent fashion (P = 0.004). Keratinocyte therapy offered no improvements in mortality above I/R. Histologic profiles were equivalent between ischemic groups, regardless of the application of hBMSCs or keratinocytes. Cellular therapy yielded significantly decreased murine intestinal levels of soluble activin receptor-like kinase 1, betacellulin, and endothelin, whereas increasing levels of eotaxin, monokine induced by gamma interferon (MIG), monocyte chemoattractant protein 1, IL-6, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein 10 (IP-10) from ischemia were appreciated. hBMSC therapy yielded significantly higher expression of murine intestinal VEGF and lower levels of intestinal MIG compared to keratinocyte therapy. Application of hBMSCs after ischemia yielded significantly lower murine levels of hepatic MIG, IP-10, and G-CSF compared to keratinocyte therapy. Conclusions Human BMSCs produce multiple beneficial growth factors. Direct application of hBMSCs to the peritoneal cavity after intestinal I/R decreased mortality by 60%. Improved outcomes with hBMSC therapy were not associated with improved histologic profiles in this model. hBMSC therapy was associated with higher VEGF in intestines and lower levels of proinflammtory MIG, IP-10, and G-CSF in liver tissue after ischemia, suggesting that reperfusion with hBMSC therapy may alter survival by modulating the systemic inflammatory response to ischemia.