Aims To investigate the mechanisms underlying the beneficial effect of hypoxia preconditioning (HPC) on mesenchymal stromal cells (MSCs) and optimize novel non-invasive methods to assess the effect of biological interventions aimed to increased cell survival. Main methods MSCs from rat femur, with or without HPC, were exposed to hypoxic conditions in cell culture (1% O2 for 24 h) and cell survival (by the LDH release assay and Annexin-V staining) was measured. Oxidant status (conversion of dichloro-fluorescein-DCF- and dihydro-ethidium-DHE-, protein expression of oxidant enzymes) was characterized, together with the mobility pattern of cells under stress. Furthermore, cell survival was assessed non-invasively using state-of-the-art molecular imaging. Key findings Compared to controls, Hypoxia resulted in increased expression of the oxidative stress enzyme NAD(P)H oxidase (subunit 67phox: 0.05 ± 0.01 AU and 0.48 ± 0.02 AU, respectively, p<0.05) and in the amount of ROS (DCF: 13 ± 1 and 42 ± 3 RFU/µg protein, respectively, p<0.05) which led to a decrease in stem cell viability. Hypoxia preconditioning preserved cell biology, as evidenced by preservation of oxidant status (16 ± 1 RFU/µg protein, p<0.05 vs. hypoxia), and cell viability. Most importantly, the beneficial effect of HPC can be assessed non-invasively using molecular imaging. Significance HPC preserves cell viability and function, in part through preservation of oxidant status, and its effects can be assessed using state-of-the-art molecular imaging. Understanding of the mechanisms underlying the fate of stem cells will be critical for the advancement of the field of stem cell therapy.

Low phytic acid (LPA) genotypes of wheat (Triticum aestivum L.) improve the nutritional quality of wheat by reducing the concentration of phytic acid (PA) in the aleurone layer, thus reducing the chelation of nutritionally important minerals and improving the bioavailability of phosphorus. Field studies were conducted at Aberdeen and Tetonia, ID, in 2003 and to evaluate the effects of the LPA genotype on the agronomic performance of wheat. These studies included wildtype (WT) and LPA genotypes in hard red spring, hard white spring, and soft white spring wheat genetic backgrounds. In the hard red spring genetic background, LPA genotypes had delayed development and reduced grain yield (8-25%) in the high yield environment, in part due to reduced kernel size (up to 3 mg kernel 21 ). In the hard white spring genetic background, differences in crop development and grain yield were not observed; however, in the high yield environment LPA genotypes produced smaller kernels (2.0-2.4 mg kernel 21 ). In the soft white spring genetic background, LPA genotypes developed earlier, but the grain yield of LPA genotypes was reduced 20 to 24% in the high yield environment. However, LPA kernels, on average, were heavier and larger in diameter than WT kernels. The absence of consistent effects of the LPA genotype across the three genetic backgrounds suggests that deleterious effects of the LPA genotype may be mitigated by plant breeding.

Objective Polycystic kidney disease (PKD) is a common cause of end stage renal failure and many of these patients suffer vascular dysfunction and hypertension. It remains unclear whether PKD is associated with abnormal microvascular structure. Thus, this study examined the renovascular structure in PKD. Methods PKD rats (PCK model) and controls were studied at 10 weeks of age, and mean arterial pressure (MAP), renal blood flow and creatinine clearance were measured. Microvascular architecture and cyst number and volume were assessed using micro-computed tomography, and angiogenic pathways evaluated. Results Compared to controls, PKD animals had an increase in MAP (126.4±4.0 vs. 126.2±2.7mmHg) and decreased clearance of creatinine (0.39±0.09 vs. 0.30±0.05ml/min), associated with a decrease in microvascular density, both in the cortex (256±22 vs. 136±20 vessels per cm2) and medullar (114±14 vs. 50±9 vessels/cm2) and an increase in the average diameter of glomeruli (104.14±2.94 vs. 125.76±9.06 mm). PKD animals had increased fibrosis (2.2±0.2 fold vs. control) and a decrease in the cortical expression in hypoxia inducible factor 1-α and vascular endothelial growth factor. Conclusion PKD animals have impaired renal vascular architecture, which can have significant functional consequences. The PKD microvasculature could represent a therapeutic target to decrease the impact of this disease.

Objectives We aimed to validate a pathway-specific reporter gene that could be used to noninvasively image the oxidative status of progenitor cells. Background In cell therapy studies, reporter gene imaging plays a valuable role in the assessment of cell fate in living subjects. After myocardial injury, noxious stimuli in the host tissue confer oxidative stress to transplanted cells that may influence their survival and reparative function. Methods Rat mesenchymal stromal cells (MSCs) were studied for phenotypic evidence of increased oxidative stress under in vitro stress. On the basis of their upregulation of the pro-oxidant enzyme NAD(P)H oxidase p67phox, an oxidative stress sensor was constructed, comprising the firefly luciferase (Fluc) reporter gene driven by the NAD(P)H p67phox promoter. MSCs co-transfected with NAD(P)H p67phox-Fluc and a cell viability reporter gene (CMV-Renilla luciferase) were studied under in vitro and in vivo pro-oxidant conditions. Results After in vitro validation of the sensor during low serum culture, transfected MSCs were transplanted into a rat model of myocardial ischemia/reperfusion (IR) and monitored by bioluminescence imaging. Compared to sham controls (no IR), cardiac Fluc intensity was significantly higher in IR rats (3.5-fold at 6 h, 2.6-fold at 24 h, 5.4-fold at 48 h, P<0.01), indicating increased cellular oxidative stress. This was corroborated by ex vivo luminometry after correcting for renilla luciferase (Rluc) activity as a measure of viable MSC number (Fluc:Rluc ratio 0.011 ± 0.003 for sham versus 0.026 ± 0.004 for IR at 48 h, P<0.05). Furthermore, in IR animals that received MSCs preconditioned with an anti-oxidant agent (tempol), Fluc signal was strongly attenuated, substantiating the specificity of the oxidative stress sensor. Conclusions Pathway-specific reporter gene imaging allows assessment of changes in the oxidative status of MSCs after delivery to ischemic myocardium, providing a template to monitor key biological interactions between transplanted cells and their host environment in living subjects.

Polycystic kidney disease (PKD) is a common cause of end stage renal failure, for which there is no accepted treatment. Progenitor and stem cells have been shown to restore renal function in a model of renovascular disease, a disease that shares many features with PKD. The objective of this study was to examine the potential of adult stem cells to restore renal structure and function in PKD. Bone marrow-derived mesenchymal stromal cells (MSCs, 2.5×105) were intrarenally infused in 6 week-old PCK rats. At 10 weeks of age, PCK rats had an increase in systolic blood pressure (SBP) vs. controls (126.22±2.74 vs. 116.45±3.53mmHg, p<0.05) and decreased creatinine clearance (3.76±0.31 vs. 6.10±0.48µl/min/g, p<0.01), which were improved in animals that received MSCs (SBP: 114.67±1.34mmHg, and creatinine clearance: 4.82±0.24µl/min/g, p=0.001 and p=0.003 vs. PKD, respectively). MSCs preserved vascular density and glomeruli diameter, measured using micro-computed tomography. PCK animals had increased urine osmolality (843.9±54.95 vs. 605.6±45.34mOsm, p<0.01 vs. control), which was improved after MSC infusion and not different from control (723.75±56.6mOsm, p=0.13 vs. control). Furthermore, MSCs reduced fibrosis and preserved the expression of the pro-angiogenic molecules, while cyst size and number were unaltered by MSCs. Delivery of exogenous MSCs improved vascular density and renal function in PCK animals, and the benefit was observed up to four weeks after a single infusion. Cell-based therapy constitutes a novel approach in PKD.