The ability of cells to detect changes in the microenvironment is important in cell signaling and responsiveness to environmental fluctuations. Our interest is in understanding how human bone marrow stromal-derived cells (MSC) and their relatives, vascular smooth muscle cells (VSMC), interact with their environment through novel receptors. We found, through a proteomics screen, that MSC express the bitter taste receptor, TAS2R46, a protein more typically localized to the taste bud. Expression was also confirmed in VSMCs. A prototypical bitter compound that binds to the bitter taste receptor class, denatonium, increased intracellular calcium release and decreased cAMP levels as well as increased the extracellular release of ATP in human MSC. Denatonium also bound and activated rodent VSMC with a change in morphology upon compound exposure. Finally, rodents given denatonium in vivo had a significant drop in blood pressure indicating a vasodilator response. This is the first description of chemosensory detection by MSC and VSMCs via a taste receptor. These data open a new avenue of research into discovering novel compounds that operate through taste receptors expressed by cells in the marrow and vascular microenvironments.

Background Mesenchymal Stromal Cells (MSC) are gaining in popularity as an experimental therapy for a number of conditions that often require expansion ex vivo prior to use. Data comparing clinical-grade MSC from various ages of donors is scant. We hypothesized that MSC from older donors may display differences in cellular fitness when expanded for clinical use. Methods We evaluated the expression of several markers of aging, oxidative stress, and growth kinetics, and telomere length in MSCs obtained from a wide age range (8 months to 58 years). Results To evaluate cellular fitness we compared MSC expanded from younger (8 months - 6 years) versus older (38 - 58 years) donors in terms of selected cell surface markers, lipofuscin, migration ability, telomere length, and expression of iNOS, PGE2, p16INK, and SOD. Results did not differ between these groups. Neither SOD activity (0.025 vs 0.028 U/ml) nor death after oxidative challenge were significantly different (1% vs 1.5%, p=0.14). We did find that although MSC from older individuals produced slightly fewer cells over a 28-day culture period and have slightly longer doubling time (54 hrs vs 42 hrs), a satisfactory clinical product can still be ob tained regardless of age cohort. Discussion Collectively, these data show that MSC can be expanded without significant alterations in expansile properties or obvious changes in parameters associated with senescence. Because cellular fitness was equivalent in these cohorts, MSC from donors up to age 58 years can be used as a source of cells for cellular therapy.

938 The ability of cells to detect molecules in the microenvironment is important in cell signaling and cell responsiveness to environmental changes. Through an isobaric tag for relative and absolute quantitation (iTRAQ) based proteomic screen of human bone marrow stroma-derived cells (MSC) at the third passage we identified the novel expression of a bitter taste receptor, TAS2R46 in undifferentiated MSC and MSC differentiated into osteocytes, adipocytes, and cartilage. The relative amount of the receptor by iTRAQ and qRT-PCR was equivalent in all cell types. TAS2R46 expression was verified by flow cytometry, immunohistochemistry, and RT-PCR. Expression of TAS2R46 was also found in freshly obtained bone marrow mononuclear cells, and sorting by flow cytometry showed that the taste receptor positive cells had the ability to become MSC in vitro, while significantly fewer cells became MSC in the taste receptor negative/low fraction. Other members of the bitter taste receptor family including TAS2R4, TAS2R5, TAS2R1, TAS2R38 were negative by flow cytometry and iTRAQ. Bitter compounds, of which the prototypical molecule is denatonium, were found to increase intracellular human MSC calcium levels in a dose-responsive manner up to a level of 200% above baseline in fluorescent intracellular calcium detection assays. Other bitter compounds tested were caffeine, thujone, and salicin, and quinine gave rise to increased intracellular calcium levels. The specificity of the calcium response was verified through transgenic overexpression experiments, antibody inhibition, and a novel newly developed direct labeling method in which we were to directly label denatonium and show binding to the cell surface. Evaluation of downstream signaling events showed that interaction with this receptor caused a decrease in cAMP levels of 40% with exposure to 3 mM denatonium. Finally, to reveal the potential that this receptor may have, a native physiologically relevant function related to small peptide binding, a casein hydrosylate was used as a substrate. Remarkably, casein hydrosylate caused a similar increase in intracellular calcium as denatonium. Furthermore, this effect was augmented when TAS2R46 was overexpressed as a transgene. Current studies are working towards revealing the specific peptide that binds to this receptor. This is the first description of chemosensory detection by MSC. Our data show that this environmental detection occurs through a novel expression of a bitter taste receptor, TAS2R46. This ability may allow MSC to sample changes in their microenvironment triggered by small molecules, which could include either toxins or hormones as true natural ligands of this receptor class. The expression of bitter taste receptors may influence the response of MSCs to noxious materials or pathological insults to the organism that are detected as bitter substrates. Future studies will examine the directed mobility of MSCs to such stimuli which may offer new insights as to how MSCs sense organism injury. Disclosures: No relevant conflicts of interest to declare.

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