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Reduced Activation of RhoA Mediates Altered Differentiation of Human Mesenchymal Stem Cells in MMG.  V. E. Meyers1, M. Zayzafoon1, J.M. McDonald1 1Department of Pathology, University of Alabama at Birmingham, Birmingham, AL USA.

Bone loss in microgravity has been documented since the inception of human spaceflight.  However, the mechanisms responsible for this phenomenon remain elusive.  We have previously demonstrated reduced ostoeblastogenesis and increased adipogenesis of human mesenchymal stem cells (hMSC) cultured in modeled microgravity (MMG).  The small GTPase, RhoA, has recently been shown to mediate the lineage decision of hMSC.  RhoA is recognized primarily for its role in stress fiber formation and cytoskeletal organization.  Therefore, we examined the effects of MMG on RhoA activation and cytoskeletal organization in hMSC.  Cells were seeded onto polystyrene microcarrier beads, and aggregates were allowed to form for one week in DMEM containing 10% FBS.  Aggregates were then transferred to a rotary cell culture system or maintained at normal gravity under osteogenic conditions for 7 days.  Disruption of stress fibers occurs in hMSC as early as 3 hours after induction of MMG, as determined by Alexa594-conjugated phalloidin staining.  After 7 days of MMG, stress fibers are completely absent.  Likewise, 7 days of MMG led to an 88 + 2% reduction in activation of RhoA.  Consistent with decreased RhoA activity and our previous data indicating an increase in adipocytic markers, intracellular lipid staining increased substantially in cells cultured in MMG.  In summary, the data presented here provide a novel mechanism for decreased osteoblastic and increased adipocytic differentiation of hMSC in MMG, namely disruption of the actin cytoskeleton and reduced Rho activity.