Low Intensity Vibrations Augment Mesenchymal Stem Cell Proliferation and Differentiation Capacity During \u3ci\u3ein vitro\u3c/i\u3e Expansion

A primary component of exercise, mechanical signals, when applied in the form of low intensity vibration (LIV), increases mesenchymal stem cell (MSC) osteogenesis and proliferation. While it is generally accepted that exercise effectively combats the deleterious effects of aging in the musculoskeletal system, how long-term exercise affects stem cell aging, which is typified by reduced proliferative and differentiative capacity, is not well explored. As a first step in understanding the effect of long-term application of mechanical signals on stem cell function, we investigated the effect of LIV during in vitro expansion of MSCs. Primary MSCs were subjected to either a control or to a twice-daily LIV regimen for up to sixty cell passages (P60) under in vitro cell expansion conditions. LIV effects were assessed at both early passage (EP) and late passage (LP). At the end of the experiment, P60 cultures exposed to LIV maintained a 28% increase of cell doubling and a 39% reduction in senescence-associated β-galactosidase activity (p \u3c 0.01) but no changes in telomere lengths and p16INK4a levels were observed. Prolonged culture-associated decreases in osteogenic and adipogenic capacity were partially protected by LIV in both EP and LP groups (p \u3c 0.05). Mass spectroscopy of late passage MSC indicated a synergistic decrease of actin and microtubule cytoskeleton-associated proteins in both control and LIV groups while LIV induced a recovery of proteins associated with oxidative reductase activity. In summary, our findings show that the application of long-term mechanical challenge (+LIV) during in vitro expansion of MSCs for sixty passages significantly alters MSC proliferation, differentiation and structure. This suggests LIV as a potential tool to investigate the role of physical activity during aging

Cell Mechanosensitivity to Extremely Low Magnitude Signals is Enabled by a LINCed Nucleus

A cell's ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSCs), high-magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of focal adhesion (FA) kinase (FAK)/mTORC2/Akt signaling generated at FAs. Physiologic systems also rely on a persistent barrage of low-level signals to regulate behavior. Exposing MSC to extremely low-magnitude mechanical signals (LMS) suppresses adipocyte formation despite the virtual absence of substrate strain (<0.001%), suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here, we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating FAK and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the FAs, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting linker of nucleoskeleton and cytoskeleton (LINC) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by HMS was unaffected by LINC decoupling, consistent with signal initiation at the FA mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with "outside-in" signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent "inside-inside" signaling

Cell Mechanosensitivity to Extremely Low-Magnitude Signals Is Enabled by a LINCed Nucleus: LINC Enables Sensing of Low-Intensity Vibrations

A cell’s ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSC), high magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of FAK/mTORC2/Akt signaling generated at focal adhesions [1]. Physiologic systems also rely on a persistent barrage of low level signals to regulate behavior [2]. Exposing MSC to extremely low magnitude mechanical signals (LMS) suppresses adipocyte formation [3] despite the virtual absence of substrate strain (<0.001%) [2], suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating focal adhesion kinase (FAK) and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the focal adhesions, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting LINC (Linker of Nucleoskeleton and Cytoskeleton) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by high magnitude strain (HMS) was unaffected by LINC decoupling, consistent with signal initiation at the focal adhesion (FA) mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with “outside-in” signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent “inside-inside” signaling

Sun-Mediated Mechanical LINC Between Nucleus and Cytoskeleton Regulates βcatenin Nuclear Access

βcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathways to control cell function and fate. Regulation of βcatenin in the cytoplasm has been well studied but βcatenin nuclear trafficking and function remains unclear. In a previous study we showed that, in mesenchymal stem cells (MSC), mechanical blockade of adipogenesis relied on inhibition of βcatenin destruction complex element GSK3β (glycogen synthase kinase 3β) to increase nuclear βcatenin as well as the function of Linker of Cytoskeleton and Nucleoskeleton (LINC) complexes, suggesting that these two mechanisms may be linked. Here we show that shortly after inactivation of GSK3β due to either low intensity vibration (LIV), substrate strain or pharmacologic inhibition, βcatenin associates with the nucleoskeleton, defined as the insoluble nuclear fraction that provides structure to the integrated nuclear envelope, nuclear lamina and chromatin. Co-depleting LINC elements Sun-1 and Sun-2 interfered with both nucleoskeletal association and nuclear entry of βcatenin, resulting in decreased nuclear βcatenin levels. Our findings reveal that the insoluble structural nucleoskeleton actively participates in βcatenin dynamics. As the cytoskeleton transmits applied mechanical force to the nuclear surface to influence the nucleoskeleton and its LINC mediated interaction, our results suggest a pathway by which LINC mediated connectivity may play a role in signaling pathways that depend on nuclear access of βcatenin

Role of Simulated Microgravity on Mechanically-induced Nuclear Shuttling of of YAP/TAZ in Mesenchymal Stem Cells

Bone deterioration in spaceflight is in part driven by reduced functionality of mesenchymal stem cells (MSC) that replace and regenerate musculoskeletal tissues by sensing and responding to environmental cues. In MSCs, mechanotransducers YAP and TAZ play critical roles in regulating growth and differentiation. The functionality of YAP/TAZ signaling requires them to shuttle into the nucleus to activate their target genes. Recent work from our group shows that altered gravity conditions in simulated microgravity (sMG) significantly decreased cell proliferation and compromised nuclear structure. This suggests that loss of form in sMG can compromise YAP/TAZ signaling in MSCs. Therefore, our main motivation is to identify the microgravity-mediated alterations in YAP/TAZ levels, compartmentalization and nuclear shuttling in response to mechanical stimuli. Here we hypothesize that sMG will decrease YAP/TAZ shuttling into nucleus in response to low intensity vibration (LIV, 90Hz, 0.7g) and mechanical strain (0.2Hz, 2%). YAP/TAZ compartmentalization will be compared between sMG treated MSCs and non-sMG controls after either acute single session of LIV or strain using cell fractionation and western blot analysis. Findings from this study will be critical for understanding the effects of spaceflight on MSC growth and differentiation via YAP/TAZ signaling

Low Intensity Vibrations Augment Proliferation and Differentiation in Aging Mesenchymal Stem Cells

Poor musculoskeletal health is one of the primary contributors to disability among aged individuals. Stem cell aging, typified by reduced proliferative and differentiative capacity, is in-part driven by decreased mechanosensory capabilities of cells –observed as attenuated exercise efficacy in older individuals. A principal source of mechanical signals that are universally recognized to maintain a healthy musculoskeletal system is exercise. A primary component of exercise, mechanical signals, when applied in the form of low intensity vibration (LIV), increases Mesenchymal Stem Cell (MSC) osteogenesis and decreases adipogenesis. Therefore, we asked if continuous application of LIV would increase proliferation and differentiation in an in vitro aging model. Two identical set of MSCs were kept sub-confluent (\u3c60%) and passaged twice weekly, while one set was LIV treated (Indicated as P5L, P7L etc…) twice daily (20min at 90Hz, 0.7g) the other set was not vibrated (indicated as P5, P7 etc...). LIV promoted proliferation rates, increasing cell-doubling 81% (p\u3c0.05) over 40 passages which resulted in 47% shorter telomeres (p\u3c0.05). Osteogenic capacity measured by ALP (Alkaline Phosphatase) expression at P30 against young P6 MSCs remained 58% higher in LIV-treated P30L MSCs (p\u3c0.05). In summary our findings that despite having shorter telomeres, long term LIV-treated MSCs remain proliferative, pro-osteogenic. We are currently investigating effects of long term LIV on acute FAK signaling, osteogenesis, adipogenesis and extracellular matrix deposition

The Role of Low Intensity Vibrations in Adipocyte Inflammatory Response

Adipocytes found in bone marrow and adipose tissues functions an energy depot for excess energy. Excessive lipid storage in adipocytes in cases like obesity leads to secretion of pro-inflammatory cytokines, causing chronic inflammation associated with type II diabetes and other metabolic disorders. Low intensity vibration (LIV) – an exercise mimetic – decreases fat accumulation in bone marrow and adipose tissues. While we have shown that LIV suppression of adipogenesis relies on activation of F-actin regulatory signaling pathways, the role of LIV in adipocyte inflammatory cytokine secretion is unknown.Using adipocyte progenitor mesenchymal stem cells (MSC), the goal of this study is to understand whether LIV can lower the inflammatory marker secretion of adipocytes. We hypothesize that the LIV sustains higher F-actin levels during MSC differentiation to adipocytes and decrease secretion of pro-inflammatory signaling molecule monocyte chemoattractant protein-1 (MCP-1). To test this hypothesis MSCs will be subjected to adipogenic differentiation media for five days. During differentiation MSCs will be subjected to LIV (90Hz, 0.7g) twice daily for 20 minutes, MCP-1 expression and protein levels will be measured. An additional group will be used to measure F-actin levels via immunostaining. Findings will reveal if LIV can decrease inflammation in adipose tissues

Low Intensity Vibrations Augment Mesenchymal Stem Cell Proliferation and Differentiation Capacity during in vitro Expansion

Abstract A primary component of exercise, mechanical signals, when applied in the form of low intensity vibration (LIV), increases mesenchymal stem cell (MSC) osteogenesis and proliferation. While it is generally accepted that exercise effectively combats the deleterious effects of aging in the musculoskeletal system, how long-term exercise affects stem cell aging, which is typified by reduced proliferative and differentiative capacity, is not well explored. As a first step in understanding the effect of long-term application of mechanical signals on stem cell function, we investigated the effect of LIV during in vitro expansion of MSCs. Primary MSCs were subjected to either a control or to a twice-daily LIV regimen for up to sixty cell passages (P60) under in vitro cell expansion conditions. LIV effects were assessed at both early passage (EP) and late passage (LP). At the end of the experiment, P60 cultures exposed to LIV maintained a 28% increase of cell doubling and a 39% reduction in senescence-associated β-galactosidase activity (p < 0.01) but no changes in telomere lengths and p16INK4a levels were observed. Prolonged culture-associated decreases in osteogenic and adipogenic capacity were partially protected by LIV in both EP and LP groups (p < 0.05). Mass spectroscopy of late passage MSC indicated a synergistic decrease of actin and microtubule cytoskeleton-associated proteins in both control and LIV groups while LIV induced a recovery of proteins associated with oxidative reductase activity. In summary, our findings show that the application of long-term mechanical challenge (+LIV) during in vitro expansion of MSCs for sixty passages significantly alters MSC proliferation, differentiation and structure. This suggests LIV as a potential tool to investigate the role of physical activity during aging