Osteocyte specific responses to soluble and mechanical stimuli in a stem cell derived culture model.

Studying osteocyte behavior in culture has proven difficult because these embedded cells require spatially coordinated interactions with the matrix and surrounding cells to achieve the osteocyte phenotype. Using an easily attainable source of bone marrow mesenchymal stem cells, we generated cells with the osteocyte phenotype within two weeks. These "stem cell derived-osteocytes" (SCD-O) displayed stellate morphology and lacunocanalicular ultrastructure. Osteocytic genes Sost, Dmp1, E11, and Fgf23 were maximally expressed at 15 days and responded to PTH and 1,25(OH)2D3. Production of sclerostin mRNA and protein, within 15 days of culture makes the SCD-O model ideal for elucidating regulatory mechanisms. We found sclerostin to be regulated by mechanical factors, where low intensity vibration significantly reduced Sost expression. Additionally, this model recapitulates sclerostin production in response to osteoactive hormones, as PTH or LIV repressed secretion of sclerostin, significantly impacting Wnt-mediated Axin2 expression, via ß-catenin signaling. In summary, SCD-O cells produce abundant matrix, rapidly attain the osteocyte phenotype, and secrete functional factors including sclerostin under non-immortalized conditions. This culture model enables ex vivo observations of osteocyte behavior while preserving an organ-like environment. Furthermore, as marrow-derived mesenchymal stem cells can be obtained from transgenic animals; our model enables study of genetic control of osteocyte behaviors.

Mechanically activated Fyn utilizes mTORC2 to regulate RhoA and adipogenesis in mesenchymal stem cells.

Mechanical strain provides an anti-adipogenic, pro-osteogenic stimulus to mesenchymal stem cells (MSC) through generating intracellular signals and via cytoskeletal restructuring. Recently, mTORC2 has been shown to be a novel mechanical target critical for the anti-adipogenic signal leading to preservation of ß-catenin. As mechanical activation of mTORC2 requires focal adhesions (FAs), we asked whether proximal signaling involved Src and FAK, which are early responders to integrin-FA engagement. Application of mechanical strain to marrow-derived MSCs was unable to activate mTORC2 when Src family kinases were inhibited. Fyn, but not Src, was specifically required for mechanical activation of mTORC2 and was recruited to FAs after strain. Activation of mTORC2 was further diminished following FAK inhibition, and as FAK phosphorylation (Tyr-397) required Fyn activity, provided evidence of Fyn/FAK cooperativity. Inhibition of Fyn also prevented mechanical activation of RhoA as well as mechanically induced actin stress fiber formation. We thus asked whether RhoA activation by strain was dependent on mTORC2 downstream of Fyn. Inhibition of mTORC2 or its downstream substrate, Akt, both prevented mechanical RhoA activation, indicating that Fyn/FAK affects cytoskeletal structure via mTORC2. We then sought to ascertain whether this Fyn-initiated signal pathway modulated MSC lineage decisions. siRNA knockdown of Fyn, but not Src, led to rapid attainment of adipogenic phenotype with significant increases in adipocyte protein 2, peroxisome proliferator-activated receptor gamma, adiponectin, and perilipin. As such, Fyn expression in mdMSCs contributes to basal cytoskeletal architecture and, when associated with FAs, functions as a proximal mechanical effector for environmental signals that influence MSC lineage allocation.