Mechanical activation of β‐catenin regulates phenotype in adult murine marrow‐derived mesenchymal stem cells

Abstract

Regulation of skeletal remodeling appears to influence the differentiation of multipotent mesenchymal stem cells (MSC) resident in the bone marrow. As murine marrow cultures are contaminated with hematopoietic cells, they are problematic for studying direct effects of mechanical input. Here we use a modified technique to isolate marrow-derived MSC (mdMSC) from adult mice, yielding a population able to differentiate into adipogenic and osteogenic phenotypes that is devoid of hematopoietic cells. In pure mdMSC populations, a daily strain regimen inhibited adipogenic differentiation, suppressing expression of PPAR␥ and adiponectin. Strain increased ␤-catenin and inhibition of adipogenesis required this effect. Under osteogenic conditions, strain activated ␤-catenin signaling and increased expression of WISP1 and COX2. mdMSC were also generated from mice lacking caveolin-1, a protein known to sequester ␤-catenin: caveolin-1 (−/−) mdMSC exhibited retarded differentiation along both adipogenic and osteogenic lineages but retained mechanical responses that involved ␤-catenin activation. Interestingly, caveolin-1 (−/−) mdMSC failed to express bone sialoprotein and did not form mineralized nodules. In summary, mdMSC from adult mice respond to both soluble factors and mechanical input, with mechanical activation of ␤-catenin influencing phenotype. As such, these cells offer a useful model for studies of direct mechanical regulation of MSC differentiation and function. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J. Orthop. Res. 28: 1531Res. 28: -1538Res. 28: , 2010 Keywords: stem cell; osteoblast; adipocyte; mechanical loading; caveolin-1Maintenance of the skeleton is a process which must balance diverse inputs that importantly include mechanical information. This information not only influences differentiated cells resident within the bone tissue but may also have critical effects on the fate of multipotent mesenchymal stem cells (MSC) located in the bone marrow. For example, Wnt/␤-catenin signaling, associated with promoting bone formation, 1 appears to primarily impact early events during MSC lineage determination.2 Similarly, mechanical input activates ␤-catenin and promotes expression of Wnt/␤-catenin target genes in bone cells, 3−6 suggesting that loading effects may be most relevant during MSC differentiation. Indeed, recent studies support a mechanical influence on MSC fate in animals: daily exposure to extremely low magnitude mechanical stimulation increases MSC proliferation, with a biasing of the MSC population toward osteogenesis, 7 and mice subjected to a climbing regimen have more osteoblasts and fewer adipocytes in the marrow cavity.8 Conversely, hindlimb unloading increases the potential for adipogenesis in ex vivo marrow cultures. In vitro experimentation has shown that mechanical information promotes the osteogenic potential of precursor cells. 10 In contrast, daily loading of murine embryonic MSC inhibits adipocyte differentiation. 11Loading of primary marrow ce...

Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells

“…First, the multipotential nature of the MSCs allows an investigation of cellular transition into several different cell types using the same starting material. Second, marrow-derived MSCs can be isolated from transgenic or knock-out animal models, permitting investigations of the contributions of the genetic manipulations to the growth and differentiation of the cells (34). Finally, due to their accessibility from patients, the differentiation and reprogramming of MSCs ex vivo have become a focus in clinical therapeutics and drug discovery.…”

“…Cell Culture and Differentiation-Marrow-derived MSCs were isolated from female C57bl/6 mice, cryopreserved, and expanded through no more than 10 passages (34,35). The MSCs were cultured in minimum Eagle's medium ␣ modification supplemented with 10% fetal bovine serum from Hyclone (Logan, UT) and 1% penicillin-streptomycin from Invitrogen.…”

“…As outlined in Fig. 1 and detailed under "Experimental Procedures," marrow-derived cells were isolated from C57bl/6 mice, and selected MSCs were cultured and validated as described previously (34,35). MSCs were then expanded for differentiation, staining, ChIP-seq, and RNA-seq analyses.…”

Mechanisms of Enhancer-mediated Hormonal Control of Vitamin D Receptor Gene Expression in Target Cells

“…UAMS-32PB (UAMS-PB) cells, which are mouse UAMS-32 stromal/osteoblastic cells expressing blasticidin-resistant PTH receptor (31), were kindly provided by Charles O'Brien (University of Arkansas Medical School). Mesenchymal stem cells (MSCs) were derived from C57BL/6 mice (Harlan Teklad) as reported previously (32). The three cell lines mentioned above and UAMS-PB cells in which genomes were edited by the CRISPR/Cas9 system as described below were cultured in minimum Eagle's medium ␣ modification supplemented with 10% FBS and 1% penicillin/streptomycin.…”

“…Importantly, a VDR enhancer profile similar to that obtained in MC3T3-E1 cells was also identified for the VDR in the UAMS-PB cell line, a finding that strongly supports the validity of each of these three enhancers in this cell type despite their cell line nature. As regulatory enhancers are frequently established early on within the development of a cell lineage, we also examined in a separate analysis whether these three individual Vdr gene enhancers were similarly located within an MSC precursor with the proven potential to give rise to not only osteoblastic cells but to additional mesenchymal lineage-derived cells such as chondrocytes and adipocytes as well (32). As seen in Fig.…”