Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

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Multilineage cells from human adipose tissue: implications for cell-based therapies.

The last decade has provided a virtual explosion of data on the molecular biology and function of osteocytes. Far from being the “passive placeholder in bone,” this cell has been found to have numerous functions, such as acting as an orchestrator of bone remodeling through regulation of both osteoclast and osteoblast activity and also functioning as an endocrine cell. The osteocyte is a source of soluble factors not only to target cells on the bone surface but also to target distant organs, such as kidney, muscle, and other tissues. This cell plays a role in both phosphate metabolism and calcium availability and can remodel its perilacunar matrix. Osteocytes compose 90% to 95% of all bone cells in adult bone and are the longest lived bone cell, up to decades within their mineralized environment. As we age, these cells die, leaving behind empty lacunae that frequently micropetrose. In aged bone such as osteonecrotic bone, empty lacunae are associated with reduced remodeling. Inflammatory factors such as tumor necrosis factor and glucocorticoids used to treat inflammatory disease induce osteocyte cell death, but by different mechanisms with potentially different outcomes. Therefore, healthy, viable osteocytes are necessary for proper functionality of bone and other organs. © 2011 American Society for Bone and Mineral Research.

/pdf/the-amazing-osteocyte-3wma8pwcxq.pdf

The Amazing Osteocyte

https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(10)62349-5.pdf

Osteoporosis: now and the future

Palladium-catalyzed amination reactions of aryl halides have undergone rapid development in the last 12 years, largely driven by the implementation of new classes of ligands. Biaryl phosphanes have proven to provide especially active catalysts in this context. This Review discusses the application of these catalysts in C-N cross-coupling reactions in the synthesis of heterocycles and pharmaceuticals, in materials science, and in natural product synthesis.

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Biaryl Phosphane Ligands in Palladium‐Catalyzed Amination

Mesenchymal stem cells: clinical applications and biological characterization

SOST is a target gene for PTH in bone.

β-Catenin-dependent canonical Wnt signaling plays an important role in bone metabolism by controlling differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. To investigate its function in osteocytes, the cell type constituting the majority of bone cells, we generated osteocyte-specific β-catenin-deficient mice (Ctnnb1loxP/loxP; Dmp1-Cre). Homozygous mutants were born at normal Mendelian frequency with no obvious morphological abnormalities or detectable differences in size or body weight, but bone mass accrual was strongly impaired due to early-onset, progressive bone loss in the appendicular and axial skeleton with mild growth retardation and premature lethality. Cancellous bone mass was almost completely absent, and cortical bone thickness was dramatically reduced. The low-bone-mass phenotype was associated with increased osteoclast number and activity, whereas osteoblast function and osteocyte density were normal. Cortical bone Wnt/β-catenin target gene expression was reduced, and of the known regulators of osteoclast differentiation, osteoprotegerin (OPG) expression was significantly downregulated in osteocyte bone fractions of mutant mice. Moreover, the OPG levels expressed by osteocytes were higher than or comparable to the levels expressed by osteoblasts during skeletal growth and at maturity, suggesting that the reduction in osteocytic OPG and the concomitant increase in osteocytic RANKL/OPG ratio contribute to the increased number of osteoclasts and resorption in osteocyte-specific β-catenin mutants. Together, these results reveal a crucial novel function for osteocyte β-catenin signaling in controlling bone homeostasis.

https://journals.asm.org/doi/pdf/10.1128/MCB.01428-09

Osteocyte Wnt/β-Catenin Signaling Is Required for Normal Bone Homeostasis

Mutations in distant regulatory elements can negatively impact human development and health, yet due to the difficulty of detecting these critical sequences we predominantly focus on coding sequences for diagnostic purposes We have undertaken a comparative sequence-based approach to characterize a large noncoding region deleted in patients affected by Van Buchem disease (VB), a severe sclerosing bone dysplasia Using BAC recombination and transgenesis we characterized the expression of human sclerostin (sost) from normal (hSOSTwt) or Van Buchem(hSOSTvb D) alleles Only the hSOSTwt allele faithfully expressed high levels of human sost in the adult bone and impacted bone metabolism, consistent with the model that the VB noncoding deletion removes a sost specific regulatory element By exploiting cross-species sequence comparisons with in vitro and in vivo enhancer assays we were able to identify a candidate enhancer element that drives human sost expression in osteoblast-like cell lines in vitro and in the skeletal anlage of the E145 mouse embryo, and discovered a novel function for sclerostin during limb development Our approach represents a framework for characterizing distant regulatory elements associated with abnormal human phenotypes

/pdf/genomic-deletion-of-a-long-range-bone-enhancer-misregulates-1c5xq8fodh.pdf

Genomic deletion of a long-range bone enhancer misregulates sclerostin in Van Buchem disease

Intermittent parathyroid hormone (PTH) treatment is a potent bone anabolic principle that suppresses expression of the bone formation inhibitor Sost. We addressed the relevance of Sost suppression for PTH-induced bone anabolism in vivo using mice with altered Sost gene dosage. Six-month-old Sost overexpressing and 2-month-old Sost deficient male mice and their wild-type littermates were subjected to daily injections of 100 microg/kg PTH(1-34) or vehicle for a 2-month period. A follow-up study was performed in Sost deficient mice using 40 and 80 microg/kg PTH(1-34). Animals were sacrificed 4 hours after the final PTH administration and Sost expression in long bone diaphyses was determined by qPCR. Bone changes were analyzed in vivo in the distal femur metaphysis by pQCT and ex vivo in the tibia and lumbar spine by DXA. Detailed ex vivo analyses of the femur were performed by pQCT, microCT, and histomorphometry. Overexpression of Sost resulted in osteopenia and Sost deletion in high bone mass. As shown before, PTH suppressed Sost in wild-type mice. PTH treatment induced substantial increases in bone mineral density, content, and cortical thickness and in aging wild-type mice also led to cancellous bone gain owing to amplified bone formation rates. PTH-induced bone gain was blunted at all doses and skeletal sites in Sost overexpressing and deficient mice owing to attenuated bone formation rates, whereas bone resorption was not different from that in PTH-treated wild-type controls. These data suggest that suppression of the bone formation inhibitor Sost by intermittent PTH treatment contributes to PTH bone anabolism.

Parathyroid hormone (PTH)–induced bone gain is blunted in SOST overexpressing and deficient mice

Expression of the osteocyte-derived bone formation inhibitor sclerostin in adult bone requires a distant enhancer. We show that MEF2 transcription factors control this enhancer and mediate inhibition of sclerostin expression by PTH.



Introduction: Sclerostin encoded by the SOST gene is a key regulator of bone formation. Lack of SOST expression is the cause for the progressive bone overgrowth disorders sclerosteosis and Van Buchem disease. We have previously identified a distant enhancer within the 52-kb Van Buchem disease deletion downstream of the SOST gene that is essential for its expression in adult bone. Furthermore, we and others have reported that SOST expression is suppressed by PTH. The aim of this study was to identify transcription factors involved in SOST bone enhancer activity and mediating PTH responsiveness.



Materials and Methods: Regulation of the SOST enhancer and promoter was studied by luciferase reporter gene assays. Transcription factor binding sites were mapped by footprint analysis and functional mutation analyses using transient transfections of osteoblast-like UMR-106 cells that exhibit endogenous SOST expression. Specific transcription factor binding was predicted by sequence analysis and shown by gel retardation assays and antibody-induced supershifts. Expression of myocyte enhancer factors 2 (MEF2) was detected by in situ hybridization, quantitative RT-PCR (qPCR), and immunohistochemistry. The role of MEF2s in SOST expression was assessed by reporter gene assays and siRNA-mediated RNA knockdown.



Results: PTH completely suppressed the transcriptional activity of the SOST bone enhancer but did not affect the SOST promoter. A MEF2 response element was identified in the bone enhancer. It was essential for transcriptional activation, bound MEF2 transcription factors, and mediated PTH responsiveness. Expression of MEF2s in bone was shown by qPCR, in situ hybridization, and immunohistochemistry. MEF2s and sclerostin co-localized in osteocytes. Enhancer activity was stimulated by MEF2C overexpression and inhibited by co-expression of a dominant negative MEF2C mutant. Finally, siRNA-mediated knockdown of MEF2A, C, and D suppressed endogenous SOST expression in UMR-106 osteoblast-like cells.



Conclusions: These data strongly suggest that SOST expression in osteocytes of adult bone and its inhibition by PTH is mediated by MEF2A, C, and D transcription factors controlling the SOST bone enhancer. Hence, MEF2s are implicated in the regulation of adult bone mass.

Hansjoerg Keller

Papers

SOST is a target gene for PTH in bone.

Osteocyte Wnt/β-Catenin Signaling Is Required for Normal Bone Homeostasis

Genomic deletion of a long-range bone enhancer misregulates sclerostin in Van Buchem disease

Parathyroid hormone (PTH)–induced bone gain is blunted in SOST overexpressing and deficient mice

Control of the SOST bone enhancer by PTH using MEF2 transcription factors.