Hypoxia impacts human MSC response to substrate stiffness during chondrogenic differentiation

Tissue engineering strategies often aim to direct tissue formation by mimicking conditions progenitor cells experience within native tissues. For example, to create cartilage in vitro, researchers often aim to replicate the biochemical and mechanical milieu cells experience during cartilage formation in the developing limb bud. This includes stimulating progenitors with TGF-β1/3, culturing under hypoxic conditions, and regulating mechanosensory pathways using biomaterials that control substrate stiffness and/or cell shape. However, as progenitors differentiate down the chondrogenic lineage, the pathways that regulate their responses to mechanotransduction, hypoxia and TGF-β may not act independently, but rather also impact one another, influencing overall cell response. Here, to better understand hypoxia's influence on mechanoregulatory-mediated chondrogenesis, we cultured human marrow stromal/mesenchymal stem cells (hMSC) on soft (0.167 kPa) or stiff (49.6 kPa) polyacrylamide hydrogels in chondrogenic medium containing TGF-β3. We then compared cell morphology, phosphorylated myosin light chain 2 staining, and chondrogenic gene expression under normoxic and hypoxic conditions, in the presence and absence of pharmacological inhibition of cytoskeletal tension. We show that on soft compared to stiff substrates, hypoxia prompts hMSC to adopt more spread morphologies, assemble in compact mesenchymal condensation-like colonies, and upregulate NCAM expression, and that inhibition of cytoskeletal tension negates hypoxia-mediated upregulation of molecular markers of chondrogenesis, including COL2A1 and SOX9. Taken together, our findings support a role for hypoxia in regulating hMSC morphology, cytoskeletal tension and chondrogenesis, and that hypoxia's effects are modulated, at least in part, by mechanosensitive pathways. Our insights into how hypoxia impacts mechanoregulation of chondrogenesis in hMSC may improve strategies to develop tissue engineered cartilage

Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells

This article has been made available through the Brunel Open Access Publishing Fund and is available from the specified link - 2010 by The American Society of HematologyThe directed differentiation of human pluripotent stem cells offers the unique opportunity to generate a broad spectrum of human cell types and tissues for transplantation, drug discovery, and studying disease mechanisms. Here, we report the stepwise generation of bone-resorbing osteoclasts from human embryonic and induced pluripotent stem cells. Generation of a primitive streak-like population in embryoid bodies, followed by specification to hematopoiesis and myelopoiesis by vascular endothelial growth factor and hematopoietic cytokines in serum-free media, yielded a precursor population enriched for cells expressing the monocyte-macrophage lineage markers CD14, CD18, CD11b, and CD115. When plated in monolayer culture in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand (RANKL), these precursors formed large, multinucleated osteoclasts that expressed tartrate-resistant acid phosphatase and were capable of resorption. No tartrate-resistant acid phosphatase-positive multinucleated cells or resorption pits were observed in the absence of RANKL. Molecular analyses confirmed the expression of the osteoclast marker genes NFATc1, cathepsin K, and calcitonin receptor in a RANKL-dependent manner, and confocal microscopy demonstrated the coexpression of the alpha v beta 3 integrin, cathepsin K and F-actin rings characteristic of active osteoclasts. Generating hematopoietic and osteoclast populations from human embryonic and induced pluripotent stem cells will be invaluable for understanding embryonic bone development and postnatal bone disease. (Blood. 2010; 115(14): 2769-2776)This study was supported (in part) by research funding from the Nuffield Foundation-Oliver Bird Rheumatism Program to A.E.G., Arthritis Research Campaign (ARC ref 18197) to G.S., National Institutes of Health grants R01 HL080627 and P20 GM075019 to G.M.K., and Austrian Science Fund (FWF) and Anabonos EU-FP7 to E.F.W

Induction of neural crest stem cells from Bardet–Biedl syndrome patient derived hiPSCs

Neural crest cells arise in the embryo from the neural plate border and migrate throughout the body, giving rise to many different tissue types such as bones and cartilage of the face, smooth muscles, neurons, and melanocytes. While studied extensively in animal models, neural crest development and disease have been poorly described in humans due to the challenges in accessing embryonic tissues. In recent years, patient-derived human induced pluripotent stem cells (hiPSCs) have become easier to generate, and several streamlined protocols have enabled robust differentiation of hiPSCs to the neural crest lineage. Thus, a unique opportunity is offered for modeling neurocristopathies using patient specific stem cell lines. In this work, we make use of hiPSCs derived from patients affected by the Bardet–Biedl Syndrome (BBS) ciliopathy. BBS patients often exhibit subclinical craniofacial dysmorphisms that are likely to be associated with the neural crest-derived facial skeleton. We focus on hiPSCs carrying variants in the BBS10 gene, which encodes a protein forming part of a chaperonin-like complex associated with the cilium. Here, we establish a pipeline for profiling hiPSCs during differentiation toward the neural crest stem cell fate. This can be used to characterize the differentiation properties of the neural crest-like cells. Two different BBS10 mutant lines showed a reduction in expression of the characteristic neural crest gene expression profile. Further analysis of both BBS10 mutant lines highlighted the inability of these mutant lines to differentiate toward a neural crest fate, which was also characterized by a decreased WNT and BMP response. Altogether, our study suggests a requirement for wild-type BBS10 in human neural crest development. In the long term, approaches such as the one we describe will allow direct comparison of disease-specific cell lines. This will provide valuable insights into the relationships between genetic background and heterogeneity in cellular models. The possibility of integrating laboratory data with clinical phenotypes will move us toward precision medicine approaches

Recurrent rearrangements of FOS and FOSB define osteoblastoma.

The transcription factor FOS has long been implicated in the pathogenesis of bone tumours, following the discovery that the viral homologue, v-fos, caused osteosarcoma in laboratory mice. However, mutations of FOS have not been found in human bone-forming tumours. Here, we report recurrent rearrangement of FOS and its paralogue, FOSB, in the most common benign tumours of bone, osteoblastoma and osteoid osteoma. Combining whole-genome DNA and RNA sequences, we find rearrangement of FOS in five tumours and of FOSB in one tumour. Extending our findings into a cohort of 55 cases, using FISH and immunohistochemistry, provide evidence of ubiquitous mutation of FOS or FOSB in osteoblastoma and osteoid osteoma. Overall, our findings reveal a human bone tumour defined by mutations of FOS and FOSB

Sarcoma treatment in the era of molecular medicine

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.Peer reviewe

The osteogenic potential of the neural crest lineage may contribute to craniosynostosis

AbstractThe craniofacial skeleton is formed from the neural crest and mesodermal lineages, both of which contribute mesenchymal precursors during formation of the skull bones. The large majority of cranial sutures also include a proportion of neural crest derived mesenchyme. While some studies have addressed the relative healing abilities of neural crest and mesodermal bone, relatively little attention has been paid to differences in intrinsic osteogenic potential. Here we use mouse models to compare neural crest osteoblasts (from frontal bones or dura mater) to mesodermal osteoblasts (from parietal bones). Usingin vitroculture approaches we find that neural crest-derived osteoblasts readily generate bony nodules while mesodermal osteoblasts do so less efficiently. Furthermore, we find that co-culture of neural crest-derived osteoblasts with mesodermal osteoblasts is sufficient to nucleate ossification centres. All together, this suggests that the intrinsic osteogenic abilities of neural crest-derived mesenchyme may be a primary driver behind craniosynostosis.</jats:p

Podoplanin Is Regulated by AP-1 and Promotes Platelet Aggregation and Cell Migration in Osteosarcoma

Podoplanin is a type-I transmembrane sialomucin-like protein, which is expressed in a wide range of cell types and is involved in platelet aggregation and tumor metastasis. Here, we investigated the function, regulation, and expression of podoplanin in osteosarcoma. Podoplanin expression was observed in three osteosarcoma cell lines (MG-63, HOS, and U-2 OS) with platelet aggregation–inducing ability, which was blocked by podoplanin small-interfering RNA or a neutralizing antibody. Overexpression of podoplanin in nonmetastatic Dunn osteosarcoma cells promoted cell migration without attenuating cell proliferation. Both podoplanin and TGF-β1 were up-regulated by c-Fos induction in MC3T3-E1 osteoblastic cells, and were highly expressed in c-Fos transgenic mouse osteosarcomas and c-Fos–transformed osteosarcoma cell lines. Immunohistochemistry of human osteosarcoma tissue microarrays (n = 133) showed staining of tumor cells embedded in an excess of irregular neoplastic bone matrix in 100% of tumors undergoing so-called “normalization/maturation.” Podoplanin was also expressed in osteosarcoma subtypes, with 65% of osteoblastic, 100% of chondroblastic, and 79% of fibroblastic tumors. CD44 and pERM immunohistochemistry showed coexpression with podoplanin in both mouse and human osteosarcoma. Podoplanin expression was significantly higher in metastatic osteosarcomas (n = 6) than in primary osteosarcomas (n = 10). Our data suggest that podoplanin, which is not expressed in normal osteoblasts but in osteocytes, is aberrantly expressed in transformed osteoblasts and in osteosarcoma, and is under AP-1 transcriptional control. Thus podoplanin is a candidate molecule for therapeutic targeting