Enhanced chondrogenesis from human embryonic stem cells.

Human embryonic stem cells (hESCs) have great potential for the repair of damaged articular cartilage. We developed a serum-free 14-day protocol for hESC differentiation into chondrocyte progenitors, which surprisingly lacked strong cartilage matrix production in in vitro tests. In order to direct these progenitors to a more mature phenotype, we investigated substituting different members of the TGFβ family in the protocol.

Chondrocytes Derived From Mesenchymal Stromal Cells and Induced Pluripotent Cells of Patients With Familial Osteochondritis Dissecans Exhibit an Endoplasmic Reticulum Stress Response and Defective Matrix Assembly.

Unlabelled: : Familial osteochondritis dissecans (FOCD) is an inherited skeletal defect characterized by the development of large cartilage lesions in multiple joints, short stature, and early onset of severe osteoarthritis. It is associated with a heterozygous mutation in the ACAN gene, resulting in a Val-Met replacement in the C-type lectin domain of aggrecan. To understand the cellular pathogenesis of this condition, we studied the chondrogenic differentiation of patient bone marrow mesenchymal stromal cells (BM-MSCs).

The development of a mature collagen network in cartilage from human bone marrow stem cells in Transwell culture.

Damaged hyaline cartilage shows a limited capacity for innate repair. Potential sources of cells to augment the clinical repair of cartilage defects include autologous chondrocytes and mesenchymal stem cells. We have reported that culture of human bone marrow mesenchymal stem cells with specific growth and differentiation factors as shallow multilayers on Transwell permeable membranes provided ideal conditions for chondrogenesis.

PhenomeExpress: a refined network analysis of expression datasets by inclusion of known disease phenotypes.

We describe a new method, PhenomeExpress, for the analysis of transcriptomic datasets to identify pathogenic disease mechanisms. Our analysis method includes input from both protein-protein interaction and phenotype similarity networks. This introduces valuable information from disease relevant phenotypes, which aids the identification of sub-networks that are significantly enriched in differentially expressed genes and are related to the disease relevant phenotypes.

Cartilage repair using human embryonic stem cell-derived chondroprogenitors.

In initial work, we developed a 14-day culture protocol under potential GMP, chemically defined conditions to generate chondroprogenitors from human embryonic stem cells (hESCs). The present study was undertaken to investigate the cartilage repair capacity of these cells. The chondrogenic protocol was optimized and validated with gene expression profiling.

Assembly of the respiratory mucin MUC5B: a new model for a gel-forming mucin.

Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer.

Generating cartilage repair from pluripotent stem cells.

The treatment of degeneration and injury of articular cartilage has been very challenging for scientists and surgeons. As an avascular and hypocellular tissue, cartilage has a very limited capacity for self-repair. Chondrocytes are the only cell type in cartilage, in which they are surrounded by the extracellular matrix that they secrete and assemble.

Cartilage tissue engineering approaches applicable in orthopaedic surgery: the past, the present, and the future.

Tissue is frequently damaged or lost in injury and disease. There has been an increasing interest in stem cell applications and tissue engineering approaches in surgical practice to deal with damaged or lost tissue. Although there have been developments in almost all surgical disciplines, the greatest advances are being made in orthopaedics, especially in cartilage repair.

The characterisation of mesenchymal stem cells: a stem cell is not a stem cell is not a stem cell.

There has been an increasing interest in stem cell applications and tissue engineering approaches in surgical practice to deal with damaged or lost tissue. Although there have been developments in almost all surgical disciplines, the greatest advances are being made in orthopaedics. This is due to many factors including the familiarity with bone marrow derived mesenchymal stem cells.

Mesenchymal stem cells, sources of cells and differentiation potential.

Tissue is frequently damaged or lost in injury and disease. There has been an increasing interest in stem cell applications and tissue engineering approaches in surgical practice to deal with damaged or lost tissue. Tissue engineering is an exciting strategy being explored to deal with damaged or lost tissue.

Directed differentiation of human embryonic stem cells toward chondrocytes.

We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates.

Order within disorder: aggrecan chondroitin sulphate-attachment region provides new structural insights into protein sequences classified as disordered.

Structural investigation of proteins containing large stretches of sequences without predicted secondary structure is the focus of much increased attention. Here, we have produced an unglycosylated 30 kDa peptide from the chondroitin sulphate (CS)-attachment region of human aggrecan (CS-peptide), which was predicted to be intrinsically disordered and compared its structure with the adjacent aggrecan G3 domain. Biophysical analyses, including analytical ultracentrifugation, light scattering, and circular dichroism showed that the CS-peptide had an elongated and stiffened conformation in contrast to the globular G3 domain.

Fell-Muir lecture: cartilage 2010 - the known unknowns.

Over the past 40 years there have been giant steps forward in our understanding of cellular and molecular biology that have given us the framework by which to understand tissue organization and tissue function on a range of scales. However, although the progress has been great, the more we have discovered, the more we are aware of what we don't yet know. In this article, I would like to flag up some issues of cartilage biology, function and pathology where we still have significant ignorance.

Bone marrow-derived mesenchymal stem cells express the pericyte marker 3G5 in culture and show enhanced chondrogenesis in hypoxic conditions.

Bone marrow-derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in some cells. In this study, bone marrow-derived stem cells were characterized and the effects of hypoxia on chondrogenesis investigated.

The potential of stem cells in the treatment of knee cartilage defects.

Cartilage is frequently damaged but only shows a limited capacity for repair. There are a number of treatment strategies currently available for the repair of articular cartilage defects including abrasion chondroplasty, subchondral drilling, microfracture and mosaicplasty but these show variable results. For the younger patients, there is great interest in the potential of cell-based strategies to provide a biological replacement of damaged cartilage using autologous chondrocytes.

Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes.

The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471-39479, 2006).

Stem cell applications and tissue engineering approaches in surgical practice.

There has been an increasing interest in stem cell applications and tissue engineering approaches in surgical practice to deal with damaged or lost tissue. Although there have been developments in almost all surgical disciplines, the greatest advances are being made in orthopaedics, especially in bone repair. Significant hurdles however remain to be overcome before tissue engineering becomes more routinely used in surgical practice.

The influence of donor and hypoxic conditions on the assembly of cartilage matrix by osteoarthritic human articular chondrocytes on Hyalograft matrices.

Resurfacing of cartilage defects using cell-seeded, biomaterial grafts is a promising approach for articular cartilage repair and in this study we investigated the ability of human chondrocytes from osteoarthritic joints to generate cartilage tissue under standard conditions in cultured over 21 days on Hyalograft matrices under normoxic (20% O(2)) and hypoxic (5% O(2)) conditions. The results showed that constructs were more chondrogenic when cultured under hypoxic conditions, which resulted in greater production of sulphated glycosaminoglycan and collagen type II within the constructs and the cells expressed higher levels of genes encoding cartilage matrix proteins and chondrocyte transcription factors. However, there were very wide differences in the chondrogenic potential amongst donors as the weight ratio of total sulphated glycosaminoglycan to DNA in constructs varied from above 200 to below 10.

Hyaluronan binding to link module of TSG-6 and to G1 domain of aggrecan is differently regulated by pH.

The physiological functions of hyaluronan (HA) in the extracellular matrix of vertebrate tissues involve a range of specific protein interactions. In this study, the interaction of HA with the Link module from TSG-6 (Link_TSG6) and G1 domain of aggrecan (G1), were investigated by a biophysical analysis of translational diffusion in dilute solution using confocal fluorescence recovery after photobleaching (confocal FRAP). Both Link_TSG6 and G1 were shown to bind to polymeric HA and these interactions could be competed with HA(8) and HA(10) oligosaccharides, respectively.

Human infrapatellar fat pad-derived stem cells express the pericyte marker 3G5 and show enhanced chondrogenesis after expansion in fibroblast growth factor-2.

Introduction: Infrapatellar fat pad (IPFP) is a possible source of stem cells for the repair of articular cartilage defects. In this study, adherent proliferative cells were isolated from digests of IPFP tissue. The effects of the expansion of these cells in fibroblast growth factor-2 (FGF-2) were tested on their proliferation, characterisation, and chondrogenic potential.

Cellular methods in cartilage research: primary human chondrocytes in culture and chondrogenesis in human bone marrow stem cells.

Work in our laboratory has focused on the in vitro culture of both human articular chondrocytes and human mesenchymal stem cells to understand what controls their ability to synthesise an appropriate cartilage-like extracellular matrix containing a predominantly collagen type II fibrillar network embedded in an aggrecan-rich ECM. This review focuses on the methodologies that we have found to be successful with cartilage and bone marrow sources of human cells and comments on the many factors which may enable improved phenotypic performance once the cells are in a fully chondrogenic environment.

SOX9 transduction increases chondroitin sulfate synthesis in cultured human articular chondrocytes without altering glycosyltransferase and sulfotransferase transcription.

The transcription factor SOX9 (Sry-type high-mobility-group box 9) is expressed in all chondrocytes and is essential for the expression of aggrecan, which during biosynthesis is substituted with more than 10 times its weight of CS (chondroitin sulfate) and is secreted by chondrocytes to form the characteristic GAG (glycosaminoglycan)-rich ECM (extracellular matrix) of cartilage. SOX9 expression rapidly falls during monolayer culture of isolated chondrocytes and this turns off aggrecan and associated CS synthesis. We therefore investigated whether SOX9 transduction of cultured human articular chondrocytes had any effect on the gene expression of the glycosyltransferases and sulfotransferases necessary for GAG biosynthesis.

Notch signaling through Jagged-1 is necessary to initiate chondrogenesis in human bone marrow stromal cells but must be switched off to complete chondrogenesis.

We investigated Notch signaling during chondrogenesis in human bone marrow stromal cells (hMSC) in three-dimensional cell aggregate culture. Expression analysis of Notch pathway genes in 14-day chondrogenic cultures showed that the Notch ligand Jagged-1 (Jag-1) sharply increased in expression, peaking at day 2, and then declined. A Notch target gene, HEY-1, was also expressed, with a temporal profile that closely followed the expression of Jag-1, and this preceded the rise in type II collagen expression that characterized chondrogenesis.

SOX9 transduction of a human chondrocytic cell line identifies novel genes regulated in primary human chondrocytes and in osteoarthritis.

The transcription factor SOX9 is important in maintaining the chondrocyte phenotype. To identify novel genes regulated by SOX9 we investigated changes in gene expression by microarray analysis following retroviral transduction with SOX9 of a human chondrocytic cell line (SW1353). From the results the expression of a group of genes (SRPX, S100A1, APOD, RGC32, CRTL1, MYBPH, CRLF1 and SPINT1) was evaluated further in human articular chondrocytes (HACs).