38 research outputs found
Rapid isolation, expansion and differentiation of osteoprogenitors from full-term umbilical cord blood
No full textThere is an urgent clinical requirement for appropriate bone substitutes that can be used for the repair and regeneration of diseased or damaged skeletal tissues. Cell-sourcing limitations in particular have affected progress, largely because of the shortage of accessible tissues capable of yielding sufficient numbers of viable osteoprogenitor cells. Previous work has suggested that umbilical cord blood (UCB) contains circulating progenitor cells (mesenchymal stem cells) capable of osteogenic differentiation, although a comparable number of reports refute this claim. From a screen of more than 20 different culture conditions, we have identified an optimal, simple, and reliable technique to generate, from full-term human UCB, stromal cells with the ability to undergo rapid osteogenic differentiation. By comparing different sorting and culture strategies, we demonstrated that early exposure of mononuclear UCB cells to medium conditioned by osteoblastic cells in the presence of osteogenic supplements and human plasma, markedly increased the frequency of stromal cell growth, the rate of osteogenic differentiation, and their attachment to and spreading on calcium phosphate scaffolds. These findings suggest that full-term UCB may act as an appropriate source of osteoprogenitor cells, which will impact significantly on the development of autologous tissue- engineered bone constructs
Real-time analysis of endogenous Wnt signalling in 3D mesenchymal stromal cells
No full textWnt signalling has been implicated in the regulation of stem cell self-renewal and differentiation; however, the majority of in vitro studies are carried out using monolayer 2D culture techniques. Here, we used mesenchymal stromal cell (MSC) EGFP reporter lines responsive to Wnt pathway activation in a 3D spheroid culture system to mimic better the in vivo environment. Endogenous Wnt signalling was then investigated under basal conditions and when MSCs were induced to undergo osteogenic and adipogenic differentiation. Interestingly, endogenous Wnt signalling was only active during 3D differentiation whereas 2D cultures showed no EGFP expression throughout an extended differentiation time-course. Furthermore, exogenous Wnt signalling in 3D adipogenic conditions inhibited differentiation compared to unstimulated controls. In addition, suppressing Wnt signalling by Dkk-1 restored and facilitated adipogenic differentiation in MSC spheroids. Our findings indicate that endogenous Wnt signalling is active and can be tracked in 3D MSC cultures where it may act as a molecular switch in adipogenesis. The identification of the signalling pathways that regulate MSCs in a 3D in vivo-like environment will advance our understanding of the molecular mechanisms that control MSC fate
Anatomy and pathology of the Texel sheep larynx
No full textLaryngeal chondritis, or “Texel throat”, is a disease affecting the upper respiratory tract of sheep with breeds like the Texel appearing to be predisposed. Previous work suggests the conformation of these breeds of sheep may be predisposing these animals to laryngeal disease. This study evaluated the anatomy of the Texel sheep larynx and describes incidental pathology. Forty-three larynges from rams of the Texel and Bluefaced Leicester breeds of sheep were measured and photographed. A larynx from each breed was submitted for computed tomography (CT) and magnetic resonance imaging (MRI). Measurements, photography, CT, and MRI demonstrated a difference in the anatomy of the larynx between breeds and a higher proportion of Texel sheep had laryngeal lesions. This study supports the hypothesis that the anatomy of the Texel sheep could be pre-disposing the breed to laryngeal chondritis
Glycan profiling shows unvaried N-glycomes in MSC clones with distinct differentiation potentials
No full textDifferent cell types have different N-glycomes in mammals. This means that cellular differentiation is accompanied by changes in the N-glycan profile. Yet when the N-glycomes of cell types with differing fates diverge is unclear. We have investigated the N-glycan profiles of two different clonal populations of mesenchymal stromal cells (MSCs). One clone (Y101), when differentiated into osteoblasts, showed a marked shift in the glycan profile towards a higher abundance of complex N-glycans and more core fucosylation. Yet chemical inhibition of complex glycan formation during osteogenic differentiation did not prevent the formation of functional osteoblasts. However, the N-glycan profile of another MSC clone (Y202), which cannot differentiate into osteoblasts, was not significantly different from that of the clone that can. Interestingly, incubation of Y202 cells in osteogenic medium caused a similar reduction of oligomannose glycan content in this non-differentiating cell line. Our analysis implies that the N glycome changes seen upon differentiation do not have direct functional links to the differentiation process. Thus N-glycans may instead be important for self-renewal rather than for cell fate determination
CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations
No full textHeterogeneity of bone marrow mesenchymal stromal cells (MSCs, frequently referred to as “mesenchymal stem cells”) clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterized by CD317 expression (CD317(pos) (29.77 ± 3.00% of the total MSC population), comprising CD317(dim) (28.10 ± 4.60%) and CD317(bright) (1.67 ± 0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317(pos) MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317(neg) MSCs had no effect. Only CD317(neg) MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317(pos) MSCs increased polarization towards pro-inflammatory Th1 cells and CD317(neg) cell lines did not. Using an in vivo peritonitis model, we found that CD317(neg) and CD317(pos) MSCs suppressed leukocyte recruitment but only CD317(neg) MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317(neg) MSC lines, but not CD317(pos) MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs
Function of genes significantly down-regulated by Fgf4 overexpression and Cic knockdown.
No full textFunction of genes significantly down-regulated by Fgf4 overexpression and Cic knockdown.</p
Overlapping effects on gene expression in response to Cic knockdown in <i>Xenopus tropicalis</i> and ectodermal wounding in <i>Xenopus laevis</i>.
No full textXenopus laevis wounding datasets from [50] were filtered to only include genes annotated in the Xenopus tropicalis genome (12992 genes). Datasets for 30–90 minutes post-wounding were combined and filtered according to the following criteria, fold up-regulation ≥1.75 and p-adj ≤0.1. Duplicate gene entries were removed from the Xenopus laevis dataset. A, shows the highly significant overlap of genes up-regulated by Cic-knockdown in Xenopus tropicalis and wounding in Xenopus laevis. B, Genes up-regulated by Cic-knockdown and ectodermal wounding. Orange shading indicates genes analysed at the site of wounding in this study (Fig 5). (JPG)</p
Genes significantly down-regulated by Fgf4 overexpression and Cic knockdown.
No full textGenes significantly down-regulated by Fgf4 overexpression and Cic knockdown.</p
Genes significantly up-regulated by Fgf4 overexpression and Cic knockdown.
No full textGenes significantly up-regulated by Fgf4 overexpression and Cic knockdown.</p
Raw data for beta catenin western blot in Fig 1D.
No full textActivation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. We have proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor. Here we investigate the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. We characterise Xenopus Cic and show that it is widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. In keeping with our hypothesis, we show that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. We investigate two of these genes as putative targets of the proposed Fgf/Erk/Cic axis: fos and rasl11b, which encode a leucine zipper transcription factor and a ras family GTPase, respectively. We identify Cic consensus binding sites in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. We show that expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, we show that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. Our data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding.</div
