scholarly journals Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis

2020 ◽  
Vol 79 (12) ◽  
pp. 1625-1634
Author(s):  
Anke J Roelofs ◽  
Karolina Kania ◽  
Alexandra J Rafipay ◽  
Meike Sambale ◽  
Stephanie T Kuwahara ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Functional Disability ◽  
Articular Chondrocytes ◽  
In Situ Hybridisation ◽  
Cell Labelling ◽  
Synovial Lining ◽  
Mesenchymal Progenitors ◽  
Reporter Mice ◽  
Cell Subpopulations

ObjectivesOsteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA.MethodsFluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations.ResultsArticular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone.ConclusionOur findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA.

scholarly journals A non-invasive method for in situ quantification of subpopulation behaviour in mixed cell culture

2005 ◽  
Vol 3 (6) ◽  
pp. 63-69 ◽  
Author(s):  
Ben D MacArthur ◽  
Rahul S Tare ◽  
Colin P Please ◽  
Philip Prescott ◽  
Richard O.C Oreffo
Keyword(s):  
Quantitative Methods ◽  
Cell Number ◽  
Cell Labelling ◽  
Specific Cell ◽  
Mixed Cell ◽  
Cell Behaviour ◽  
Invasive Method ◽  
Cell Subpopulations ◽  
Natural Tissue

Ongoing advances in quantitative molecular- and cellular-biology highlight the need for correspondingly quantitative methods in tissue-biology, in which the presence and activity of specific cell-subpopulations can be assessed in situ . However, many experimental techniques disturb the natural tissue balance, making it difficult to draw realistic conclusions concerning in situ cell behaviour. In this study, we present a widely applicable and minimally invasive method which combines fluorescence cell labelling, retrospective image analysis and mathematical data processing to detect the presence and activity of cell subpopulations, using adhesion patterns in STRO-1 immunoselected human mesenchymal populations and the homogeneous osteoblast-like MG63 continuous cell line as an illustration. Adhesion is considered on tissue culture plastic and fibronectin surfaces, using cell area as a readily obtainable and individual cell specific measure of spreading. The underlying statistical distributions of cell areas are investigated and mappings between distributions are examined using a combination of graphical and non-parametric statistical methods. We show that activity can be quantified in subpopulations as small as 1% by cell number, and outline behaviour of significant subpopulations in both STRO-1 +/− fractions. This method has considerable potential to understand in situ cell behaviour and thus has wide applicability, for example in developmental biology and tissue engineering.

scholarly journals A Zebrafish Model of Cooperating C and N Terminal CEBPA Mutations Reveals Defects in Early Myelopoeisis and HSPCs Leading to Leukaemogenesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1343-1343
Author(s):  
Catherine Hockings ◽  
Victoria Deaner ◽  
Yvette Hoade ◽  
Phoebe Dace ◽  
Alex Lubin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Juvenile Fish ◽  
In Situ Hybridisation ◽  
Stem Cell Marker ◽  
C And N

Abstract Acute Myeloid Leukaemia (AML) is thought to occur due to stepwise accumulation of mutations in haematopoietic stem and progenitor cells (HSPCs) or early myeloid progenitor cells (EMPs) to either block differentiation or increase proliferation. In around 10% of sporadic AML cases and several known germline predisposition kindreds the myeloid transcription factor CEBPA is mutated. 50% of cases possess biallelic mutations in both C and N terminae (dm) and 50% monoallelic mutation (sm). Using TALENs we have created both C and N terminal mutants in zebrafish to study their cooperating effects in the development of AML. Our first observation was of striking defects in mature myelocytes and monocytes in all double mutants (cebpamut/mut) as assessed by Sudan black staining at 5 days post fertilisation (dpf) and apoeb whole mount in situ hybridisation (WISH) at 3dpf. In situ hybridisation revealed defects in myelopoeisis in all cebpamut/mut fry as early as 28hpf, with markedly decreased expression of coronin and l-plastin. We then interrogated the model further to assess when and where the differentiation block was occurring. We utilised the transcription factor pu.1 both as an in vivo transgenic marker Tg(pu.1:GFP) and WISH probe at earlier time points. In primitive haematopoiesis (until 22hpf) pu.1 expression showed no defects in dm mutants. However, pu.1 expressing cells were markedly reduced in dm mutants in establishment of myelopoeisis in the caudal haematopoietic tissue (CHT), fetal liver equivalent. Following formation of the CHT at around 30hpf, differing patterns of pu.1 expression were seen in sm mutants, with cebpaWT/Cterm having significantly increased staining, which was reduced in cebpaWT/Nterm (see figure 1). HSPC numbers in cebpamut/mut were seen to be normal by WISH for gata2b and runx1 and flow cytometry in Tg(CD41:GFP) fry during early haematopoeisis (2-5dpf). However, myb expression was markedly increased at 36hpf and 3 days but only in cebpaCterm/Nterm and cebpaNterm/Nterm dm mutants (see figure 2). This suggests an accumulation of early progenitors with myeloid potential but not commitment, as implied by absence of pu.1 expression but normal CD41lo numbers and other stem cell marker expression. Absence of MCherry expression in the transgenic LysC:MCherry, which highlights mature myeloid cells in vivo, was sufficiently reliable to genotype all cebpamut/mut fry and observe them for impaired survival compared to wild-type and heterozygous siblings and for the development of leukaemia. Similarly to the increase in myb staining, leukaemic transformation was only observed in dm mutants with at least one N terminal mutation, as assessed by development of anaemia and florid Tg(CD41:GFP) expression. Flow cytometry in juvenile fish (4-6weeks) also identifies a developing pre-leukaemic phenotype with moderate yet significant expansion of HSPCs and continuing absence LysC expression in cebpaNterm/Cterm and cebpaNTerm/NTerm. However, survival is poor in all dm fish, likely due to the metabolic role of cebpa and vulnerability to infection secondary to the severe myelomonocytic defect. Our results show that absence of WT cebpa has dramatic effects myelopoiesis from early stages of differentiation in definitive haematopoiesis. In addition, accumulation of myb expressing progenitors occurs as they arise from the aorta in dm mutants, identifying this as a sub-population of HSPC vulnerable to further leukaemogenic hits. Ongoing work will define the mechanism of these effects in sm and dm mutants and include comparative expression profiling in myb and pu.1 positive cells. Disclosures No relevant conflicts of interest to declare.

Bio-P and non-bio-P bacteria identification by a novel microbial approach

1999 ◽  
Vol 39 (6) ◽  
pp. 13-20 ◽  
Author(s):  
Philip L. Bond ◽  
Jürg Keller ◽  
Linda L. Blackall
Keyword(s):  
In Situ Hybridisation ◽  
Microbial Populations ◽  
Biological Phosphorus Removal ◽  
Gram Positive Bacteria ◽  
P Content ◽  
Identification Of Bacteria ◽  
Widespread Belief ◽  
Biological Phosphorus ◽  
P Removal

Culturing bacteria from activated sludge with enhanced biological phosphorus removal (EBPR) has strongly implicated Acinetobacter with the process. However, using fluorescent in-situ hybridisation (FISH) probing to analyse microbial populations, we have shown evidence opposing this widespread belief. We describe the phosphorus (P) removing performance and microbial population analyses of sludges obtained in a laboratory scale EBPR reactor. Two sludges with extremely high P removing capabilities were examined, the P content of these sludges was 8.6% (P sludge) and 12.3% (S sludge) of the MLSS. Identification of bacteria using FISH probing indicated both sludges were dominated by microbes from the beta proteobacteria and high mol% G+C Gram positive bacteria. Acinetobacter could make up only a small proportion of the cells in these sludges. Sludge with extremely poor P removal (P content of 1.5%, referred to as T sludge) was then generated by reducing the P in the influent. Bacteria resembling the G-bacteria became abundant in this sludge and these were identified using FISH probing. The anaerobic transformations of the T and P sludges correlated well with that of the non-EBPR and EBPR biological models respectively, indicating that bacteria in the T sludge have the potential to inhibit P removal in EBPR systems.

scholarly journals Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 250
Author(s):  
Rebecca E O’Connor ◽  
Lucas G Kiazim ◽  
Claudia C Rathje ◽  
Rebecca L Jennings ◽  
Darren K Griffin
Keyword(s):  
Semen Analysis ◽  
In Situ Hybridisation ◽  
Economic Loss ◽  
Environmental Damage ◽  
Fluorescence In Situ Hybridisation ◽  
Reciprocal Translocations ◽  
Chromosome Translocations ◽  
Farrowing Rate ◽  
Significant Economic Loss

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

scholarly journals Adult Renal Stem/Progenitor Cells Can Modulate T Regulatory Cells and Double Negative T Cells

2020 ◽  
Vol 22 (1) ◽  
pp. 274
Author(s):  
Claudia Curci ◽  
Angela Picerno ◽  
Nada Chaoul ◽  
Alessandra Stasi ◽  
Giuseppe De Palma ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Double Negative ◽  
Chronic Injury ◽  
Peripheral Blood Mononuclear ◽  
Cell Subpopulations ◽  
T Cell Subpopulations

Adult Renal Stem/Progenitor Cells (ARPCs) have been recently identified in the human kidney and several studies show their active role in kidney repair processes during acute or chronic injury. However, little is known about their immunomodulatory properties and their capacity to regulate specific T cell subpopulations. We co-cultured ARPCs activated by triggering Toll-Like Receptor 2 (TLR2) with human peripheral blood mononuclear cells for 5 days and 15 days and studied their immunomodulatory capacity on T cell subpopulations. We found that activated-ARPCs were able to decrease T cell proliferation but did not affect CD8+ and CD4+ T cells. Instead, Tregs and CD3+ CD4- CD8- double-negative (DN) T cells decreased after 5 days and increased after 15 days of co-culture. In addition, we found that PAI1, MCP1, GM-CSF, and CXCL1 were significantly expressed by TLR2-activated ARPCs alone and were up-regulated in T cells co-cultured with activated ARPCs. The exogenous cocktail of cytokines was able to reproduce the immunomodulatory effects of the co-culture with activated ARPCs. These data showed that ARPCs can regulate immune response by inducing Tregs and DN T cells cell modulation, which are involved in the balance between immune tolerance and autoimmunity.

scholarly journals α-Fetoprotein mRNA in situ hybridisation is a highly specific marker of hepatocellular carcinoma: a multi-centre study

2021 ◽  
Author(s):  
Shi-Xun Lu ◽  
Yu-Hua Huang ◽  
Li-Li Liu ◽  
Chris Zhiyi Zhang ◽  
Xia Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
In Situ Hybridisation ◽  
Diagnostic Value ◽  
Detection Sensitivity ◽  
Specific Marker ◽  
Specific Method ◽  
Multi Centre Study ◽  
Arginase 1 ◽  
Afp Mrna

Abstract Background Pathologic diagnosis of hepatocellular carcinoma (HCC) can be challenging in differentiating from benign and non-hepatocytic malignancy lesions. The aim of this study was to investigate the potential utility of α-fetoprotein (AFP) mRNA RNAscope, a sensitive and specific method, in the diagnosis of HCC. Methods Three independent retrospective cohorts containing 2216 patients with HCC, benign liver lesions, and non-hepatocytic tumours were examined. AFP was detected using ELISA, IHC (Immunohistochemistry), and RNAscope. Glypican3 (GPC3), hepatocyte paraffin-1 (HepPar-1), and arginase-1 (Arg-1) proteins were detected using IHC. Results AFP RNAscope improved the HCC detection sensitivity by 24.7–32.7% compared with IHC. In two surgical cohorts, a panel of AFP RNAscope and GPC3 provided the best diagnostic value in differentiating HCC from benign hepatocytic lesions (AUC = 0.905 and 0.811), and a panel including AFP RNAscope, GPC3, HepPar-1, and Arg-1 yielded the best AUC (0.971 and 0.977) when distinguishing HCC from non-hepatocytic malignancies. The results from the liver biopsy cohort were similar, and additional application of AFP RNAscope improved the sensitivity by 18% when distinguishing HCC from benign hepatocytic lesions. Conclusions AFP mRNA detected by RNAscope is highly specific for hepatocytic malignancy and may serve as a novel diagnostic biomarker for HCC.

scholarly journals Abnormal mucus in cap polyposis

Gut ◽  
1998 ◽  
Vol 42 (1) ◽  
pp. 135-138 ◽  
Author(s):  
M P Buisine ◽  
J F Colombel ◽  
M Lecomte-Houcke ◽  
P Gower ◽  
J P Aubert ◽  
...  
Keyword(s):  
In Situ Hybridisation ◽  
Clinical Manifestations ◽  
Goblet Cells ◽  
Biopsy Specimens ◽  
Mucin Genes ◽  
Cap Polyposis ◽  
Abnormal Glycosylation ◽  
Histochemical Examination ◽  
Mrna In Situ Hybridisation

Background—Cap polyposis is a rare disease characterised by mucoid and bloody diarrhoea, with polyps covered by a cap of mucoid and fibrinopurulent exudate. The pathogenesis is not known.Aims—To pour some light on cap polyposis pathogenesis, by examining the mucus of patients and analysing the expression of five mucin genes, MUC2, MUC3,MUC4, MUC5AC, and MUC5B.Patient and methods—The study was performed on biopsy specimens taken from a patient with recurrent cap polyposis. Histochemical examination, electron microscopy, and mRNA in situ hybridisation were used.Results—The mucus of cap polyposis differed in three respects from that of normal adult colon: abnormal ultrastructure of the mucus in the goblet cells, predominance of non-sulphated mucins, abnormal expression of the MUC4, MUC3, andMUC5AC genes.Conclusions—Most of these abnormalities have been reported for other pathological situations, suggesting that the abnormalities observed in the mucus of this patient with cap polyposis are probably secondary phenomena rather than primary. However, the mucin abnormalities detected, which reflect deregulation of the expression of three apomucin genes, abnormal glycosylation, and abnormalities of the secretion process, are also probably involved in the clinical manifestations of cap polyposis.

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