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 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 Characterization of Polycaprolactone Nanohydroxyapatite Composites with Tunable Degradability Suitable for Indirect Printing

Polymers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 295
Author(s):  
Stephanie E. Doyle ◽  
Lauren Henry ◽  
Ellen McGennisken ◽  
Carmine Onofrillo ◽  
Claudia Di Bella ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Large Scale ◽  
Three Dimensional ◽  
Cell Number ◽  
Compressive Modulus ◽  
Degradation Rates ◽  
Scaffold Materials ◽  
Natural Tissue ◽  
Complete Regeneration

Degradable bone implants are designed to foster the complete regeneration of natural tissue after large-scale loss trauma. Polycaprolactone (PCL) and hydroxyapatite (HA) composites are promising scaffold materials with superior mechanical and osteoinductive properties compared to the single materials. However, producing three-dimensional (3D) structures with high HA content as well as tuneable degradability remains a challenge. To address this issue and create homogeneously distributed PCL-nanoHA (nHA) scaffolds with tuneable degradation rates through both PCL molecular weight and nHA concentration, we conducted a detailed characterisation and comparison of a range of PCL-nHA composites across three molecular weight PCLs (14, 45, and 80 kDa) and with nHA content up to 30% w/w. In general, the addition of nHA results in an increase of viscosity for the PCL-nHA composites but has little effect on their compressive modulus. Importantly, we observe that the addition of nHA increases the rate of degradation compared to PCL alone. We show that the 45 and 80 kDa PCL-nHA groups can be fabricated via indirect 3D printing and have homogenously distributed nHA even after fabrication. Finally, the cytocompatibility of the composite materials is evaluated for the 45 and 80 kDa groups, with the results showing no significant change in cell number compared to the control. In conclusion, our analyses unveil several features that are crucial for processing the composite material into a tissue engineered implant.

scholarly journals Development, Sex Steroid Regulation, and Phenotypic Characterization of RFamide-Related Peptide (Rfrp) Gene Expression and RFamide Receptors in the Mouse Hypothalamus

Endocrinology ◽  
2012 ◽  
Vol 153 (4) ◽  
pp. 1827-1840 ◽  
Author(s):  
Matthew C. Poling ◽  
Joshua Kim ◽  
Sangeeta Dhamija ◽  
Alexander S. Kauffman
Keyword(s):  
In Situ Hybridization ◽  
Estrogen Receptor Α ◽  
Cell Number ◽  
Phenotypic Characterization ◽  
Related Peptide ◽  
Adult Mice ◽  
Gnrh Neurons ◽  
Double Label ◽  
Hormonal Milieu

Arginine-phenylalanine-amide (RFamide)-related peptide 3 (RFRP-3, encoded by the Rfrp gene) is the mammalian ortholog of gonadotropin-inhibiting hormone and can inhibit GnRH neuronal activity and LH release. However, the development and regulation of the RFRP-3 system in both sexes is poorly understood. Using in situ hybridization, we examined changes in Rfrp-expressing neurons in mice of both sexes during development and under different adulthood hormonal milieus. We found no sex differences in Rfrp expression or cell number in adult mice. Interestingly, we identified two interspersed subpopulations of Rfrp cells (high Rfrp-expressing, HE; low Rfrp-expressing, LE), which have unique developmental and steroidal regulation characteristics. The number of LE cells robustly decreases during postnatal development, whereas HE cell number increases significantly before puberty. Using Bax knockout mice, we determined that the dramatic developmental decrease in LE Rfrp cells is not due primarily to BAX-dependent apoptosis. In adults, we found that estradiol and testosterone moderately repress Rfrp expression in both HE and LE cells, whereas the nonaromatizable androgen dihydrotestosterone has no effect. Using double-label in situ hybridization, we determined that approximately 25% of Rfrp neurons coexpress estrogen receptor-α in each sex, whereas Rfrp cells do not readily express androgen receptor in either sex, regardless of hormonal milieu. Lastly, when we looked at RFRP-3 receptors, we detected some coexpression of Gpr147 but no coexpression of Gpr74 in GnRH neurons of both intact and gonadectomized males and females. Thus, RFRP-3 may exert its effects on reproduction either directly, via Gpr147 in a subset of GnRH neurons, and/or indirectly, via upstream regulators of GnRH.

scholarly journals MECHANISM OF ACTION OF MIGRATION INHIBITORY FACTOR (MIF)

1972 ◽  
Vol 136 (3) ◽  
pp. 589-603 ◽  
Author(s):  
Richard W. Leu ◽  
A. L. W. F. Eddleston ◽  
John W. Hadden ◽  
Robert A. Good
Keyword(s):  
Alveolar Macrophage ◽  
Peritoneal Macrophage ◽  
Migration Inhibitory Factor ◽  
Immune Modulation ◽  
Peritoneal Macrophages ◽  
Cell Number ◽  
Inhibitory Factor ◽  
Cell Surface Receptor ◽  
Specific Cell ◽  
Surface Receptor

The initial interaction between migration inhibitory factor (MIF) and the guinea pig alveolar and peritoneal macrophage was studied. MIF-containing supernatants were generated from sensitized lymph node lymphocytes obtained from guinea pigs immunized with bovine gamma globulin in complete Freund's adjuvant. MIF-containing supernatants were markedly inhibitory for the migration of the peritoneal macrophage but had no effect on the alveolar macrophage. A linear relationship was observed between per cent inhibition of migration and serial twofold dilution of supernatant. Reexpressed in arbitrary MIF units, this relationship reflects a dose-response relationship with saturation characteristics. Pulse exposure of peritoneal macrophages to MIF resulted in adsorption of MIF onto both viable and nonviable cells with corresponding depletion of supernatant MIF. The alveolar macrophage did not adsorb MIF. Pulse adsorption of MIF onto the peritoneal macrophage is dependent on time, temperature, and cell number. Pretreatment of the cells with proteolytic enzyme prevents the adsorption of MIF while leaving migration unaffected. These observations support the existence of a specific cell surface receptor for MIF. The existence of such a receptor provides selectivity of immune modulation of macrophage populations by lymphocytes in delayed hypersensitivity reactions.

scholarly journals Cytokines and Growth Factors Promote Airway Smooth Muscle Cell Proliferation

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
R. Stamatiou ◽  
E. Paraskeva ◽  
K. Gourgoulianis ◽  
P.-A. Molyvdas ◽  
A. Hatziefthimiou
Keyword(s):  
Smooth Muscle ◽  
Growth Factors ◽  
Smooth Muscle Cell ◽  
Airway Smooth Muscle ◽  
Muscle Cell ◽  
Thymidine Incorporation ◽  
Cell Number ◽  
Airway Smooth Muscle Cell ◽  
Specific Cell ◽  
Dependent Manner

Chronic airway diseases, such as asthma or chronic obstructive pulmonary disease, are characterized by the presence in the airways of inflammation factors, growth factors and cytokines, which promote airway wall remodelling. The aim of this study was to investigate the effect of cytokines and growth factors on airway smooth muscle cell (ASMC) proliferation, phenotype and responsiveness. Incubation of serum starved human bronchial ASMCs with TNF-α, TGF, bFGF, and PDGF, but not IL-1β, increased methyl-[3H]thymidine incorporation and cell number, mediated by the PI3K and MAPK signalling pathways. Regarding rabbit tracheal ASMC proliferation, TNF-α, IL-1β, TGF, and PDGF increased methyl-[3H]thymidine incorporation in a PI3K- and MAPK-dependent manner. bFGF increased both methyl-[3H]thymidine incorporation and cell number. Moreover, incubation with TGF, bFGF and PDGF appears to drive human ASMCs towards a synthetic phenotype, as shown by the reduction of the percentage of cells expressing SM-α actin. In addition, the responsiveness of epithelium-denuded rabbit tracheal strips to carbachol was not significantly altered after 3-day treatment with bFGF. In conclusion, all the tested cytokines and growth factors increased ASMC proliferation to a different degree, depending on the specific cell type, with bronchial ASMCs being more prone to proliferation than tracheal ASMCs.

Temporal changes in the expression of the insulin-like growth factor II gene associated with tissue maturation in the human fetus

Development ◽  
1989 ◽  
Vol 106 (3) ◽  
pp. 543-554 ◽  
Author(s):  
A.L. Brice ◽  
J.E. Cheetham ◽  
V.N. Bolton ◽  
N.C. Hill ◽  
P.N. Schofield
Keyword(s):  
Growth Factor ◽  
Cell Types ◽  
Specific Cell ◽  
Insulin Like Growth Factor ◽  
Vitro Fertilization ◽  
Age Related ◽  
Factor Ii ◽  
Metanephric Blastema

The insulin-like growth factors are broadly distributed in the human conceptus and are thought to play a role in the growth and differentiation of tissues during development. Using in situ hybridization we have shown that a wide variety of specific cell types within tissues express the gene for insulin-like growth factor II at times of development from 18 days to 14 weeks of gestation. Examination of blastocysts produced by in vitro fertilization showed no expression, thus bracketing the time of first accumulation of IGF-II mRNA to between 5 and 18 days postfertilization. The pattern of IGF-II expression shows specific age-related differences in different tissues. In the kidney, for example, expression is found in the cells of the metanephric blastema which is dramatically reduced as the blastema differentiates. The reverse is also seen, and we have noted an increase in expression of IGF-II in the cytotrophoblast layer of the placenta with gestational age. The sites of expression do not correlate with areas of either high mitotic activity or specific types of differentiation, but the observed pattern of expression in the kidney, adrenal glands and liver suggests an explanation for the abnormally high IGF-II mRNA expression in developmental tumours such as Wilms' tumour.

3d Confocal Microscopy and Image Analysis for Measurement of Genetic Instability

1999 ◽  
Vol 5 (S2) ◽  
pp. 1022-1023
Author(s):  
C. Ortiz de Solorzano ◽  
K. Chin ◽  
D. Knowles ◽  
A. Jones ◽  
E. Garcia ◽  
...  
Keyword(s):  
Image Analysis ◽  
Confocal Microscopy ◽  
Genetic Instability ◽  
Copy Number ◽  
3D Image ◽  
3D Images ◽  
Tissue Sections ◽  
Total Dna ◽  
Natural Tissue

Solid tumors frequently contain cells that are heterogeneous in the copy number of DNA loci. This fact implies the existence of genetic instability, which may be associated with disease aggressiveness. Accurate measurement of this phenomenon requires analysis of intact nuclei within their natural tissue context. We perform these measurements by preparing >30μm thick tissue sections, labeling them with fluorescent labels for total DNA and for specific DNA loci using fluorescence in situ hybridization (FISH) which retain the transparency of the tissue and acquiring 3D images of the tissue using confocal microscopy (figure 1). In this study, we combined automated 3D image analysis (IA) algorithms for segmenting individual nuclei based on the total DNA stain1 and for segmenting the punctuate FISH signals of DNA loci. This enables us to efficiently enumerate the copy number of specific DNA loci in individual cells and as a function of the cell's location in the tissue.

scholarly journals Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections.

1993 ◽  
Vol 41 (7) ◽  
pp. 1023-1030 ◽  
Author(s):  
R Gold ◽  
M Schmied ◽  
G Rothe ◽  
H Zischler ◽  
H Breitschopf ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Specific Cell ◽  
Nick Translation ◽  
Proliferating Cells ◽  
Cell Surface Antigens ◽  
Simultaneous Application ◽  
Tissue Sections ◽  
In Situ Nick Translation

Since DNA fragmentation is a key feature of programmed cell death (PCD) and also occurs in certain stages of necrosis, we have adapted the methodology of in situ nick-translation (ISNT) to detect DNA fragmentation on a single-cell level. We first established the technique for cell preparations. Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes. After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-dUTP or with digoxigenin-labeled 11-dUTP and DNA polymerase I. The enzymatic incorporation of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly or indirectly with fluorescein-conjugated anti-digoxigenin. The quantitative results demonstrated close correlation with morphological essays for apoptosis, DNA gel electrophoresis, and ISNT. Proliferating cells determined by bromodeoxyuridine immunofluorescence were not labeled by ISNT. Immunocytochemistry for cell surface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD. Furthermore, the simultaneous application of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still intact membranes. Extending ISNT to tissue sections of paraformaldehyde-fixed, paraffin-embedded material reliably revealed labeling of cells with typical morphological features of apoptosis. However, this technique was not useful in detecting early stages of necrotic cell death.

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