scholarly journals Quantitative Single-Cell Transcript Assessment of Biomarkers Supports Cellular Heterogeneity in the Bovine IVD

2019 ◽  
Vol 6 (2) ◽  
pp. 42 ◽  
Author(s):  
Kangning Li ◽  
Devin Kapper ◽  
Sumona Mondal ◽  
Thomas Lufkin ◽  
Petra Kraus
Keyword(s):  
In Situ Hybridization ◽  
Single Cell ◽  
Gaussian Mixture ◽  
Cellular Heterogeneity ◽  
Rna In Situ Hybridization ◽  
Ivd Degeneration

Severe and chronic low back pain is often associated with intervertebral disc (IVD) degeneration. While imposing a considerable socio-economic burden worldwide, IVD degeneration is also severely impacting on the quality of life of affected individuals. Cell-based regenerative medicine approaches have moved into clinical trials, yet IVD cell identities in the mature disc remain to be fully elucidated and tissue heterogeneity exists, requiring a better characterization of IVD cells. The bovine coccygeal IVD is an accepted research model to study IVD mechano-biology and disc homeostasis. Recently, we identified novel IVD biomarkers in the outer annulus fibrosus (AF) and nucleus pulposus (NP) of the mature bovine coccygeal IVD through RNA in situ hybridization (AP-RISH) and z-proportion test. Here we follow up on Lam1, Thy1, Gli1, Gli3, Noto, Ptprc, Scx, Sox2 and Zscan10 with fluorescent RNA in situ hybridization (FL-RISH) and confocal microscopy. This permits sub-cellular transcript localization and the addition of quantitative single-cell derived values of mRNA expression levels to our previous analysis. Lastly, we used a Gaussian mixture modeling approach for the exploratory analysis of IVD cells. This work complements our earlier cell population proportion-based study, confirms the previously proposed biomarkers and indicates even further heterogeneity of cells in the outer AF and NP of a mature IVD.

scholarly journals Simultaneous fluorescence immunophenotyping and interphase cytogenetics: a contribution to the characterization of tumor cells.

1992 ◽  
Vol 40 (2) ◽  
pp. 171-175 ◽  
Author(s):  
K Weber-Matthiesen ◽  
M Winkemann ◽  
A Müller-Hermelink ◽  
B Schlegelberger ◽  
W Grote
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Tumor Cells ◽  
Interphase Cytogenetics ◽  
Normal Cells ◽  
Interphase Cells ◽  
Immunocytochemical Studies

In immunocytochemical studies, the phenotypic evaluation of tumor cells is often complicated by accompanying normal cells, representing the original tissue or infiltrating leukocytes. This holds particularly true for tissues with a great morphological and immunophenotypical variability, such as bone marrow. A method that identifies mitotic tumor cells by chromosomal aberrations and permits the subsequent immunophenotypical analysis was a first progress, demonstrated by Teerenhovi et al. However, the results are usually hampered by the low number of analyzable mitoses. We demonstrate here a method that simultaneously combines immunophenotyping and in situ hybridization with centromere-specific probes. Using our method, numerically aberrant tumor cells can be identified by interphase cytogenetics and subsequently analyzed immunophenotypically. Since all interphase cells can be analyzed, we are not limited by the number and banding quality of analyzable mitoses.

scholarly journals Cell population characterization and discovery using single-cell technologies in endocrine systems

2020 ◽  
Vol 65 (2) ◽  
pp. R35-R51
Author(s):  
Leonard Y M Cheung ◽  
Karine Rizzoti
Keyword(s):  
Single Cell ◽  
Cell Types ◽  
Reproductive Organs ◽  
Cellular Heterogeneity ◽  
Disease Mechanisms ◽  
Cell Technologies ◽  
Endocrine Organs ◽  
Source Materials

In the last 15 years, single-cell technologies have become robust and indispensable tools to investigate cell heterogeneity. Beyond transcriptomic, genomic and epigenome analyses, technologies are constantly evolving, in particular toward multi-omics, where analyses of different source materials from a single cell are combined, and spatial transcriptomics, where resolution of cellular heterogeneity can be detected in situ. While some of these techniques are still being optimized, single-cell RNAseq has commonly been used because the examination of transcriptomes allows characterization of cell identity and, therefore, unravel previously uncharacterized diversity within cell populations. Most endocrine organs have now been investigated using this technique, and this has given new insights into organ embryonic development, characterization of rare cell types, and disease mechanisms. Here, we highlight recent studies, particularly on the hypothalamus and pituitary, and examine recent findings on the pancreas and reproductive organs where many single-cell experiments have been performed.

A single-cell atlas of mouse lung development

Development ◽  
2021 ◽  
Vol 148 (24) ◽  
Author(s):  
Nicholas M. Negretti ◽  
Erin J. Plosa ◽  
John T. Benjamin ◽  
Bryce A. Schuler ◽  
A. Christian Habermann ◽  
...  
Keyword(s):  
Single Cell ◽  
Spatial Organization ◽  
Expression Patterns ◽  
Mesenchymal Cell ◽  
Mouse Lung ◽  
Rna In Situ Hybridization ◽  
And Function ◽  
Parenchymal Cells

ABSTRACT Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages – wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.

scholarly journals Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

2012 ◽  
Vol 14 (3) ◽  
pp. 443-451 ◽  
Author(s):  
Xiaozhu Wang ◽  
Shin-ichiro Takebayashi ◽  
Evans Bernardin ◽  
David M. Gilbert ◽  
Ravindran Chella ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cell ◽  
Cell Nuclei ◽  
Chromosomal Dna

scholarly journals RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

2021 ◽  
Vol 4 (1) ◽  
pp. 20
Author(s):  
Mujeeb Shittu ◽  
Tessa Steenwinkel ◽  
William Dion ◽  
Nathan Ostlund ◽  
Komal Raja ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Expression Patterns ◽  
Drosophila Species ◽  
Gene Expression Patterns ◽  
Probe Design ◽  
Tissue Preparation ◽  
Design And Synthesis ◽  
Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

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