Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity

Ruf-Zamojski, Frederique; Ge, Yongchao; Nair, Venugopalan D.; Zamojski, Michel; Pinças, Hanna; Toufaily, Chirine; Tomé-García, Jessica; Stoeckius, Marlon; Stephenson, William; Smith, Gregory R.; Bernard, Daniel J.; Tsankova, Nadejda M.; Hartmann, Boris; Fribourg, Miguel; Smibert, Peter; Swerdlow, Harold; Turgeon, Judith L; Sealfon, Stuart C.

doi:10.1093/nar/gky991

Cited by 22 publications

(15 citation statements)

References 47 publications

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“…Elegant in vivo imaging helped to identify networks of FSC and HPC (20), whereas single-cell imaging and patch clamp experiments showed the cell type-specific electrical and calcium signaling patterns in unstimulated and stimulated cells (17). RNA-sequencing analysis in cultured rat anterior pituitary cells was used to identify upregulated genes and a GnRH controlled gene network in G (21), and single cell RNA-sequencing (scRNA-seq) was used to characterize gene expression in LβT2 gonadotroph cells (22, 23), purified mouse gonadotrophs (24), and dispersed male mouse pituitary cells (25). Despite these advances, the gene expression patterns underlying the identity of anterior pituitary cell types, their functions, and their interrelationships remain incompletely understood.…”

Section: Introductionmentioning

confidence: 99%

Cell Type- and Sex-Dependent Transcriptome Profiles of Rat Anterior Pituitary Cells

et al. 2019

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Understanding the physiology and pathology of an organ composed of a variety of cell populations depends critically on genome-wide information on each cell type. Here, we report single-cell transcriptome profiling of over 6,800 freshly dispersed anterior pituitary cells from postpubertal male and female rats. Six pituitary-specific cell types were identified based on known marker genes and characterized: folliculostellate cells and hormone-producing corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Also identified were endothelial and blood cells from the pituitary capillary network. The expression of numerous developmental and neuroendocrine marker genes in both folliculostellate and hormone-producing cells supports that they have a common origin. For several genes, the validity of transcriptome analysis was confirmed by qRT-PCR and single cell immunocytochemistry. Folliculostellate cells exhibit impressive transcriptome diversity, indicating their major roles in production of endogenous ligands and detoxification enzymes, and organization of extracellular matrix. Transcriptome profiles of hormone-producing cells also indicate contributions toward those functions, while also clearly demonstrating their endocrine function. This survey highlights many novel genetic markers contributing to pituitary cell type identity, sexual dimorphism, and function, and points to relationships between hormone-producing and folliculostellate cells.

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Section: Introductionmentioning

confidence: 99%

Cell Type- and Sex-Dependent Transcriptome Profiles of Rat Anterior Pituitary Cells

et al. 2019

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“…However, some cell types are more vulnerable to freezing than others, for example in human endometrium biopsies stromal cells survive freezing better than epithelial cells (23) . Fixation of cells with traditional cross-linking fixatives (24) , reversible-crosslinkers (25) , non-crosslinking alternatives such as methanol (26) and other novel stabilization reagents (27) has also been tested. Fixation halts transcriptional change and stabilizes cell types, although it usually creates 3' bias.…”

Section: Introductionmentioning

confidence: 99%

Lung, spleen and oesophagus tissue remains stable for scRNAseq in cold preservation

Madissoon

Wilbrey-Clark

Miragaia

et al. 2019

Preprint

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BackgroundThe Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single cell RNA sequencing can generate high quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design. Despite this, there has not yet been a systematic assessment of the effect of cold storage time on the quality of scRNAseq ResultsThis study assessed the effect of cold storage on fresh healthy spleen, oesophagus and lung from ≥5 donors over 72 hours. We collected 240,000 high quality single cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these three organs and will allow cross-organ comparison of cell types.We see little effect of cold ischaemic time on cell viability, yield, total number of reads per cell and other quality control metrics in any of the tissues within the first 24 hours. However, we observed higher percentage of mitochondrial reads, indicative of cellular stress, and

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“…After 1 day of culture, coverslips were placed in racks and incubated overnight in low-serum (1% FBS) medium (day 2). On day 3, cells were exposed to 5-minute duration pulses of 2 nM GnRH every 2 hours (a frequency that is more favorable to Fshb induction than to Lhb ) in low-serum medium, and temporal gene responses were assessed after the fourth or fifth pulse, as previously described [21, 22]. For each time point, a minimum of four biological replicates ( i.e., independent coverslips) were collected.…”

Section: Methodsmentioning

confidence: 99%

Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines

Ruf-Zamojski

Pinças

et al. 2019

Journal of the Endocrine Society

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L β T2 and α T3-1 are important, widely studied cell line models for the pituitary gonadotropes that were generated by targeted tumorigenesis in transgenic mice. L β T2 cells are more mature gonadotrope precursors than α T3-1 cells. Microsatellite authentication patterns, chromosomal characteristics, and their intercellular variation have not been reported. We performed microsatellite and cytogenetic analysis of both cell types at early passage numbers. Short tandem repeat (STR) profiling was consistent with a mixed C57BL/6J × BALB/cJ genetic background, with distinct patterns for each cell type. Spectral karyotyping in α T3-1 cells revealed cell-to-cell variation in chromosome composition and pseudodiploidy. In L β T2 cells, chromosome counting and karyotyping demonstrated pseudotriploidy and high chromosomal variation among cells. Chromosome copy number variation was confirmed by single-cell DNA sequencing. Chromosomal compositions were consistent with a male sex for α T3-1 and a female sex for L β T2 cells. Among L β T2 stocks used in multiple laboratories, we detected two genetically similar but distinguishable lines via STR authentication, L β T2a and L β T2b. The two lines differed in morphological appearance, with L β T2a having significantly smaller cell and nucleus areas. Analysis of immediate early gene and gonadotropin subunit gene expression revealed variations in basal expression and responses to continuous and pulsatile GnRH stimulation. L β T2a showed higher basal levels of Egr1 , Fos , and Lhb but lower Fos induction. Fshb induction reached significance only in L β T2b cells. Our study highlights the heterogeneity in gonadotrope cell line genomes and provides reference STR authentication patterns that can be monitored to improve experimental reproducibility and facilitate comparisons of results within and across laboratories.

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Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity

Cited by 22 publications

References 47 publications

Cell Type- and Sex-Dependent Transcriptome Profiles of Rat Anterior Pituitary Cells

Cell Type- and Sex-Dependent Transcriptome Profiles of Rat Anterior Pituitary Cells

Lung, spleen and oesophagus tissue remains stable for scRNAseq in cold preservation

Cytogenetic, Genomic, and Functional Characterization of Pituitary Gonadotrope Cell Lines

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