Isolation of Highly Viable Thymic Epithelial Cells for Use in In Vitro and In Vivo Experiments

Intrathymic T-cell differentiation is under the control of the thymic microenvironment, which acts on maturing thymocytes via membrane as well as soluble products. Increasing data show that this process can be modulated by classical hormones, as exemplified herein by prolactin (PRL) and growth hormone (GH), largely secreted by the pituitary gland.Both PRL and GH stimulate the secretion of thymulin, a thymic hormone produced by thymic epithelial cells. Conversely, low levels of circulating thymulin parallel hypopituitary states. Interestingly, the enhancing effects of GH on thymulin seem to be mediated by insulinlike growth factor (IGF-1) since they can be abrogated with anti-IGF-1 or anti-IGF-l-receptor antibodies. The influence of PRL and GH on the thymic epithelium is pleiotropic: PRL enhancesin vivothe expression of high-molecular-weight cytokeratins and stimulatesin vitroTEC proliferation, an effect that is shared by GH and IGF-1.Differentiating T cells are also targets for the intrathymic action of PRL and GH.In vivoinoculation of a rat pituitary cell line into old rats results in restoration of the thymus, including differentiation of CD4-CD8-thymocytes into CD4+CD8+cells. Furthermore, PRL may regulate the maintenance of thymocyte viability during the double-positive stage of thymocyte differentiation.Injections of GH into aging mice increase total thymocyte numbers and the percentage of CD3-bearing cells, as well as the Concanavalin-A mitogenic response and IL-6 production by thymocytes. Interestingly, similar findings are observed in animals treated with IGF-1. Lastly, the thymic hypoplasia observed in dwarf mice can be reversed with GH treatment.In keeping with the data summarized earlier is the detection of receptors for PRL and GH on both thymocytes and thymic epithelial cells. Importantly, recent studies indicate that both cell types can produce PRL and GH intrathymically. Similarly, production of IGF-1 and expression of a corresponding receptor has also been demonstrated.In conclusion, these data strongly indicate that the thymus is physiologically under control of pituitary hormones PRL and GH. In addition to the classical endocrine pathway, paracrine and autocrine circuits are probably implicated in such control.

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Differential addressing of 5-HT1A and 5-HT1B receptors in epithelial cells and neurons

Journal of Cell Science ◽

10.1242/jcs.112.6.967 ◽

1999 ◽

Vol 112 (6) ◽

pp. 967-976

Author(s):

A. Ghavami ◽

K.L. Stark ◽

M. Jareb ◽

S. Ramboz ◽

L. Segu ◽

...

Keyword(s):

Epithelial Cells ◽

Neuronal Cultures ◽

Striatal Neurons ◽

Primary Neuronal Cultures ◽

Axon Terminals ◽

In Vivo Experiments ◽

In Vitro Systems ◽

The Central Nervous System

The 5-HT1A and 5-HT1B serotonin receptors are expressed in a variety of neurons in the central nervous system. While the 5-HT1A receptor is found on somas and dendrites, the 5-HT1B receptor has been suggested to be localized predominantly on axon terminals. To study the intracellular addressing of these receptors, we have used in vitro systems including Madin-Darby canine kidney (MDCK II) epithelial cells and primary neuronal cultures. Furthermore, we have extended these studies to examine addressing in vivo in transgenic mice. In epithelial cells, 5-HT1A receptors are found on both apical and basolateral membranes while 5-HT1B receptors are found exclusively in intracellular vesicles. In hippocampal neuronal cultures, 5-HT1A receptors are expressed on somatodendritic membranes but are absent from axons. In contrast, 5-HT1B receptors are found on both dendritic and axonal membranes, including growth cones where they accumulate. Using 5-HT1A and 5-HT1B knockout mice and the binary tTA/tetO system, we generated mice expressing these receptors in striatal neurons. These in vivo experiments demonstrate that, in striatal medium spiny neurons, the 5-HT1A receptor is restricted to the somatodendritic level, while 5-HT1B receptors are shipped exclusively toward axon terminals. Therefore, in all systems we have examined, there is a differential sorting of the 5-HT1A and 5-HT1B receptors. Furthermore, we conclude that our in vivo transgenic system is the only model that reconstitutes proper sorting of these receptors.

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Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire

Journal of Experimental Medicine ◽

10.1084/jem.20070795 ◽

2007 ◽

Vol 204 (11) ◽

pp. 2521-2528 ◽

Cited By ~ 249

Author(s):

Daniel Gray ◽

Jakub Abramson ◽

Christophe Benoist ◽

Diane Mathis

Keyword(s):

Epithelial Cells ◽

Flow Cytometric Analysis ◽

Tolerance Induction ◽

Brdu Incorporation ◽

Thymic Epithelial Cells ◽

High Turnover ◽

Central Tolerance ◽

Cross Presentation

Expression of autoimmune regulator (Aire) by thymic medullary epithelial cells (MECs) is critical for central tolerance of self. To explore the mechanism by which such a rare cell population imposes tolerance on the large repertoire of differentiating thymocytes, we examined the proliferation and turnover of Aire+ and Aire− MEC subsets through flow cytometric analysis of 5-bromo-2′deoxyuridine (BrdU) incorporation. The Aire+ MEC subset was almost entirely postmitotic and derived from cycling Aire− precursors. Experiments using reaggregate thymic organ cultures revealed the presence of such precursors among Aire− MECs expressing low levels of major histocompatibility complex class II and CD80. The kinetics of BrdU decay showed the Aire+ population to have a high turnover. Aire did not have a direct impact on the division of MECs in vitro or in vivo but, rather, induced their apoptosis. We argue that these properties strongly favor a “terminal differentiation” model for Aire function in MECs, placing strict temporal limits on the operation of any individual Aire+ MEC in central tolerance induction. We further speculate that the speedy apoptosis of Aire-expressing MECs may be a mechanism to promote cross-presentation of the array of peripheral-tissue antigens they produce.

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Experimental study of tissue-engineered urethra with collagen / chitosan composite as scaffolds

10.1101/2020.04.30.070284 ◽

2020 ◽

Author(s):

Zhai Hongfeng ◽

Qiu Changhong ◽

Jin Jun ◽

Shao Xin

Keyword(s):

New Zealand ◽

Epithelial Cells ◽

Cell Activity ◽

Three Dimensional ◽

Volume Ratio ◽

In Vivo Experiments ◽

High Area ◽

Chitosan Composite

AbstractIn this article we investigated the preparation of tissue-engineered urethra by using the urethral epithelial subculture cells of male New Zealand young rabbits. We inoculated the epithelial cells of urinary mucosa of male New Zealand young rabbits on collagen, chitosan and collagen chitosan composite as scaffolds to prepare tissue-engineered urethra. The results of inverted phase contrast microscope, HE staining and scanning electron microscope of three kinds of tissue-engineered urethra were compared. What’s more, we reported a new method for quantitative and rapid detection of epithelial cell activity of urinary mucosa in situ by Interactive Laser Cytometer. The collagen chitosan composite was more similar to the extracellular matrix of mammalian. Its three-dimensional porous structure had a high area volume ratio, which was conducive to cell adhesion, growth and metabolism. In vitro, the urethral epithelial cells had been cultured on collagen chitosan composite, and the tissue-engineered urethra was successfully prepared, which laid a solid foundation for further in vivo experiments.

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Autoimmune regulator act in synergism with thymocyte adhesion in the control of lncRNAs in medullary thymic epithelial cells

10.1101/2021.07.14.452339 ◽

2021 ◽

Author(s):

Max J Duarte ◽

Romario S Mascarenhas ◽

Amanda F Assis ◽

Pedro P Tanaka ◽

Cesar A Speck-Hernandez ◽

...

Keyword(s):

Epithelial Cells ◽

Model Systems ◽

Thymic Epithelial Cells ◽

Large Set ◽

Rna Pol Ii ◽

Pol Ii ◽

Autoimmune Regulator ◽

Medullary Thymic Epithelial Cells

The autoimmune regulator (Aire) gene in medullary thymic epithelial cells (mTECs) encodes the AIRE protein, which interacts with its partners within the nucleus. This Aire complex induces stalled RNA Pol II on chromatin to proceed with transcription elongation of a large set of messenger RNAs and microRNAs. Considering that RNA Pol II also transcribes long noncoding RNAs (lncRNAs), we hypothesized that Aire might be implicated in the upstream control of this RNA species. To test this, we employed a loss-of-function approach in which Aire knockout mTECs were compared to Aire wild-type mTECs for lncRNA transcriptional profiling both in vitro and in vivo model systems. RNA sequencing enables the differential expression profiling of lncRNAs when these cells adhere in vitro to thymocytes or do not adhere to them as a way to test the effect of cell adhesion. Sets of lncRNAs that are unique and that are shared in vitro and in vivo were identified. Among these, we found the Aire-dependent lncRNAs as for example, Platr28, Ifi30, Morrbid, Malat1, and Xist. This finding represents the first evidence that Aire mediates the transcription of lncRNAs in mTECs. Microarray hybridizations enabled us to observe that temporal thymocyte adhesion modulates the expression levels of such lncRNAs as Morrbid, Xist, and Fbxl12o after 36h of adhesion. This finding shows the existence of a synergistic mechanism involving a link between thymocyte adhesion, Aire, and lncRNAs in mTECs that might be important for immune self-representation.

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Production of anti-thymulin (FTS) monoclonal antibodies by immunization against human thymic epithelial cells.

Journal of Histochemistry & Cytochemistry ◽

10.1177/32.4.6200531 ◽

1984 ◽

Vol 32 (4) ◽

pp. 432-438 ◽

Cited By ~ 16

Author(s):

S Berrih ◽

W Savino ◽

M Azoulay ◽

M Dardenne ◽

J F Bach

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Epithelial Cells ◽

Thymic Epithelial Cells ◽

Mouse Serum ◽

Thymic Hormone ◽

Double Labeling ◽

Natural Hormone

A monoclonal antibody specific for thymulin (FTS), a thymic hormone initially isolated from serum, was obtained by cell fusion using spleen cells from BALB/c mice immunized with cultured human thymic epithelial cells. Hybridomas were selected according to their capacity to produce antibodies binding specifically to thymic epithelial cells in culture (as assessed by indirect immunofluorescence) and their ability to absorb in vitro the biological activity of synthetic and natural hormone preparations and to induce in vivo the disappearance of endogenous circulating thymulin. In this way monoclonal antibodies were obtained that recognized a subpopulation of nonlymphoid cells on frozen sections of mouse and human thymuses. The epithelial nature of these cells was assessed using an antikeratin antiserum. The binding of the antibodies to thymic cells was completely abolished by its absorption with the synthetic hormone or normal (but not of thymectomized) mouse serum. The thymic specificity of the antibody was further confirmed by the complete absence of binding to sections of all the various lymphoid and epithelial organs examined (from both humans and mice). Double labeling experiments using the monoclonal antibody described above and a monoclonal antibody prepared by immunization with the synthetic peptide showed that the two antibodies bound to the same cell. These results provide further evidence for the exclusive presence of the thymic hormone thymulin in thymic epithelial cells.

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