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Article: An improved clonogenic culture method for thymic epithelial cells

Sekai, Miho / Jianwei Wang / Nagahiro Minato / Yoko Hamazaki

Journal of immunological methods. 2019 Feb. 05,

2019

Abstract: A clonogenic assay system for thymic epithelial cells (TECs) is of crucial importance for identifying thymic epithelial stem and/or progenitor cells, evaluating their activities, and understanding the mechanisms of thymic involution. However, current ... ...

Abstract	A clonogenic assay system for thymic epithelial cells (TECs) is of crucial importance for identifying thymic epithelial stem and/or progenitor cells, evaluating their activities, and understanding the mechanisms of thymic involution. However, current systems are not sufficiently sensitive at detecting and quantifying TEC colonies from the adult thymus. Here, we optimized the culture condition to detect visible colonies from adult TECs by modifying our previous culture methods. Epidermal growth factor and leukemia inhibitory factor significantly enhanced the colony-forming efficiency of total TECs from embryo as well as adult mice when added 3 days after plating. Importantly, characteristics of the TEC colonies formed by the improved condition were almost equivalent to those by the original culture condition with respect to self-renewal and the expression of cell surface markers and intracellular keratins. Furthermore, the colonies derived from total TECs showed immature phenotypes and generated both mature cortical TECs and medullary TECs upon implantation in vivo. These data indicate a more sensitive clonogenic assay system for TECs was established and suggest the improved culture condition supports the colony formation of stem/progenitor cells for cTECs, mTECs and/or bipotent TECs.
Keywords	adults ; embryo (animal) ; epidermal growth factor ; epithelial cells ; epithelium ; leukemia inhibitory factor ; mice ; phenotype ; stem cells ; thymus gland
Language	English
Dates of publication	2019-0205
Size	p. .
Publishing place	Elsevier B.V.
Document type	Article
Note	Pre-press version
ZDB-ID	120142-6
ISSN	1872-7905 ; 0022-1759
ISSN (online)	1872-7905
ISSN	0022-1759
DOI	10.1016/j.jim.2019.02.003
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Claudin 4 in pancreatic β cells is involved in regulating the functional state of adult islets

Hongtu Li / Abraham Neelankal John / Takahiro Nagatake / Yoko Hamazaki / Fang‐Xu Jiang

FEBS Open Bio, Vol 10, Iss 1, Pp 28-

2020 Volume 40

Abstract: The functional state (FS) of adult pancreatic islets is regulated by a large array of regulatory molecules including numerous transcription factors. Whether any islet structural molecules play such a role has not been well understood. Here, multiple ... ...

Abstract	The functional state (FS) of adult pancreatic islets is regulated by a large array of regulatory molecules including numerous transcription factors. Whether any islet structural molecules play such a role has not been well understood. Here, multiple technologies including bioinformatics analyses were used to explore such molecules. The tight junction family molecule claudin 4 (Cldn4) was the highest enriched amongst over 140 structural genes analysed. Cldn4 expression was ~75‐fold higher in adult islets than in exocrine tissues and was mostly up‐regulated during functional maturation of developing islet cells. Cldn4 was progressively down‐regulated in functionally compromised, dedifferentiating insulin‐secreting β cells and in db/db type 2 diabetic islets. Furthermore, the genetic deletion of Cldn4 impaired significantly the FS without apparently affecting pancreas morphology, islet architectural structure and cellular distribution, and secretion of enteroendocrine hormones. Thus, we suggest a previously unidentified role for Cldn4 in regulating the FS of islets, with implications in translational research for better diabetes therapies.
Keywords	β cells ; bioinformatics analysis ; claudin 4 ; dedifferentiation ; functional state ; Biology (General) ; QH301-705.5
Subject code	570
Language	English
Publishing date	2020-01-01T00:00:00Z
Publisher	Wiley
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Enteroendocrine cells are specifically marked by cell surface expression of claudin-4 in mouse small intestine.

Takahiro Nagatake / Harumi Fujita / Nagahiro Minato / Yoko Hamazaki

PLoS ONE, Vol 9, Iss 6, p e

2014 Volume 90638

Abstract: Enteroendocrine cells are solitary epithelial cells scattered throughout the gastrointestinal tract and produce various types of hormones, constituting one of the largest endocrine systems in the body. The study of these rare epithelial cells has been ... ...

Abstract	Enteroendocrine cells are solitary epithelial cells scattered throughout the gastrointestinal tract and produce various types of hormones, constituting one of the largest endocrine systems in the body. The study of these rare epithelial cells has been hampered by the difficulty in isolating them because of the lack of specific cell surface markers. Here, we report that enteroendocrine cells selectively express a tight junction membrane protein, claudin-4 (Cld4), and are efficiently isolated with the use of an antibody specific for the Cld4 extracellular domain and flow cytometry. Sorted Cld4+ epithelial cells in the small intestine exclusively expressed a chromogranin A gene (Chga) and other enteroendocrine cell-related genes (Ffar1, Ffar4, Gpr119), and the population was divided into two subpopulations based on the activity of binding to Ulex europaeus agglutinin-1 (UEA-1). A Cld4+UEA-1- cell population almost exclusively expressed glucose-dependent insulinotropic polypeptide gene (Gip), thus representing K cells, whereas a Cld4+UEA-1+ cell population expressed other gut hormone genes, including glucagon-like peptide 1 (Gcg), pancreatic polypeptide-like peptide with N-terminal tyrosine amide (Pyy), cholecystokinin (Cck), secretin (Sct), and tryptophan hydroxylase 1 (Tph1). In addition, we found that orally administered luminal antigens were taken up by the solitary Cld4+ cells in the small intestinal villi, raising the possibility that enteroendocrine cells might also play a role in initiation of mucosal immunity. Our results provide a useful tool for the cellular and functional characterization of enteroendocrine cells.
Keywords	Medicine ; R ; Science ; Q
Subject code	571
Language	English
Publishing date	2014-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: CD153/CD30 signaling promotes age-dependent tertiary lymphoid tissue expansion and kidney injury

Yuki Sato / Akiko Oguchi / Yuji Fukushima / Kyoko Masuda / Naoya Toriu / Keisuke Taniguchi / Takahisa Yoshikawa / Xiaotong Cui / Makiko Kondo / Takeshi Hosoi / Shota Komidori / Yoko Shimizu / Harumi Fujita / Li Jiang / Yingyi Kong / Takashi Yamanashi / Jun Seita / Takuya Yamamoto / Shinya Toyokuni /

Yoko Hamazaki / Masakazu Hattori / Yasunobu Yoshikai / Peter Boor / Jürgen Floege / Hiroshi Kawamoto / Yasuhiro Murakawa / Nagahiro Minato / Motoko Yanagita

The Journal of Clinical Investigation, Vol 132, Iss

2022 Volume 2

Abstract: Tertiary lymphoid tissues (TLTs) facilitate local T and B cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here, we identified TNF superfamily CD153/CD30 signaling ... ...

Abstract	Tertiary lymphoid tissues (TLTs) facilitate local T and B cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here, we identified TNF superfamily CD153/CD30 signaling between 2 unique age-dependent lymphocyte subpopulations, CD153+PD-1+CD4+ senescence-associated T (SAT) cells and CD30+T-bet+ age-associated B cells (ABCs), as a driver for TLT expansion. SAT cells, which produced ABC-inducing factors IL-21 and IFN-γ, and ABCs progressively accumulated within TLTs in aged kidneys after injury. Notably, in kidney injury models, CD153 or CD30 deficiency impaired functional SAT cell induction, which resulted in reduced ABC numbers and attenuated TLT formation with improved inflammation, fibrosis, and renal function. Attenuated TLT formation after transplantation of CD153-deficient bone marrow further supported the importance of CD153 in immune cells. Clonal analysis revealed that SAT cells and ABCs in the kidneys arose from both local differentiation and recruitment from the spleen. In the synovium of aged rheumatoid arthritis patients, T peripheral helper/T follicular helper cells and ABCs also expressed CD153 and CD30, respectively. Together, our data reveal a previously unappreciated function of CD153/CD30 signaling in TLT formation and propose targeting the CD153/CD30 signaling pathway as a therapeutic target for slowing kidney disease progression.
Keywords	Inflammation ; Nephrology ; Medicine ; R
Subject code	570
Language	English
Publishing date	2022-01-01T00:00:00Z
Publisher	American Society for Clinical Investigation
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The potential role of Osteopontin in the maintenance of commensal bacteria homeostasis in the intestine.

Koyu Ito / Akira Nakajima / Yuji Fukushima / Keiichiro Suzuki / Keiko Sakamoto / Yoko Hamazaki / Kouetsu Ogasawara / Nagahiro Minato / Masakazu Hattori

PLoS ONE, Vol 12, Iss 3, p e

2017 Volume 0173629

Abstract: Osteopontin (Opn), a multifunctional extracellular matrix protein, is implicated in the pathogenesis of various inflammatory disorders. Under physiologic conditions, its expression is restricted to certain tissues including bone and kidney tubule. ... ...

Abstract	Osteopontin (Opn), a multifunctional extracellular matrix protein, is implicated in the pathogenesis of various inflammatory disorders. Under physiologic conditions, its expression is restricted to certain tissues including bone and kidney tubule. However, cellular activation during disease development induces Opn expression in various immune cells. In this study, using Opn-EGFP knock-in (KI) mice we found that CD8α+ T cells in the intestinal tissues, including Peyer's patch, lamina propria and epithelium, express Opn under steady state conditions. Therefore, we examined the role of Opn-expressing CD8α+ T cells in intestinal homeostasis. Interestingly, Opn knockout (KO) mice had altered fecal microflora concordant with a reduction of TCRγδ+ intraepithelial lymphocytes (IELs). Consistent with this result, both treatment with anti-Opn blocking antibody and deficiency of Opn resulted in decreased survival of TCRγδ+ and TCRαβ+ IELs. This data suggests that a possibility that Opn may function as a survival factor for IELs in the intestinal tissue. Collectively, these data suggest the possibility that Opn might regulate the homeostasis of intestinal microflora through maintenance of TCRγδ+ IELs, possibly by support of IEL survival.
Keywords	Medicine ; R ; Science ; Q
Subject code	616 ; 570
Language	English
Publishing date	2017-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Claudin-4 deficiency results in urothelial hyperplasia and lethal hydronephrosis.

Harumi Fujita / Yoko Hamazaki / Yumi Noda / Masanobu Oshima / Nagahiro Minato

PLoS ONE, Vol 7, Iss 12, p e

2012 Volume 52272

Abstract: Claudin (Cld)-4 is one of the dominant Clds expressed in the kidney and urinary tract, including selective segments of renal nephrons and the entire urothelium from the pelvis to the bladder. We generated Cldn4(-/-) mice and found that these mice had ... ...

Abstract	Claudin (Cld)-4 is one of the dominant Clds expressed in the kidney and urinary tract, including selective segments of renal nephrons and the entire urothelium from the pelvis to the bladder. We generated Cldn4(-/-) mice and found that these mice had increased mortality due to hydronephrosis of relatively late onset. While the renal nephrons of Cldn4(-/-) mice showed a concomitant diminution of Cld8 expression at tight junction (TJ), accumulation of Cld3 at TJ was markedly enhanced in compensation and the overall TJ structure was unaffected. Nonetheless, Cldn4(-/-) mice showed slightly yet significantly increased fractional excretion of Ca(2+) and Cl(-), suggesting a role of Cld4 in the specific reabsorption of these ions via a paracellular route. Although the urine volume tended to be increased concordantly, Cldn4(-/-) mice were capable of concentrating urine normally on dehydration, with no evidence of diabetes insipidus. In the urothelium, the formation of TJs and uroplaques as well as the gross barrier function were also unaffected. However, intravenous pyelography analysis indicated retarded urine flow prior to hydronephrosis. Histological examination revealed diffuse hyperplasia and a thickening of pelvic and ureteral urothelial layers with markedly increased BrdU uptake in vivo. These results suggest that progressive hydronephrosis in Cldn4(-/-) mice arises from urinary tract obstruction due to urothelial hyperplasia, and that Cld4 plays an important role in maintaining the homeostatic integrity of normal urothelium.
Keywords	Medicine ; R ; Science ; Q
Subject code	616
Language	English
Publishing date	2012-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Impaired airway mucociliary function reduces antigen-specific IgA immune response to immunization with a claudin-4-targeting nasal vaccine in mice

Hidehiko Suzuki / Takahiro Nagatake / Ayaka Nasu / Huangwenxian Lan / Koji Ikegami / Mitsutoshi Setou / Yoko Hamazaki / Hiroshi Kiyono / Kiyohito Yagi / Masuo Kondoh / Jun Kunisawa

Scientific Reports, Vol 8, Iss 1, Pp 1-

2018 Volume 9

Abstract: Abstract Vaccine delivery is an essential element for the development of mucosal vaccine, but it remains to be investigated how physical barriers such as mucus and cilia affect vaccine delivery efficacy. Previously, we reported that C-terminal fragment ... ...

Abstract	Abstract Vaccine delivery is an essential element for the development of mucosal vaccine, but it remains to be investigated how physical barriers such as mucus and cilia affect vaccine delivery efficacy. Previously, we reported that C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) targeted claudin-4, which is expressed by the epithelium associated with nasopharynx-associated lymphoid tissue (NALT), and could be effective as a nasal vaccine delivery. Mice lacking tubulin tyrosine ligase-like family, member 1 (Ttll1-KO mice) showed mucus accumulation in nasal cavity due to the impaired motility of respiratory cilia. Ttll1-KO mice nasally immunized with C-CPE fused to pneumococcal surface protein A (PspA-C-CPE) showed reduced PspA-specific nasal IgA responses, impaired germinal center formation, and decreased germinal center B-cells and follicular helper T cells in the NALT. Although there was no change in the expression of claudin-4 in the NALT epithelium in Ttll1-KO mice, the epithelium was covered by a dense mucus that prevented the binding of PspA-C-CPE to NALT. However, administration of expectorant N-acetylcysteine removed the mucus and rescued the PspA-specific nasal IgA response. These results show that the accumulation of mucus caused by impaired respiratory cilia function is an interfering factor in the C-CPE-based claudin-4-targeting nasal vaccine.
Keywords	Medicine ; R ; Science ; Q
Subject code	570
Language	English
Publishing date	2018-02-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Live-Cell FRET Imaging Reveals a Role of Extracellular Signal-Regulated Kinase Activity Dynamics in Thymocyte Motility

Yoshinobu Konishi / Kenta Terai / Yasuhide Furuta / Hiroshi Kiyonari / Takaya Abe / Yoshihiro Ueda / Tatsuo Kinashi / Yoko Hamazaki / Akifumi Takaori-Kondo / Michiyuki Matsuda

iScience, Vol 10, Iss , Pp 98-

2018 Volume 113

Abstract: Summary: Extracellular signal-regulated kinase (ERK) plays critical roles in T cell development in the thymus. Nevertheless, the dynamics of ERK activity and the role of ERK in regulating thymocyte motility remain largely unknown due to technical ... ...

Abstract	Summary: Extracellular signal-regulated kinase (ERK) plays critical roles in T cell development in the thymus. Nevertheless, the dynamics of ERK activity and the role of ERK in regulating thymocyte motility remain largely unknown due to technical limitations. To visualize ERK activity in thymocytes, we here developed knockin reporter mice expressing a Förster/fluorescence resonance energy transfer (FRET)-based biosensor for ERK from the ROSA26 locus. Live imaging of thymocytes isolated from the reporter mice revealed that ERK regulates thymocyte motility in a subtype-specific manner. Negative correlation between ERK activity and motility was observed in CD4/CD8 double-positive thymocytes and CD8 single-positive thymocytes, but not in CD4 single-positive thymocytes. Interestingly, however, the temporal deviations of ERK activity from the average correlate with the motility of CD4 single-positive thymocytes. Thus, live-cell FRET imaging will open a window to understanding the dynamic nature and the diverse functions of ERK signaling in T cell biology. : Biological Sciences; Components of the Immune System; Molecular Biology Subject Areas: Biological Sciences, Components of the Immune System, Molecular Biology
Keywords	Science ; Q
Subject code	570
Language	English
Publishing date	2018-12-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Adult Thymic Medullary Epithelium Is Maintained and Regenerated by Lineage-Restricted Cells Rather Than Bipotent Progenitors

Izumi Ohigashi / Saulius Zuklys / Mie Sakata / Carlos E. Mayer / Yoko Hamazaki / Nagahiro Minato / Georg A. Hollander / Yousuke Takahama

Cell Reports, Vol 13, Iss 7, Pp 1432-

2015 Volume 1443

Abstract: Medullary thymic epithelial cells (mTECs) play an essential role in establishing self-tolerance in T cells. mTECs originate from bipotent TEC progenitors that generate both mTECs and cortical TECs (cTECs), although mTEC-restricted progenitors also have ... ...

Abstract	Medullary thymic epithelial cells (mTECs) play an essential role in establishing self-tolerance in T cells. mTECs originate from bipotent TEC progenitors that generate both mTECs and cortical TECs (cTECs), although mTEC-restricted progenitors also have been reported. Here, we report in vivo fate-mapping analysis of cells that transcribe β5t, a cTEC trait expressed in bipotent progenitors, during a given period in mice. We show that, in adult mice, most mTECs are derived from progenitors that transcribe β5t during embryogenesis and the neonatal period up to 1 week of age. The contribution of adult β5t+ progenitors was minor even during injury-triggered regeneration. Our results further demonstrate that adult mTEC-restricted progenitors are derived from perinatal β5t+ progenitors. These results indicate that the adult thymic medullary epithelium is maintained and regenerated by mTEC-lineage cells that pass beyond the bipotent stage during early ontogeny.
Keywords	Biology (General) ; QH301-705.5
Subject code	572
Language	English
Publishing date	2015-11-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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