Primary explants of the postnatal thymus allow the expansion of clonogenic thymic epithelial cells that constitute thymospheres.

BACKGROUND: Thymic epithelial cells (TECs) are responsible for shaping the repertoires of T cells, where their postnatal regeneration depends on a subset of clonogenic TECs. Despite the implications for regenerative medicine, their cultivation and expansion remain challenging. Primary explant cell culture is a technique that allows the seeding and expansion of difficult-to-culture cells. Here, we report a reliable and simple culture system to obtain functional TECs and thymic interstitial cells (TICs). METHODS: To establish primary thymic explants, we harvested 1 mm cleaned fragments of thymus from 5-week-old C57/BL6 mice. Tissue fragments of a complete thymic lobe were placed in the center of a Petri dish with 1 mL of DMEM/F-12 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin‒streptomycin. To compare, thymic explants were also cultivated by using serum-free DMEM/F-12 medium supplemented with 10% KnockOut™. RESULTS: We obtained high numbers of functional clonogenic TECs and TICs from primary thymic explants cultivated with DMEM/F-12 with 20% FBS. These cells exhibited a highly proliferative and migration profile and were able to constitute thymospheres. Furthermore, all the subtypes of medullary TECs were identified in this system. They express functional markers to shape T-cell and type 2 innate lymphoid cells repertoires, such as Aire, IL25, CCL21 and CD80. Finally, we also found that ≥ 70% of lineage negative TICs expressed high amounts of Aire and IL25. CONCLUSION: Thymic explants are an efficient method to obtain functional clonogenic TECs, all mTEC subsets and different TICs Aire+IL25+ with high regenerative capacity.

[Mechanism of pregnancy-induced thymus involution and regeneration and medication rules of postpartum prescriptions].

In order to prevent the maternal immune defenses to the semi-allogeneic fetus, the maternal body will present a special adaptive immune system change represented by acute thymic involution(ATI) during pregnancy, which can be quickly regenerated after delivery. The ATI during pregnancy is related to the level of sex hormones, which is mainly caused by progesterone. Pregnancy-induced ATI is manifested as the continuous shrinkage of thymus volume, especially the cortex, and the wrinkle and phagocytosis of the subcapsular cortical thymic epithelial cells(cTECs), while other thymic epithelial cells(TECs) remain unchanged. The postpartum thymus is regenerated by the co-mediation of forkhead box N1(FOXN1) as well as its target genes chemokine(C-C motif) ligand 25(CCL25), chemokine(C-X-C motif) ligand 12(CXCL12), δ-like ligand 4(DLL4), cathepsin L(CTSL), and serine protease 16(PRSS16). Once the postpartum thymus is poorly repaired, immune dysfunction of the maternal body and several puerperal diseases will be induced, seriously endangering the survival of the mother and the newborn. In traditional Chinese medicine(TCM), Qi and blood are the cornerstone of pregnancy, and the thymus plays a key role in regulating Qi and blood. The deficiency of Qi and blood during pregnancy and childbirth is closely related to the abnormal ATI during pregnancy and the poor regeneration of the postpartum thymus. Based on this theory, TCM has profound academic ideas and rich clinical experience in postpartum recuperation. Based on the systematic description of the mechanism of ATI regeneration during pregnancy, as well as data mining and analysis of two classic gynecological works of TCM, Wan's Gynecology and Fu Qing-zhu's Treatise on Gynecology, this study found that the commonly used TCM for postpartum included Angelicae Sinensis Radix, Ginseng Radix et Rhizoma, Glycyrrhizae Radix et Rhizoma, and Chuanxiong Rhizoma. Among them, Ginseng Radix et Rhizoma, Angelicae Sinensis Radix, and Chuanxiong Rhizoma are high-frequency TCMs with positive effects on postpartum recovery.However, the mechanism of these TCMs in promoting postpartum thymus regeneration needs further investigation.

Damage-induced pyroptosis drives endog thymic regeneration via induction of Foxn1 by purinergic receptor activation.

Endogenous thymic regeneration is a crucial process that allows for the renewal of immune competence following stress, infection or cytoreductive conditioning. Fully understanding the molecular mechanisms driving regeneration will uncover therapeutic targets to enhance regeneration. We previously demonstrated that high levels of homeostatic apoptosis suppress regeneration and that a reduction in the presence of damage-induced apoptotic thymocytes facilitates regeneration. Here we identified that cell-specific metabolic remodeling after ionizing radiation steers thymocytes towards mitochondrial-driven pyroptotic cell death. We further identified that a key damage-associated molecular pattern (DAMP), ATP, stimulates the cell surface purinergic receptor P2Y2 on cortical thymic epithelial cells (cTECs) acutely after damage, enhancing expression of Foxn1, the critical thymic transcription factor. Targeting the P2Y2 receptor with the agonist UTPγS promotes rapid regeneration of the thymus in vivo following acute damage. Together these data demonstrate that intrinsic metabolic regulation of pyruvate processing is a critical process driving thymus repair and identifies the P2Y2 receptor as a novel molecular therapeutic target to enhance thymus regeneration.

Mechanism of pregnancy-induced thymus involution and regeneration and medication rules of postpartum prescriptions / 中国中药杂志

In order to prevent the maternal immune defenses to the semi-allogeneic fetus, the maternal body will present a special adaptive immune system change represented by acute thymic involution(ATI) during pregnancy, which can be quickly regenerated after delivery. The ATI during pregnancy is related to the level of sex hormones, which is mainly caused by progesterone. Pregnancy-induced ATI is manifested as the continuous shrinkage of thymus volume, especially the cortex, and the wrinkle and phagocytosis of the subcapsular cortical thymic epithelial cells(cTECs), while other thymic epithelial cells(TECs) remain unchanged. The postpartum thymus is regenerated by the co-mediation of forkhead box N1(FOXN1) as well as its target genes chemokine(C-C motif) ligand 25(CCL25), chemokine(C-X-C motif) ligand 12(CXCL12), δ-like ligand 4(DLL4), cathepsin L(CTSL), and serine protease 16(PRSS16). Once the postpartum thymus is poorly repaired, immune dysfunction of the maternal body and several puerperal diseases will be induced, seriously endangering the survival of the mother and the newborn. In traditional Chinese medicine(TCM), Qi and blood are the cornerstone of pregnancy, and the thymus plays a key role in regulating Qi and blood. The deficiency of Qi and blood during pregnancy and childbirth is closely related to the abnormal ATI during pregnancy and the poor regeneration of the postpartum thymus. Based on this theory, TCM has profound academic ideas and rich clinical experience in postpartum recuperation. Based on the systematic description of the mechanism of ATI regeneration during pregnancy, as well as data mining and analysis of two classic gynecological works of TCM, Wan's Gynecology and Fu Qing-zhu's Treatise on Gynecology, this study found that the commonly used TCM for postpartum included Angelicae Sinensis Radix, Ginseng Radix et Rhizoma, Glycyrrhizae Radix et Rhizoma, and Chuanxiong Rhizoma. Among them, Ginseng Radix et Rhizoma, Angelicae Sinensis Radix, and Chuanxiong Rhizoma are high-frequency TCMs with positive effects on postpartum recovery.However, the mechanism of these TCMs in promoting postpartum thymus regeneration needs further investigation.

Key Factors for Thymic Function and Development.

The thymus is the organ responsible for T cell development and the formation of the adaptive immunity function. Its multicellular environment consists mainly of the different stromal cells and maturing T lymphocytes. Thymus-specific progenitors of epithelial, mesenchymal, and lymphoid cells with stem cell properties represent only minor populations. The thymic stromal structure predominantly determines the function of the thymus. The stromal components, mostly epithelial and mesenchymal cells, form this specialized area. They support the consistent developmental program of functionally distinct conventional T cell subpopulations. These include the MHC restricted single positive CD4+ CD8- and CD4- CD8+ cells, regulatory T lymphocytes (Foxp3+), innate natural killer T cells (iNKT), and γδT cells. Several physiological causes comprising stress and aging and medical treatments such as thymectomy and chemo/radiotherapy can harm the thymus function. The present review summarizes our knowledge of the development and function of the thymus with a focus on thymic epithelial cells as well as other stromal components and the signaling and transcriptional pathways underlying the thymic cell interaction. These critical thymus components are significant for T cell differentiation and restoring the thymic function after damage to reach the therapeutic benefits.

Thymus Functionality Needs More Than a Few TECs.

The thymus, a primary lymphoid organ, produces the T cells of the immune system. Originating from the 3rd pharyngeal pouch during embryogenesis, this organ functions throughout life. Yet, thymopoiesis can be transiently or permanently damaged contingent on the types of systemic stresses encountered. The thymus also undergoes a functional decline during aging, resulting in a progressive reduction in naïve T cell output. This atrophy is evidenced by a deteriorating thymic microenvironment, including, but not limited, epithelial-to-mesenchymal transitions, fibrosis and adipogenesis. An exploration of cellular changes in the thymus at various stages of life, including mouse models of in-born errors of immunity and with single cell RNA sequencing, is revealing an expanding number of distinct cell types influencing thymus functions. The thymus microenvironment, established through interactions between immature and mature thymocytes with thymus epithelial cells (TEC), is well known. Less well appreciated are the contributions of neural crest cell-derived mesenchymal cells, endothelial cells, diverse hematopoietic cell populations, adipocytes, and fibroblasts in the thymic microenvironment. In the current review, we will explore the contributions of the many stromal cell types participating in the formation, expansion, and contraction of the thymus under normal and pathophysiological processes. Such information will better inform approaches for restoring thymus functionality, including thymus organoid technologies, beneficial when an individuals' own tissue is congenitally, clinically, or accidentally rendered non-functional.

Recent Advancements in Regenerative Approaches for Thymus Rejuvenation.

The thymus plays a key role in adaptive immunity by generating a diverse population of T cells that defend the body against pathogens. Various factors from disease and toxic insults contribute to the degeneration of the thymus resulting in a fewer output of T cells. Consequently, the body is prone to a wide host of diseases and infections. In this review, first, the relevance of the thymus is discussed, followed by thymic embryological organogenesis and anatomy as well as the development and functionality of T cells. Attempts to regenerate the thymus include in vitro methods, such as forming thymic organoids aided by biofabrication techniques that are transplantable. Ex vivo methods that have shown promise in enhancing thymic regeneration are also discussed. Current regenerative technologies have not yet matched the complexity and functionality of the thymus. Therefore, emerging techniques that have shown promise and the challenges that lie ahead are explored.

Thymic Engraftment by in vitro-Derived Progenitor T Cells in Young and Aged Mice.

T cells play a critical role in mediating antigen-specific and long-term immunity against viral and bacterial pathogens, and their development relies on the highly specialized thymic microenvironment. T cell immunodeficiency can be acquired in the form of inborn errors, or can result from perturbations to the thymus due to aging or irradiation/chemotherapy required for cancer treatment. Hematopoietic stem cell transplant (HSCT) from compatible donors is a cornerstone for the treatment of hematological malignancies and immunodeficiency. Although it can restore a functional immune system, profound impairments exist in recovery of the T cell compartment. T cells remain absent or low in number for many months after HSCT, depending on a variety of factors including the age of the recipient. While younger patients have a shorter refractory period, the prolonged T cell recovery observed in older patients can lead to a higher risk of opportunistic infections and increased predisposition to relapse. Thus, strategies for enhancing T cell recovery in aged individuals are needed to counter thymic damage induced by radiation and chemotherapy toxicities, in addition to naturally occurring age-related thymic involution. Preclinical results have shown that robust and rapid long-term thymic reconstitution can be achieved when progenitor T cells, generated in vitro from HSCs, are co-administered during HSCT. Progenitor T cells appear to rely on lymphostromal crosstalk via receptor activator of NF-κB (RANK) and RANK-ligand (RANKL) interactions, creating chemokine-rich niches within the cortex and medulla that likely favor the recruitment of bone marrow-derived thymus seeding progenitors. Here, we employed preclinical mouse models to demonstrate that in vitro-generated progenitor T cells can effectively engraft involuted aged thymuses, which could potentially improve T cell recovery. The utility of progenitor T cells for aged recipients positions them as a promising cellular therapy for immune recovery and intrathymic repair following irradiation and chemotherapy, even in a post-involution thymus.

Thymus Regeneration and Future Challenges.

Thymus regenerative therapy implementation is severely obstructed by the limited number and expansion capacity in vitro of tissue-specific thymic epithelial stem cells (TESC). Current solutions are mostly based on growth factors that can drive differentiation of pluripotent stem cells toward tissue-specific TESC. Target-specific small chemical compounds represent an alternative solution that could induce and support the clonal expansion of TESC and reversibly block their differentiation into mature cells. These compounds could be used both in the composition of culture media designed for TESC expansion in vitro, and in drugs development for thymic regeneration in vivo. It should allow reaching the ultimate objective - autologous thymic tissue regeneration in paediatric patients who had their thymus removed in the course of cardiac surgery.

Interplay between Follistatin, Activin A, and BMP4 Signaling Regulates Postnatal Thymic Epithelial Progenitor Cell Differentiation during Aging.

A key feature of immune functional impairment with age is the progressive involution of thymic tissue responsible for naive T cell production. In this study, we identify two major phases of thymic epithelial cell (TEC) loss during aging: a block in mature TEC differentiation from the pool of immature precursors, occurring at the onset of puberty, followed by impaired bipotent TEC progenitor differentiation and depletion of Sca-1lo cTEC and mTEC lineage-specific precursors. We reveal that an increase in follistatin production by aging TECs contributes to their own demise. TEC loss occurs primarily through the antagonism of activin A signaling, which we show is required for TEC maturation and acts in dissonance to BMP4, which promotes the maintenance of TEC progenitors. These results support a model in which an imbalance of activin A and BMP4 signaling underpins the degeneration of postnatal TEC maintenance during aging, and its reversal enables the transient replenishment of mature TECs.

Aging of lymphoid organs: Can photobiomodulation reverse age-associated thymic involution via stimulation of extrapineal melatonin synthesis and bone marrow stem cells?

Thymic atrophy and the subsequent reduction in T-cell production are the most noticeable age-related changes affecting lymphoid organs in the immune system. In fact, thymic involution has been described as "programmed aging." New therapeutic approaches, such as photobiomodulation (PBM), may reduce or reverse these changes. PBM (also known as low-level laser therapy) involves the delivery of non-thermal levels of red or near-infrared light that are absorbed by mitochondrial chromophores, in order to prevent tissue death and stimulate healing and regeneration. PBM may reverse or prevent thymic involution due to its ability to induce extrapineal melatonin biosynthesis via cyclic adenosine monophosphate (AMP) or NF-kB activation, or alternatively by stimulating bone marrow stem cells that can regenerate the thymus. This perspective puts forward a hypothesis that PBM can alter thymic involution, improve immune functioning in aged people and even extend lifespan.

Native thymic extracellular matrix improves in vivo thymic organoid T cell output, and drives in vitro thymic epithelial cell differentiation.

Although the thymus is a primary lymphoid organ, its function is compromised by an age-induced loss of resident epithelial cells, which results in reduced naïve T cell output. This has important implications for immune recovery in aged and elderly patients following damage from cytoablative therapies. As thymic architecture plays a crucial role in naïve T cell development, a tissue specific scaffold that provides essential supporting matrix may assist in stem cell-based thymus regeneration to recreate complex organoids. Here we investigate thymus decellularization approaches that preserve major extracellular matrix components and support thymic epithelial cells for the generation of a functional thymic microenvironment with improved T cell output. We also established an in vitro, serum-free culture system that both maintains a progenitor thymic epithelial cell pool and drives their differentiation in the presence of decellularized thymic matrix. This approach enables further dissection of key cellular and niche components involved in thymic epithelial stem cell maintenance and T cell production.

The Thymus: A Forgotten, But Very Important Organ.

Medical science seems to be on the threshold of a revolution: It seems possible that in twenty years, doctors will be able to replace organs in the human body like parts in a car. This is thanks to the recent achievement of a team from the Medical Research Council Center for Regenerative Medicine in Edinburgh, Scotland - the group of researchers tried to regenerate the thymus gland in mice. The thymus gland is an essential organ for the development of the immune system, but very few people have any idea that it exists. In the literature and also in people's awareness, the fact is often that the thymus controls and harmonizes the entire immune system and the immune functioning of the organism. It is the primary donor of cells for the lymphatic system, much as bone marrow is the cell donor for the cardiovascular system. It is within the thymus that progenitor cells are created and then undergo maturation and differentiation into mature T cells. The thymus gland is located in the mediastinum, behind the sternum. It is composed of two identical lobes. Each lobe is divided into a central medulla and a peripheral cortex. The thymus is at its largest and most active during the neonatal and pre-adolescent periods. After this period the organ gradually disappears and is replaced by fat. In elderly individuals the thymus weighs 5 g. The aim of this work is to shed new light on this important immune defense organ, whose function is not confined to the destruction of nonfunctional T cells.

Characterization of the expression of cytokeratins 5, 8, and 14 in mouse thymic epithelial cells during thymus regeneration following acute thymic involution.

The thymus is a central lymphoid organ for T cell development. Thymic epithelial cells (TECs) constitute a major component of the thymic stroma, which provides a specialized microenvironment for survival, proliferation, and differentiation of immature T cells. In this study, subsets of TECs were examined immunohistochemically to investigate their cytokeratin (CK) expression patterns during thymus regeneration following thymic involution induced by cyclophosphamide treatment. The results demonstrated that both normal and regenerating mouse thymuses showed a similar CK expression pattern. The major medullary TECs (mTEC) subset, which is stellate in appearance, exhibited CK5 and CK14 staining, and the minor mTEC subset, which is globular in appearance, exhibited CK8 staining, whereas the vast majority of cortical TECs (cTECs) expressed CK8 during thymus regeneration. Remarkably, the levels of CK5 and CK14 expression were enhanced in mTECs, and CK8 expression was upregulated in cTECs during mouse thymus regeneration after cyclophosphamide-induced acute thymic involution. Of special interest, a relatively high number of CK5(+)CK8(+) TEC progenitors occurred in the thymic cortex during thymus regeneration. Taken together, these findings shed more light on the role of CK5, CK8, and CK14 in the physiology of TECs during mouse thymus regeneration, and on the characterization of TEC progenitors for restoration of the epithelial network and for concomitant regeneration of the adult thymus.

Expression of IL-17A during Thymus Regeneration in the Rat / 체질인류학회지

IL-17A is a pro-inflammatroy cytokine secreted by activated T cells. The IL-17 family consist of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F. IL-17A and IL-17F are produced primarily in activated T cells. In contrast, IL-17B, IL-17C, IL-17D and IL-17E are expressed in a wide assortment of tissues. Their functions partially overlap those of IL-17A, although they have not been as thoroughly investigated. The receptor for IL-17A (IL-17R) is widely expressed in a variety of tissues. IL-17A and IL-17E mRNAs were expressed in only EL4 cells. IL-17C mRNA expression was observed in the thymic subcapsular/cortex epithelial cells (SNEC), cortex or cortical reticular cells (CREC), medullary epithelial cells (MEC), medullary interdigitating-like cells (MDC), thymocytes and EL4 cells. However, IL-17C mRNA was not expressed in RAW 264.7 cells. Immunohistochemical study also demonstrated not only the presence of IL-17A mainly in the thymic epithelial cells, but also the upregulated expression of IL-17A in the thymic epithelial cells of the regenerating thymus. Thus, the results of the present study suggest that IL-17A expressed in the thymocytes and thymic epithelial cells could play an important role in the development of new T cells to replace T cells damaged by cyclophosphamide treatment during thymus regeneration.

Characterization of the expression of cytokeratins 5, 8, and 14 in mouse thymic epithelial cells during thymus regeneration following acute thymic involution / 대한해부학회지

The thymus is a central lymphoid organ for T cell development. Thymic epithelial cells (TECs) constitute a major component of the thymic stroma, which provides a specialized microenvironment for survival, proliferation, and differentiation of immature T cells. In this study, subsets of TECs were examined immunohistochemically to investigate their cytokeratin (CK) expression patterns during thymus regeneration following thymic involution induced by cyclophosphamide treatment. The results demonstrated that both normal and regenerating mouse thymuses showed a similar CK expression pattern. The major medullary TECs (mTEC) subset, which is stellate in appearance, exhibited CK5 and CK14 staining, and the minor mTEC subset, which is globular in appearance, exhibited CK8 staining, whereas the vast majority of cortical TECs (cTECs) expressed CK8 during thymus regeneration. Remarkably, the levels of CK5 and CK14 expression were enhanced in mTECs, and CK8 expression was upregulated in cTECs during mouse thymus regeneration after cyclophosphamide-induced acute thymic involution. Of special interest, a relatively high number of CK5+CK8+ TEC progenitors occurred in the thymic cortex during thymus regeneration. Taken together, these findings shed more light on the role of CK5, CK8, and CK14 in the physiology of TECs during mouse thymus regeneration, and on the characterization of TEC progenitors for restoration of the epithelial network and for concomitant regeneration of the adult thymus.

Expression of the p75NTR Neurotrophin Receptor in the Thymic Medullary Epithelial Cells and Interdigitating Dendritic Cells during Rat Thymus Regeneration Following Acute Thymic Involution / 대한해부학회지

The existence of a functional link between the nervous and immune systems has been well established. The present study was to characterize the expression of p75NTR during thymus regeneration from acute involution induced by cyclophosphamide in the rat. Immunohistochemical and double immunofluorescence analyses demonstrated that expression of the p75NTR was decreased in the thymic medullary epithelial cells and interdigitating dendritic cells during thymus regeneration. The presence of p75NTR protein in extracts from the control and regenerating rat thymus was confirmed by western blot. Furthermore, RT-PCR analysis supported these results by demonstrating that thymic extracts contain p75NTR mRNA at lower levels during thymus regeneration. Thus, our results suggest that the p75NTR located on the thymic medullary epithelial cells and interdigitating dendritic cells could play a role in the development of new T cells to replace the thymocytes damaged during thymus regeneration