Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors.

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs.

Thymic epithelial organoids mediate T cell development.

Although the advent of organoids opened unprecedented perspectives for basic and translational research, immune system-related organoids remain largely underdeveloped. Here we established organoids from the thymus, the lymphoid organ responsible for T cell development. We identified conditions enabling thymic epithelial progenitor cell proliferation and development into organoids with diverse cell populations and transcriptional profiles resembling in vivo thymic epithelial cells (TECs) more closely than traditional TEC cultures. Contrary to these two-dimensional cultures, thymic epithelial organoids maintained thymus functionality in vitro and mediated physiological T cell development upon reaggregation with T cell progenitors. The reaggregates showed in vivo-like epithelial diversity and ability to attract T cell progenitors. Thymic epithelial organoids are the first organoids originating from the stromal compartment of a lymphoid organ. They provide new opportunities to study TEC biology and T cell development in vitro, paving the way for future thymic regeneration strategies in ageing or acute injuries.

An induced thymic epithelial cell-based high throughput screen for thymus extracellular matrix mimetics.

Thymic epithelial cells (TECs) are key effectors of the thymic stroma and are critically required for T-cell development. TECs comprise a diverse set of related but functionally distinct cell types that are scarce and difficult to isolate and handle. This has precluded TEC-based screening assays. We previously described induced thymic epithelial cells (iTECs), an artificial cell type produced in vitro by direct reprogramming, raising the possibility that iTECs might provide the basis for functional screens related to TEC biology. Here, we present an iTEC-based three-stage medium/high-throughput in vitro assay for synthetic polymer mimics of thymic extracellular matrix (ECM). Using this assay, we identified, from a complex library, four polymers that bind iTEC as well as or better than gelatin but do not bind mesenchymal cells. We show that these four polymers also bind and maintain native mouse fetal TECs and native human fetal TECs. Finally, we show that the selected polymers do not interfere with iTEC function or T-cell development. Collectively, our data establish that iTECs can be used to screen for TEC-relevant compounds in at least some medium/high-throughput assays and identify synthetic polymer ECM mimics that can replace gelatin or ECM components in TEC culture protocols.

Canonical Notch signaling controls the early thymic epithelial progenitor cell state and emergence of the medullary epithelial lineage in fetal thymus development.

Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T cell repertoire. During thymus organogenesis, these functionally distinct sub-lineages are thought to arise from a common thymic epithelial progenitor cell (TEPC). However, the mechanisms controlling cTEC and mTEC production from the common TEPC are not understood. Here, we show that emergence of the earliest mTEC lineage-restricted progenitors requires active NOTCH signaling in progenitor TEC and that, once specified, further mTEC development is NOTCH independent. In addition, we demonstrate that persistent NOTCH activity favors maintenance of undifferentiated TEPCs at the expense of cTEC differentiation. Finally, we uncover a cross-regulatory relationship between NOTCH and FOXN1, a master regulator of TEC differentiation. These data establish NOTCH as a potent regulator of TEPC and mTEC fate during fetal thymus development, and are thus of high relevance to strategies aimed at generating/regenerating functional thymic tissue in vitro and in vivo.

Expression of Plet1 controls interstitial migration of murine small intestinal dendritic cells.

Under homeostatic conditions, dendritic cells (DCs) continuously patrol the intestinal lamina propria. Upon antigen encounter, DCs initiate C-C motif chemokine receptor 7 (CCR7) expression and migrate into lymph nodes to direct T cell activation and differentiation. The mechanistic underpinnings of DC migration from the tissues to lymph nodes have been largely elucidated, contributing greatly to our understanding of DC functionality and intestinal immunity. In contrast, the molecular mechanisms allowing DCs to efficiently migrate through the complex extracellular matrix of the intestinal lamina propria prior to antigen encounter are still incompletely understood. Here we show that small intestinal murine CD11b+ CD103+ DCs express Placenta-expressed transcript 1 (Plet1), a glycophoshatidylinositol (GPI)-anchored surface protein involved in migration of keratinocytes during wound healing. In the absence of Plet1, CD11b+ CD103+ DCs display aberrant migratory behavior, and accumulate in the small intestine, independent of CCR7 responsiveness. RNA-sequencing indicated involvement of Plet1 in extracellular matrix-interactiveness, and subsequent in-vitro migration assays revealed that Plet1 augments the ability of DCs to migrate through extracellular matrix containing environments. In conclusion, our findings reveal that expression of Plet1 facilitates homeostatic interstitial migration of small intestinal DCs.

EuroStemCell: A European infrastructure for communication and engagement with stem cell research.

EuroStemCell is a large and growing network of organizations and individuals focused on public engagement with stem cells and regenerative medicine - a fluid and contested domain, where scientific, political, ethical, legal and societal perspectives intersect. Rooted in the European stem cell research community, this project has developed collaborative and innovative approaches to information provision and direct and online engagement, that reflect and respond to the dynamic growth of the field itself. EuroStemCell started as the communication and outreach component of a research consortium and subsequently continued as a stand-alone engagement initiative. The involvement of established European stem cell scientists has grown year-on-year, facilitating their participation in public engagement by allowing them to make high-value contributions with broad reach. The project has now had sustained support by partners and funders for over twelve years, and thus provides a model for longevity in public engagement efforts. This paper considers the evolution of the EuroStemCell project in response to - and in dialogue with - its evolving environment. In it, we aim to reveal the mechanisms and approaches taken by EuroStemCell, such that others within the scientific community can explore these ideas and be further enabled in their own public engagement endeavours.

FOXN1 in thymus organogenesis and development.

Development of the primary T-cell repertoire takes place in the thymus. The linked processes of T-cell differentiation and T-cell repertoire selection each depend on interactions between thymocytes and thymic stromal cells; in particular, with the epithelial cells of the cortical and medullary thymic compartments (cortical and medullary thymic epithelial cells; cTECs and mTECs, respectively). The importance of the thymic epithelial cell lineage in these processes was revealed in part through analysis of nude (nu/nu) mice, which are congenitally hairless and athymic. The nude phenotype results from null mutation of the forkhead transcription factor FOXN1, which has emerged as a pivotal regulator both of thymus development and homeostasis. FOXN1 has been shown to play critical roles in thymus development, function, maintenance, and even regeneration, which positions it as a master regulator of thymic epithelial cell (TEC) differentiation. In this review, we discuss current understanding of the regulation and functions of FOXN1 throughout thymus ontogeny, from the earliest stages of organogenesis through homeostasis to age-related involution, contextualising its significance through reference to other members of the wider Forkhead family.

Regeneration of the aged thymus by a single transcription factor.

Thymic involution is central to the decline in immune system function that occurs with age. By regenerating the thymus, it may therefore be possible to improve the ability of the aged immune system to respond to novel antigens. Recently, diminished expression of the thymic epithelial cell (TEC)-specific transcription factor Forkhead box N1 (FOXN1) has been implicated as a component of the mechanism regulating age-related involution. The effects of upregulating FOXN1 function in the aged thymus are, however, unknown. Here, we show that forced, TEC-specific upregulation of FOXN1 in the fully involuted thymus of aged mice results in robust thymus regeneration characterized by increased thymopoiesis and increased naive T cell output. We demonstrate that the regenerated organ closely resembles the juvenile thymus in terms of architecture and gene expression profile, and further show that this FOXN1-mediated regeneration stems from an enlarged TEC compartment, rebuilt from progenitor TECs. Collectively, our data establish that upregulation of a single transcription factor can substantially reverse age-related thymic involution, identifying FOXN1 as a specific target for improving thymus function and, thus, immune competence in patients. More widely, they demonstrate that organ regeneration in an aged mammal can be directed by manipulation of a single transcription factor, providing a provocative paradigm that may be of broad impact for regenerative biology.

Dynamics of thymus organogenesis and colonization in early human development.

The thymus is the central site of T-cell development and thus is of fundamental importance to the immune system, but little information exists regarding molecular regulation of thymus development in humans. Here we demonstrate, via spatial and temporal expression analyses, that the genetic mechanisms known to regulate mouse thymus organogenesis are conserved in humans. In addition, we provide molecular evidence that the human thymic epithelium derives solely from the third pharyngeal pouch, as in the mouse, in contrast to previous suggestions. Finally, we define the timing of onset of hematopoietic cell colonization and epithelial cell differentiation in the human thymic primordium, showing, unexpectedly, that the first colonizing hematopoietic cells are CD45(+)CD34(int/-). Collectively, our data provide essential information for translation of principles established in the mouse to the human, and are of particular relevance to development of improved strategies for enhancing immune reconstitution in patients.

Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells.

The thymus develops from the third pharyngeal pouch of the anterior gut and provides the necessary environment for thymopoiesis (the process by which thymocytes differentiate into mature T lymphocytes) and the establishment and maintenance of self-tolerance. It contains thymic epithelial cells (TECs) that form a complex three-dimensional network organized in cortical and medullary compartments, the organization of which is notably different from simple or stratified epithelia. TECs have an essential role in the generation of self-tolerant thymocytes through expression of the autoimmune regulator Aire, but the mechanisms involved in the specification and maintenance of TECs remain unclear. Despite the different embryological origins of thymus and skin (endodermal and ectodermal, respectively), some cells of the thymic medulla express stratified-epithelium markers, interpreted as promiscuous gene expression. Here we show that the thymus of the rat contains a population of clonogenic TECs that can be extensively cultured while conserving the capacity to integrate in a thymic epithelial network and to express major histocompatibility complex class II (MHC II) molecules and Aire. These cells can irreversibly adopt the fate of hair follicle multipotent stem cells when exposed to an inductive skin microenvironment; this change in fate is correlated with robust changes in gene expression. Hence, microenvironmental cues are sufficient here to re-direct epithelial cell fate, allowing crossing of primitive germ layer boundaries and an increase in potency.

Foxn1 regulates lineage progression in cortical and medullary thymic epithelial cells but is dispensable for medullary sublineage divergence.

The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC) development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development.

Thymus-associated parathyroid hormone has two cellular origins with distinct endocrine and immunological functions.

In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid Gcm2⁻/⁻ mutants. However, the developmental ontogeny and cellular identity of these "thymic" PTH-expressing cells is unknown. We found that the lethality of aparathyroid Gcm2⁻/⁻ mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2⁻/⁻ mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant "thymic PTH."

EphB-ephrin-B2 interactions are required for thymus migration during organogenesis.

Thymus organogenesis requires coordinated interactions of multiple cell types, including neural crest (NC) cells, to orchestrate the formation, separation, and subsequent migration of the developing thymus from the third pharyngeal pouch to the thoracic cavity. The molecular mechanisms driving these processes are unclear; however, NC-derived mesenchyme has been shown to play an important role. Here, we show that, in the absence of ephrin-B2 expression on thymic NC-derived mesenchyme, the thymus remains in the cervical area instead of migrating into the thoracic cavity. Analysis of individual NC-derived thymic mesenchymal cells shows that, in the absence of ephrin-B2, their motility is impaired as a result of defective EphB receptor signaling. This implies a NC-derived cell-specific role of EphB-ephrin-B2 interactions in the collective migration of the thymic rudiment during organogenesis.

Thymic epithelial cell fate and potency in early organogenesis assessed by single cell transcriptional and functional analysis.

During development, cortical (c) and medullary (m) thymic epithelial cells (TEC) arise from the third pharyngeal pouch endoderm. Current models suggest that within the thymic primordium most TEC exist in a bipotent/common thymic epithelial progenitor cell (TEPC) state able to generate both cTEC and mTEC, at least until embryonic day 12.5 (E12.5) in the mouse. This view, however, is challenged by recent transcriptomics and genetic evidence. We therefore set out to investigate the fate and potency of TEC in the early thymus. Here using single cell (sc) RNAseq we identify a candidate mTEC progenitor population at E12.5, consistent with recent reports. Via lineage-tracing we demonstrate this population as mTEC fate-restricted, validating our bioinformatics prediction. Using potency analyses we also establish that most E11.5 and E12.5 progenitor TEC are cTEC-fated. Finally we show that overnight culture causes most if not all E12.5 cTEC-fated TEPC to acquire functional bipotency, and provide a likely molecular mechanism for this changed differentiation potential. Collectively, our data overturn the widely held view that a common TEPC predominates in the E12.5 thymus, showing instead that sublineage-primed progenitors are present from the earliest stages of thymus organogenesis but that these early fetal TEPC exhibit cell-fate plasticity in response to extrinsic factors. Our data provide a significant advance in the understanding of fetal thymic epithelial development and thus have implications for thymus-related clinical research, in particular research focussed on generating TEC from pluripotent stem cells.

Identification of Plet-1 as a specific marker of early thymic epithelial progenitor cells.

The thymus is essential for a functional immune system, because the thymic stroma uniquely supports T lymphocyte development. We have previously identified the epithelial progenitor population from which the thymus arises and demonstrated its ability to generate an organized functional thymus upon transplantation. These thymic epithelial progenitor cells (TEPC) are defined by surface determinants recognized by the mAbs MTS20 and MTS24, which were also recently shown to identify keratinocyte progenitor cells in the skin. However, the biochemical nature of the MTS20 and MTS24 determinants has remained unknown. Here we show, via expression profiling of fetal mouse TEPC and their differentiated progeny and subsequent analyses, that both MTS20 and MTS24 specifically bind an orphan protein of unknown function, Placenta-expressed transcript (Plet)-1. In the postgastrulation embryo, Plet-1 expression is highly restricted to the developing pharyngeal endoderm and mesonephros until day 11.5 of embryogenesis, consistent with the MTS20 and MTS24 staining pattern; both MTS20 and MTS24 specifically bind cell lines transfected with Plet-1; and antibodies to Plet-1 recapitulate MTS20/24 staining. In adult tissues, we demonstrate expression in a number of sites, including mammary and prostate epithelia and in the pancreas, where Plet-1 is specifically expressed by the major duct epithelium, providing a specific cell surface marker for this putative reservoir of pancreatic progenitor/stem cells. Plet-1 will thus provide an invaluable tool for genetic analysis of the lineage relationships and molecular mechanisms operating in the development, homeostasis, and injury in several organ/tissue systems.

A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations.

Cellular reaggregation methods have been used to generate in vitro organotypic cultures as a means to elucidate the cellular and molecular requirements of organogenesis. However, reproducibility from experiment to experiment has remained problematic and furthermore, current protocols do not support reaggregation of many important tissues. Here, using the thymus as a model organ, we present a novel reaggregation method termed "compaction reaggregation" that offers improved kinetics of reaggregation and greatly improved efficiency. Using compaction reaggregation we have been able to reaggregate the aorta-gonad- mesonephros region, a tissue that previously proved refractory to commonly used reaggregation methods, enabling the study of hematopoietic stem cell emergence and expansion. Additionally, compaction reaggregation permits the juxtaposition of different cell layers within the aggregated structure thus providing the means to study inductive interactions between different cell populations in vitro.

A developmental look at thymus organogenesis: where do the non-hematopoietic cells in the thymus come from?

The origins of the non-hematopoietic cell types that comprise the thymic stroma remain a topic of considerable controversy. Three recent studies, using lineage analysis and other methods to determine the developmental potential of specific cell types within the thymus, have provided strong evidence of a single endodermal origin for all thymic epithelial cells. Together with other investigations that merge immunological and developmental biology approaches, these studies have suggested a new model of thymus organogenesis, and have begun to uncover the molecular pathways that control this process.

Thymus organogenesis and development of the thymic stroma.

T-cell development occurs principally in the thymus. Here, immature progenitor cells are guided through the differentiation and selection steps required to generate a complex T-cell repertoire that is both self-tolerant and has propensity to bind self major histocompatibility complex. These processes depend on an array of functionally distinct epithelial cell types within the thymic stroma, which have a common developmental origin in the pharyngeal endoderm. Here, we describe the structural and phenotypic attributes of the thymic stroma, and review current cellular and molecular understanding of thymus organogenesis.

Bmp4 and Noggin expression during early thymus and parathyroid organogenesis.

The thymus and parathyroids originate from the third pharyngeal pouches, which form as endodermal outpocketings in the pharyngeal region beginning on embryonic day 9 (E9.0) of mouse development. Using organ-specific markers, we have previously shown that thymus and parathyroid-specific organ domains are established within the primordium prior to formation of the organs proper: Gcm2 expression defines the prospective parathyroid cells in the dorsal pouch from E9.5, while Foxn1 is expressed in the thymus domain from E11.25. Bmp (bone morphogenetic protein) signaling has been implicated in thymic epithelial cell differentiation and thymus organogenesis. In the present study, we report expression patterns of Bmp4 and Noggin, a Bmp4 antagonist, in the third pharyngeal pouch using two lacZ transgenic mouse strains. Results from this gene expression study revealed localization of Bmp4 expression to the ventral region of the third pharyngeal pouch endoderm at E10.5 and E11.5, in those cells that will express Foxn1 and form the thymus. Conversely, the expression of Noggin was confined to the dorsal region of the pouch and primordium at these stages, and thus appeared to be co-expressed with Gcm2 in the parathyroid domain. This represents the first detailed study of Bmp4 and Noggin expression during the early stages of thymus and parathyroid organogenesis.