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.

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.

Describing motivation for health and treatment decisions and communication choices of calf-care workers on western United States dairies.

On large dairy farms, animal health assessments and treatments are made by farm employees. Little is known about how employees make decisions about illness detection or treatment, information critical to improving antimicrobial stewardship. The objectives of this study were to describe calf-care employee motivations for decisions associated with preweaned calf health and treatments, describe on-farm worker communication networks, and determine information sources used by these employees to support their decisions. Personal interviews were conducted with 103 calf-care employees on 28 farms in the western United States. The interview consisted of 10 motivation source type (MST) questions and questions about training, communication and educational opportunities. A latent class analysis created a summary for MST and resulted in 4 classes. Forty-three percent of calf-care employees fell into a class where responses were a combination of internal and intrinsic (personal beliefs or values and task fulfillment, respectively) and 23% were a combination of internal and goal internal (aligned with organizational goals). This latter class aligned health decisions with internal motivation and treatment decisions with goal internal. A network analysis summarized dominant communication relationships and established that feeders and treaters perceived more communication with supervisors than was reciprocated by supervisors, and that there was less communication between workers and management for tasks relative to daily work. Employee training was primarily done by herdsman, calf manager, or coworkers, and information for skill improvement and problem solving was sought from these individuals. Although veterinarians were not often involved in employee training, when they were involved, employees were likely to use them as an information source for skill improvement and problem solving. Few participants had ever used social media, but almost all had a device that could access the internet; more than 60% indicated interest in a social media platform for work-related information. Work motivation for many calf caretakers appeared to be sourced from personal beliefs, values, and job fulfillment, particularly when deciding to treat a sick calf. Investigation and incorporation of beliefs and values in training programs could help with alignment of protocols with actual treatment and further efforts to implement judicious use of antimicrobials.

Paediatric Head Injury and Traumatic Brain Injury.

Aim To determine prevalence of head injury presenting to paediatric emergency departments (PEDs) and characterise by demographics, triage category, disposition neuroimaging or re-attendance. Methods Presentations in 2014 and 2015, with diagnoses of head injury, intracranial bleed, skull fracture including single or re-attendances within 28 days post head injury to all national PEDs, were analysed. Demographics, triage score, imaging rate, admission, mechanisms and representation rate were recorded. Results Head injury was diagnosed in 13,392 of 224,860 (5.9%), median (IQR) age 3.9 (1.4 - 8.3) years. Regionally 3% of children <5 years attend each year. The total admitted/transferred was 10.8% (n=1460). Neuroimaging rate was 4.3% (n= 570). Falls predominated. Sport accounted for 12.2%. Conclusion One in twenty children PED presentations are head injury, over half in preschool children. A sizeable number were symptomatic reflected by admission, transfer, imaging or re-attendance. Observational management was favoured over imaging reflected in the higher admission versus imaging rate.

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.

Atresia Hymenalis with Haematometrocolpos: A Benefit of Teaching a Case Series and Review of the Literature.

We present four cases of atresia hymenalis with resultant haematometrocolpos diagnosed in quick succession in the emergency department with a literature review.

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.

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.

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.

Identification of a Bipotent Epithelial Progenitor Population in the Adult Thymus.

Thymic epithelial cells (TECs) are critically required for T cell development, but the cellular mechanisms that maintain adult TECs are poorly understood. Here, we show that a previously unidentified subpopulation, EpCam(+)UEA1(-)Ly-51(+)PLET1(+)MHC class II(hi), which comprises <0.5% of adult TECs, contains bipotent TEC progenitors that can efficiently generate both cortical (c) TECs and medullary (m) TECs. No other adult TEC population tested in this study contains this activity. We demonstrate persistence of PLET1(+)Ly-51(+) TEC-derived cells for 9 months in vivo, suggesting the presence of thymic epithelial stem cells. Additionally, we identify cTEC-restricted short-term progenitor activity but fail to detect high efficiency mTEC-restricted progenitors in the adult thymus. Our data provide a phenotypically defined adult thymic epithelial progenitor/stem cell that is able to generate both cTECs and mTECs, opening avenues for improving thymus function in patients.

Foxn1 Is Dynamically Regulated in Thymic Epithelial Cells during Embryogenesis and at the Onset of Thymic Involution.

Thymus function requires extensive cross-talk between developing T-cells and the thymic epithelium, which consists of cortical and medullary TEC. The transcription factor FOXN1 is the master regulator of TEC differentiation and function, and declining Foxn1 expression with age results in stereotypical thymic involution. Understanding of the dynamics of Foxn1 expression is, however, limited by a lack of single cell resolution data. We have generated a novel reporter of Foxn1 expression, Foxn1G, to monitor changes in Foxn1 expression during embryogenesis and involution. Our data reveal that early differentiation and maturation of cortical and medullary TEC coincides with precise sub-lineage-specific regulation of Foxn1 expression levels. We further show that initiation of thymic involution is associated with reduced cTEC functionality, and proportional expansion of FOXN1-negative TEC in both cortical and medullary sub-lineages. Cortex-specific down-regulation of Foxn1 between 1 and 3 months of age may therefore be a key driver of the early stages of age-related thymic involution.

Long-term persistence of functional thymic epithelial progenitor cells in vivo under conditions of low FOXN1 expression.

Normal thymus function reflects interactions between developing T-cells and several thymic stroma cell types. Within the stroma, key functions reside in the distinct cortical and medullary thymic epithelial cell (TEC) types. It has been demonstrated that, during organogenesis, all TECs can be derived from a common thymic epithelial progenitor cell (TEPC). The properties of this common progenitor are thus of interest. Differentiation of both cTEC and mTEC depends on the epithelial-specific transcription factor FOXN1, although formation of the common TEPC from which the TEC lineage originates does not require FOXN1. Here, we have used a revertible severely hypomorphic allele of Foxn1, Foxn1R, to test the stability of the common TEPC in vivo. By reactivating Foxn1 expression postnatally in Foxn1R/- mice we demonstrate that functional TEPCs can persist in the thymic rudiment until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression, suggesting that manipulation of FOXN1 activity may prove a valuable method for long term maintenance of TEPC in vitro.

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.

Generation of tissue organoids by compaction reaggregation.

Cellular reaggregation methods are commonly used to generate tissue organoids for use in biological studies. Using a modified method termed "compaction reaggregation," it is possible to establish reaggregates of reproducible size from defined input cell numbers with ease and without specialist equipment. Importantly, this method is suitable for the study of tissues that have proved refractory to reaggregation by other methods. With the option of juxtaposing cell populations, this method is useful for studies of tissue organization and structure.

Serum-free culture of mid-gestation mouse embryos: a tool for the study of endoderm-derived organs.

The experimental manipulation of mid-gestation mouse embryos is an important tool for the study of developmental biology. However, such techniques can be challenging due to difficulties accessing the embryos in utero, and therefore the ability to maintain mid-gestation mouse embryos in vitro has proved invaluable. Described here is an example of a whole embryo culture system, where a serum-free medium is used to support the development of mouse embryos in vitro from embryonic day 10.5 (E10.5) to E11.5. During this time the embryos increase in size and undergo developmental progression, as determined by morphological and molecular criteria. This makes it an ideal environment in which to support and maintain mid-gestation mouse embryos following experimental manipulations. Two applications of this whole embryo culture system are described here. In the first, protein-soaked beads are carefully positioned in the pharyngeal region of an E10.5 embryo, allowing the concentration of specific proteins to be altered within the tissue. In the second technique, morpholino oligonucleotides are electroporated into the pharyngeal region of the embryo at E10.5, creating an efficient system for the knockdown of gene function in the target cells. These techniques demonstrate the use of in vitro techniques to study organogenesis within the pharyngeal region of the mouse embryo, but with some modification they could be adapted to target any region of the endodermal gut tube.

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.