Final Report Summary - THYMISTEM (Development of Stem Cell Based Therapy for Thymic Regeneration)
Executive Summary:
ThymiStem was the European Consortium for Development of Stem Cell-Based Therapy for Thymic Regeneration. Throughout the project, our research teams worked together to lay the foundations required for improving immune reconstitution or boosting immune system function in patients, by replacing or regenerating the epithelial component of the human thymus using stem cell-based approaches. ThymiStem’s work therefore addressed the need to control differentiation and proliferation of human thymic epithelial stem cells, in order to achieve their generation or long-term expansion in culture or to stimulate their proliferation and differentiation in vivo to cause endogenous thymus regeneration. Our research aimed to develop robust protocols for the long-term in vitro culture of human thymic epithelial stem cells, including standardized quality controls, and for generating thymic epithelial stem cells from human pluripotent stem cells in vitro.
ThymiStem’s work plan was organised into 5 scientific work packages, plus two work packages dedicated specifically to training and public outreach, and project management. Our main achievements and outputs have been presented in 36 Deliverables, and have led to 17 publications in international peer-reviewed journals and many presentations at international conferences and meetings, with further peer-reviewed publications under review and in preparation. Overall, ThymiStem made substantial progress in all project areas, that has collectively advanced the field towards our specific and overarching project goals. Highlights published to date include:
• Demonstration that thymic epithelial cells can be created in culture, by molecular reprogramming of unrelated cell-types using a single transcription factor (Bredenkamp et al Nature Cell Biology 2014).
• Identification of a multipotent thymic epithelial progenitor/stem cell population in the adult thymus (Ulyanchenko et al., Cell Reports 2016).
• Elucidation of key mechanisms controlling proliferation and differentiation of thymic epithelial progenitor/stem cells during thymus development and thymus regeneration in vivo, based on modelling from bioinformatics data and genetic analyses (Verlardi et al, Blood 2014; O’Neill et al PLoS One 2016; Martin-Gayo et al, J. Exp. Med 2017; Wertheimer et al, Science Immunology 2018).
• Increased insight into mechanisms promoting and limiting thymus-dependent immune reconstitution in patients (Tuckett et al Blood 2014; Velardi et al Nature Medicine 2017; Wertheimer et al, Science Immunology 2018).
The ThymiStem Partners were: Partner 1, The University of Edinburgh (Coordinator and Principal Investigator, Professor Clare Blackburn, Principal Investigators Dr Simon Tomlinson, Dr Andrew JH Smith); Partner 2, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Spain (Principal Investigator Professor Maria Toribio); Partner 3, Rudjer Boskovic Institute, Croatia (Principal Investigator Professor Mariastefania Antica); Partner 4, Open International University of Human Development “Ukraine”, Kyiv, Ukraine (Principal Investigator Professor Valentin Shichkin); Partner 6, Nanopharma, a.s. Czech Republic (Principal Investigator Dr Katerina Vodseďálková); Partner 7, the Memorial Sloan Kettering Cancer Center (MSKCC), New York, USA (Principal Investigator, Professor Marcel van den Brink).
Project Context and Objectives:
Project Context
The European Consortium for Development of Stem Cell-Based Therapy for Thymic Regeneration, ThymiStem, was established in 2013 and brought together 8 academic research teams and 1 SME from 7 countries: UK, Spain, Croatia, Czech Republic, Turkey, Ukraine, and the USA. Collectively, the project comprised scientists and clinicians at the forefront of thymus biology, stem cell biology, and immune reconstitution, plus leading experts in bioinformatics, genetic and tissue engineering, and cell banking, working together in a single team. Our common goal was to lay the foundations for development of an improved therapy for reconstituting or boosting immune system function in patients, by replacing or regenerating the epithelial component of the human thymus using stem cell-based approaches.
Our Objectives
Responding to call HEALTH.2013.1.4-1 Controlling differentiation and proliferation in human stem cells intended for therapeutic use, ThymiStem’s research focus was on the human thymus. Specifically, our work addressed the need to control differentiation and proliferation of human thymic epithelial stem cells, in order to achieve their generation or long-term expansion in culture or to stimulate their proliferation and differentiation in vivo to cause endogenous thymus regeneration. We further aimed to use in vitro human thymic epithelial stem cells to generate thymic organoids that contain all of the differentiated epithelial cell types required for thymus function, as a step towards use of such cells for thymus transplantation.
Our research therefore aimed to develop robust protocols for the long-term in vitro culture of functional human thymic epithelial stem cells, including standardized quality controls, and for generating thymic epithelial stem cells from human pluripotent stem cells in vitro. In addition, we aimed to establish an optimized means of delivering these cells to immunocompromised recipients such that thymus function was fully restored, and to assess risks associated with this protocol. Finally, we aimed to develop optimized procedures for cryopreservation of human thymic epithelial cells.
Beyond our scientific research, ThymiStem aimed to contribute to development of the stem cell sector in Europe and to public engagement across Europe with stem cell research and regenerative medicine. For this, we aimed to provide a programme of training and exchange opportunities for researchers within the consortium, and to engage with other EU-funded research and communications projects – including by supporting the European Summer School in Stem Cells and Regenerative Medicine and the multi-lingual website www.eurostemcell.org.
Our specific measurable goals, over the four years of the project, were to develop:
• A model of the key mechanisms controlling proliferation and differentiation of thymic epithelial
progenitor/stem cells during development and homeostasis in vivo.
• An optimized method for establishment of long-term thymic epithelial stem cell cultures from ex
vivo human thymic epithelial cells and/or human pluripotent cells.
• An optimized transplantation strategy for in vitro thymic epithelial stem cell-based thymic organoids, and
assessment of the impact of the transplanted thymi on immune system function.
• Artificial matrices suitable for generating human thymus organoids in vitro, and for transplantation.
• Optimized and validated SOPs for cryopreservation and thawing of thymic stromal/epithelial cells.
• An integrated network of European human stem cell researchers with training and experience in advanced technologies and high-impact public engagement.
Project Results:
ThymiStem was structured into 5 scientific work packages, plus a work package dedicated to outreach and public engagement. Each of these work packages addressed one of the specific objectives set out above. The final work package was dedicated to project management. The main outcomes of the work performed in each of these work packages over the course of the project are summarised below.
Work Package 1 - Identity and regulation of human thymic epithelial stem cells in vivo.
ThymiStem’s overarching aim in Work Package (WP) 1 was to generate a model of the central mechanisms controlling proliferation and differentiation of thymic epithelial progenitor/stem cells (TEP/SC) during development and homeostasis in vivo, that was at least partially validated experimentally. WP1 thus had two objectives: to identify phenotypically and functionally defined epithelial stem cells within the human thymus, and to establish the molecular pathways through which human TEP/SC are maintained and expanded in vivo. The work of WP1 progressed well throughout the project, with all of the WP1 deliverable reports having been submitted.
The work related to WP1 Objective 1 was undertaken principally by Partners 1 and 2. We initially focussed on characterisation of the human fetal thymus, in order to identify human thymic epithelial cell (TEC) subpopulations equivalent to the mouse thymic epithelial progenitor/stem cells (TEP/SC) that we had previously identified. This work also entailed further characterisation of a bi-potent TEP/SC population we identified in the adult mouse thymus, that can generate both cortical and medullary TEC (the two main types of TEC in the adult thymus), in order that human cell populations taken forward for functional testing should as closely as possible resemble the most enriched mouse TEP/SC population available. We found that 2nd trimester human fetal thymi closely resembled 1 month old mouse thymi in terms of developmental stage and architecture, and were able to identify a candidate TEP/SC in the human fetal thymus at this stage that shared many similarities with the bipotent adult mouse TEP/SC we had identified. Finally, we focussed on development of an assay suitable for functional testing of candidate human TEP/SC populations. The work describing our identification of a bi-potent TEP/SC population in the adult mouse thymus has been published (Ulyanchenko et al., 2016). Deliverable 1.1 (Report on identification and functional characterisation of phenotypically defined human thymic epithelial stem/progenitor cells), which described our work on identification and characterisation of human and mouse TEP/SC, was submitted on time. Our subsequent work in WP1 Objective 1 focussed mainly on two issues: first, we further characterised the human TEC population that appeared equivalent to the bipotent mouse TEP/SC described above, showing that it had equivalent intrathymic localisation. Functional testing of this population was attempted, but so far has been hampered by the low numbers of cells from this human TEC population that can be purified for further testing. This work is on-going. Second, we performed an investigation into potential TEP/SC niche components, in order to gain insight into how human TEP/SC may be regulated. This work was reported in Deliverable 1.2 (Report on characterisation of the in vivo human TEP/SC niche.) which was also submitted on time.
The work related to WP1 Objective 2 was again performed predominantly by Partners 1 and 2, and aimed to establish the molecular pathways through which human TEP/SC are maintained and expanded in vivo. Therefore, we initially focused on collecting global transcription data, which captured the entire gene expression profile of a precisely defined TEPC population at several different stages of development. We collected three replicate datasets at each of three timepoints, and augmented these datasets with equivalent data from a series of mouse mutants that have perturbed thymus development. We also collected datasets detailing the chromatin landscape (using the approach of global chromatin immunoprecipitation and sequencing (ChIPseq) with antibodies that bind three different chromatin modifications) in cells at two of the three timepoints. Deliverable 1.3 (Report on collection of transcriptome datasets), which described this work, was delivered on time. We then performed a preliminary analysis of these ‘RNAseq’ and ‘ChIPseq’ datasets, and related to this, also performed a series of validation (or quality control) experiments which indicated the high quality of the RNAseq and ChIPseq data obtained from fetal thymus, whilst revealing problems with the RNAseq datasets obtained from adult thymus. Therefore, we principally used our fetal data, together with adult thymic epithelial cell (TEC) datasets available in the public domain, for further analyses.
In brief, we collected together into one database, called “ThymiBase” a set of TEC-derived RNAseq and ChIPseq data from public repositories as well as the above data and additional data from Thymistem groups and collaborators. We assessed and reanalyzed each of these datasets in order to provide a data resource for discovery of signalling mechanisms controlling TEC biology. This database has been continuously updated with proprietary and public domain RNAseq data, and will be made publically available once published. Partner 1 then analysed these data to identify signalling pathways implicated in maintaining mouse and human TEP/SC. These analyses were reported in Deliverable 1.4 which were delivered on time, and in later milestone reports. Subsequently, Partner 1 extended this work to form a model of signalling regulation of undifferentiated and differentiating TEPC. Overall, this analysis identified six signalling pathways as active in undifferentiated fetal TEPC. It also generated a model of TEPC differentiation, in which direct and indirect target genes regulated by the major transcription factor responsible for thymic epithelial cell differentiation were also identified, and which incorporated genetic data, obtained in WP1 by Partners 1 and 2 via extensive in vitro and in vivo analysis, regarding the cellular mechanism that may operate to control differentiation of TEP/SC and allow generation of medullary TEC during the earliest stages of thymus organogenesis. These findings were reported in Deliverables 1.5 and D1.6 which were submitted on time. In additional work in WP1, Partner 7 also tested the role of the BMP pathway in regulating TEP/SC via an extensive functional analysis. This revealed a crucial role for BMP4 during thymic regeneration after injury caused by irradiation and suggested that BMP4 potentially regulates this thymic regeneration by stimulating TEPCs.
Overall, the work of WP1 progressed well throughout the project and has provided considerable insight into fetal and adult TEP/SC regulation, meeting most if not all of the outcomes anticipated at the start of the project. The work performed in WP1 resulted in a number of peer-reviewed publications, with the major outputs and findings being: Identification of a multipotent TEP/SC in the adult mouse thymus (Ulyanchenko et al., Cell Reports 2016) and of a candidate equivalent cell population in human fetal and paediatric thymi (Blackburn et al unpublished); Identification of the Notch pathway as a key regulator of TEP/SC self-renewal and differentiation (Martin-Gayo et al, J. Exp. Med 2017; Liu...Blackburn in revision; García-León et al in preparation); Identification of the BMP pathway as a regulator of endogenous thymus regeneration (Wertheimer et al, Science Immunology 2018); Development of an ‘omics database of thymic epithelial cell datasets, with analytical capacity (ThymiBase, Kousa, Tomlinson, in preparation); and generation, via bioinformatics analysis, of a model predicting the key mechanisms controlling proliferation and differentiation of thymic epithelial progenitor/stem cells (Kousa et al, in preparation). Thus, collectively this work has provided considerable new insight into fetal and adult TEP/SC regulation, meeting most if not all the anticipated outcomes.
Work Package 2 - Generation and propagation of human thymic epithelial stem cell lines.
Our goal in WP2 was to develop an optimized and scalable method for establishment of long-term thymic epithelial stem cell (TESC) cultures from ex vivo human TEC and/or human induced pluripotent stem (iPS) cells. To achieve this, we set out to determine in vitro growth conditions that support proliferation of functionally validated human TESC lines and to generate TESC from human iPS cells, or by direct reprogramming. In the original ThymiStem proposal, we proposed to take as our starting point for culturing human TESC the method described by Bonfanti and colleagues (Nature 2010; on which Partner 1 was an author). However, Partner 1 then developed a novel method for generating TEC in the laboratory by converting (or ‘reprogramming’) into TEC an unrelated cell-type called primary embryonic fibroblasts. This work was partly supported by ThymiStem. This appeared to be a better method for producing large quantities of TEC in vitro, since in contrast to the cells cultured in the previous paper, the TEC generated by reprogramming (termed induced TEC, or iTEC), could generate a fully organized, functional thymus when transplanted (Bredenkamp et al., 2014). In addition to this advance, a subsequent report demonstrated that ex vivo mouse TEC could be propagated in vitro as spheroid cultures, termed thymospheres, and that these spheroids appear to be initiated by, and to maintain TESC (Ucar et al., 2014). Therefore, we focused the efforts of WP2 Objective 1 on development and evaluation of human iTEC and on development of human thymospheres, rather than the approach originally proposed. The work conducted progressed well in both areas.
In brief, after extensive screening of different growth factors and attachment matrices for ability to support TEPC growth in vitro (Deliverables 2.1-2.3) Partner 1 established conditions permissive for short to medium term, but not indefinite, growth of TEPC in monolayer cultures, with some cultured cells retaining capacity to contribute to thymic epithelial cell (TEC) networks for at least 3 weeks in culture. Additionally, we identified further modifications that should promote expansion of undifferentiated TEPC - and thus of an in vitro induced TESC cell type, and these remain to be tested. In parallel, Partner 3 showed that human thymic sphere cultures, that appear to be TEPC-derived, could readily be generated and propagated in vitro under conditions modified from the method described by Ucar and colleagues for mouse cells. The human thymic spheres developed by Partner 3 expressed markers of functional TEC, and at least some could support human T cell development in vitro. The above findings were reported in Deliverables 2.1 (Report on expression and functional analysis of TESC cultured on 3T3/J feeder cells), D2.2 (Report on optimization of attachment factors for maintenance of proliferating TESC in vitro), D2.3 (Report on optimization of growth factors and inhibitors for maintenance of proliferating TESC in vitro) and D2.6 (Report on generation and propagation of high efficiency human thymic epithelial stem cell lines) which were all submitted. Collectively, these data represent solid progress towards the goal of maintaining proliferating TESC in vitro. Our currently optimized conditions for the in vitro growth of TEP/SC as monolayers have been tested and validated for short term culture of mouse TEPC and, based on our evaluation of a highly similar medium (as part of WP2), are expected to apply to human. Our recent bioinformatics and functional data suggest modifications to these conditions that should specifically promote expansion of undifferentiated TEPC - and thus of an in vitro induced TESC cell type - and these conditions will be tested in the near future. Additionally, our work on human thymic spheroids indicates that the thymic spheres can readily be generated in vitro and can be propagated through at least six passages, suggesting they are based on TEPC. Importantly, the thymic spheres retain expression of functional TEC markers, and at least some can support human T cell development in vitro, indicating their promise for further development. Protection of intellectual property around this methodology is currently under discussion.
The work related to WP2 Objective 2, on generation of TEC in vitro from other cell types, was more closely related to that in Objective 1 than originally envisaged. We (Partner 1) extended our preliminary findings to show that a cell type called primary mouse embryonic fibroblasts (MEFs) can be converted to TEC by enforced expression of a single transcription factor, FOXN1 (Bredenkamp et al Nature Cell Biology 2014), and subsequently extended this finding to demonstrate that such ‘induced TEC’ could also be generated from mouse pluripotent stem cells (PS) cells. Subsequently, we focused on determining whether human PS cells could be reprogrammed into TEC using a similar strategy. For this purpose, we developed a reporter transgene suitable for introduction into human iPS cell lines, and on development of genetic constructs (transgene vectors) predicted to enable reprogramming of human iPS cells to iTEC. To this end, Partner 1 generated a bacterial artificial chromosome (BAC) carrying a human FOXN1-Venus reporter transgene suitable for introduction into human iPS cell lines, as detailed in Deliverable 2.5. However, this avenue was not pursued since, unexpectedly, we were unable to convincingly detect expression of the Venus reporter protein. In addition, Partner 1 focused on construction of vectors for promoting conversion of human iPS cell lines into iTEC, by human FOXN1 overexpression. This was reported in Deliverable 2.5. Preliminary studies indicated some difficulties with this approach. Therefore, we are now implementing a strategy to introduce the transgenes into a ‘safe harbour’ locus. The expectation is that this will enable robust transgene expression.
Overall, therefore, the work performed generated solid progress towards the two WP2 goals, and all of the WP2 deliverables were submitted. To date WP2 has resulted in one major publication (Bredenkamp et al Nature Cell Biology 2014), with additional several arising publications currently in preparation and discussion on-going around protection of the potential intellectual property arising.
Work Package 3 - Preclinical analysis of human thymic epithelial stem cell transplantation in humanized mice, and comparison to endogenous stimulation in clinical trials.
The work in WP3 aimed to assess the functional outcome of transplanting human TEP/SC-based organoids, using a preclinical humanized murine model, and also to analyse thymus data from a phase I clinical trial evaluating the effect of activating endogenous TESC. To achieve this, we addressed four specific goals. Goals 1-3 evaluated: the outcome of transplanting primary human TEP/SC in a humanized mouse model; the functional outcome of transplanting human TEP/SC-based organoids, including the longevity of the functioning grafts and the tumorigenic potential of the grafted material; and the thymus architecture and distribution of intrathymic cell types in transplanted mice; while goal 4 aimed to assess the functional outcome of activating endogenous TESC in patients.
The work performed on Aims 1-3 was performed principally by Partners 2, 3, and 6, and evaluated two alternate humanized mouse models for in vivo human T-cell reconstitution from human cord blood cells and human early thymocyte progenitors (García-Peydró et al, J Clin Inv final revision); established reproducible methods for isolation of human TEC and for generation from these cells and from human thymic sphere cultures (WP2) of reaggregate thymic organ cultures (RTOCs); and investigated the potential use of human thymic spheres for generating transplantable bioengineered thymus organoids (WP4). The results obtained indicate that implanted human thymic sphere-based RTOCs provide the appropriate thymus microenvironment for human HSPC expansion and early T-cell development, and lack tumorigenic potential. However, under current conditions, full T-cell development is precluded due to loss of architecture and collapse of the grafted thymic sphere-based RTOCs at 2-3 weeks post-implantation. These data emphasised the importance of the thymus architecture of the transplanted organoids for supporting full T-cell development, and in this regard, we have identified specific functional microenvironments for T and non-T cell development in the human thymus (Martín-Gayo et al., J Exp. Med 2017; García-León et al, in preparation). Additionally, work integrating the first-generation scaffolds produced in WP4 with human thymic-sphere-derived cells showed that the scaffolds provided an appropriate 3D structure for TEC infiltration and growth, but did not support full T cell development in vitro, suggesting that further scaffold development was required; such development was therefore undertaken in WP4. The above work was reported in Deliverables 3.1 (Report on evaluation of human TESC transplantation in humanised mice), 3.2 (Report on evaluation of thymus architecture and distribution of intrathymic cell types in the TESC-based transplanted organoids) and 3.5 (Report on the functional outcome of transplanting in vitro-expanded human TEP/SC and TESC-based organoids in humanized mice, as proof-of-principle for development of thymic regenerative immunorehabilitation therapy), all of which were submitted on time.
With respect to the fourth aim of this WP, Partner 7 set out to evaluate a new non-invasive protocol for assessing changes in thymus size and functional volume in patients after total body irradiation (TBI-based) CD34-selected allogeneic hematopoietic stem cell transplantation (allo-HSCT). This protocol, if proven useful, will be invaluable for measuring thymus regeneration in patients after activation of endogenous TESC. The first patients have been recruited to this study, which is on-going. Additionally, we have characterized the pattern of immune reconstitution in the first year after transplant, and its effects on survival and relapse, for a large retrospective study of patients who received myeloablative T cell depleted (TCD) allo-HSCT for hematologic malignancies. The data have shown that T cell recovery, which is an indirect measure of thymus regeneration and therefore most probably activation of endogenous TESC, is a predictor of outcome after TCD allo-HSCT. This study will serve as a baseline to assess effects of interventions such as KGF, Lupron and other combinations as therapeutic approaches to enhance immune recovery of patients receiving allogeneic HSCT. In this regard, we have also investigated how inhibition of sex-steroid signaling affects the outcome of HSCT after lethal TBI in mice; unexpectedly, this work uncovered a direct, sex-steroid- and thymus-independent, regulatory role for lutenizing hormone on haematopoietic stem cells (Velardi et al Nature Medicine 2018).
The work performed in WP3 has resulted in two peer-reviewed publications, with the major outputs being: Martín-Gayo et al., J Exp. Med 2017 and Velardi et al Nature Medicine 2018. A further manuscript is currently under revision (García-Peydró et al, J Clin Inv final revision) and several additional manuscripts are in preparation. All of the WP3 deliverables have been submitted.
Work Package 4 - Development of artificial matrices for generating human thymus organoids in vitro and for transplantation.
Our focus in WP4 was on the integration of tissue engineering with thymus biology, to facilitate the production of thymic organoids at the scale required for transplantation into human patients. Specifically, WP4 aimed to develop scaffold materials suitable for growth of human thymus organoids in vitro, and for transplantation. The work performed required extensive selection, fabrication and functional testing of scaffold materials and types, which was performed by Partner 6 working in conjunction with Partner 7. Overall, the focus was on development of electrospun materials. In brief, Partner 6 initially identified biocompatible polymer materials able to enhance cell attachment and proliferation, based on library searching, and then used these selected polymer materials for production of 2D scaffolds using electrospinning methods including classic, coaxial, and force spinning. Different concentrations of polymer solutions, different solvent systems, and different electrospinning conditions were tested. More than 150 electrospinning experiments were conducted to produce a range of 2D structures. Based on the electrospinning process and the quality of produced nanofibrous layers 15 samples were selected as the most promising scaffold prototypes. These were subjected to biological testing by Partner 7, were to optimization and reproducible production by Partner 6, which identified two scaffold materials as suitable for further development, as reported in Deliverable 4.1 (Report on the feasibility of enhancing TESC growth in vitro using biopolymer-based attachment matrices).
To prepare 3D tissue engineering scaffolds for testing for capacity to support TESC attachment, survival and differentiation, Partner 6 then used three different electrospinning techniques. Firstly, the technique of AC spinning was used for preparation of 3D scaffolds based on the combination of two materials. Secondly, a combination of electrospinning and linearized air flow was employed to obtain voluminous structure with the required pore diameter. Thirdly, a combination of electrospinning and melt blown technologies was used, enabling the formation of a layer containing both microfibers and nanofibers while guaranteeing good mechanical properties of the 3D scaffolds. Partner 7 assessed the adhesive properties of these biomaterials as well as cell morphology and growth of stromal cells, which Partner 6 further analysed the scaffold properties. The results of these structural studies showed that the produced tissue 3D scaffolds imitated the structure of the extracellular matrix through fibre diameters in the nanometre and micrometre dimensions and sizes of continuous pores in tens of micrometres. Thus, this structure may help recapitulate the structures formed by TEC during normal development and homeostasis in vivo. Biological tests carried out by Partner 7, demonstrated the biocompatibility of these scaffolds in vitro and in vivo and also demonstrated that the test cells (TEC) penetrated into the scaffolds and also proliferated. Partner 7 found that the resulting thymic organoids supported growth of stromal cells, with preliminary data suggesting that subcutaneous implantation of fetal thymus-derived thymic organoids resulted in generation of CD8+ and CD4+ T cells in nude mice. This work was reported in Deliverables 4.2 (Report on the use of biopolymer-based scaffolds to support generation of thymic organoids in vitro) and 4.3 (Report on the development of optimised biopolymer-based scaffolds to support generation of human thymic organoids in vitro).
Based on these results, one microfiber/nanofiber composite was identified as suitable for tissue engineering. This underwent functional testing in several partner labs, and based on this further functionalization was attempted. Technological complications were encountered with some approaches. However, promising results were obtained with two approaches: the combination of meltblown electrospun scaffolds with hydrogels, such that the hydrogels provided a superabsorbent reservoir of bioactive factors; and modification of the scaffold with polyfunctional coatings, deposited on the surface scaffold fibres, that could act as carriers of bioactive molecules. Both approaches have yielded encouraging data, with the polyfunctional coating approach appearing to provide a universal, simple and efficient method of scaffold functionalization. Of note is that meltblown technology allows for highly productive manufacturing of the scaffold, and when suitable equipment is used, also its almost unlimited size in all three axes, making this approach highly appropriate for scaling of scaffold production to the size required for clinical use. These results were reported in Deliverables 4.3 (Report on the development of optimised biopolymer-based scaffolds to support generation of human thymic organoids in vitro), 4.4 (Report on scaling of the optimized thymus organoids to a size compatible with clinical use) and 4.5 (Report on development of artificial matrices for generating human thymus organoids in vitro, and for transplantation), which have all been submitted.
Overall, the work of WP4 has provided insight into the use of tissue engineering approaches for thymic organoid production including upscaling to a size compatible with clinical use. However, some of the approaches initially identified as promising were shown through further testing to be unsuitable for production of human thymic organoids in vitro. In particular, although some evidence suggested that the MF/NF scaffolds might be useful with further optimization, we concluded that they did not represent the best option for generating human thymus organoids and that alternative bioengineered materials should be analyzed for their potential to support long-term human T-cell development, based on the outcome of suitable functional assays established, validated and then used in WP3. Both the functionalised hydrogel modified scaffolds and the coated scaffolds appear promising for this purpose. Additionally, the use of meltblown electrospinning appears suitable for generating scaffold materials at the scale required for development of transplantable human thymic organoids. Overall, substantial work was undertaken and delivered related to each of the objectives of this workpackage and, taken together, our systematic testing of molecularly tailored biomaterials and innovative scaffold designs resulted in the identification of a promising implantable platform for thymic organoid engineering. However, further development work is required to develop matrices, including those identified herein, such that they support development of a functional human thymus organoid in vitro or for transplantation.
All of the deliverables for this WP were submitted, the work has been presented at several international scientific meetings, and we anticipate that manuscripts describing the main findings will be submitted for publication in the near future.
Work Package 5 - Development of clinical grade protocols for cryopreservation and quality control of primary thymic stromal cells and cultured thymic epithelial stem cell lines.
The goal of WP5 was to enable use of TEC/TESC for research and clinical purposes, by developing standard operating protocols (SOPs) for cryopreservation and quality control of primary thymic stromal cells and cultured TESC lines. The work in WP5 was predominantly conducted by Partner 4, who performed extensive comparative testing of different conditions for tissue dissociation, freezing and thawing of human paediatric thymus tissue, and the subsequent development from these of SOPs for: thymic tissue enzymatic digestion; in vitro culture of human thymic cells; cryopreservation of human thymic cells in liquid nitrogen; thawing of human thymic cells after storage in liquid nitrogen; evaluation of human thymic cell viability by trypan blue staining; evaluation of human thymic cell viability by 7-AAD staining; evaluation of human thymic cell phenotype by flow cytometry; evaluation of the functionality of human frozen and thawed TEC by co-culturing with authologous thymocytes; and evaluation of mRNA expression in human thymic cell subpopulations. Over the course of the project, 93 paediatric thymi were collected and stored in liquid nitrogen, and used for these experiments. All of the deliverables for this WP were submitted, some has of the outcomes have been published in a peer-reviewed journal (Shichkin et al., Cryobiology 2017; Shichkin et al Trends in Transplantation 2018), with four further manuscripts currently in preparation.
Work Package 6 - Training and Dissemination.
In addition to its research programme, ThymiStem also aimed to provide training to project participants at all career stages; and to stimulate public engagement with the project aims and the wider stem cell field, since contribution in these areas is required to develop the field of human stem cell research, and also to fulfil our responsibility to European publics to enable and participate in discussion and wider consideration of our research field and related ethical and societal issues. Extensive work was therefore performed related to each of these objectives, and all of the deliverable reports from this WP were submitted. In particular, ThymiStem facilitated 9 exchange visits in which early career stage researchers visited the labs of other partners, or other internationally respected scientists in the thymus field, in order to learn new techniques and exchange knowledge. We also held two training workshops for consortium members. We also contributed to the 2014, 2015, 2016 and 2017 European Summer Schools on Stem Cells and Regenerative Medicine with a total of 19 ThymiStem researchers having attended this School, and to organising “Advances in Stem Cell Research and Regenerative Medicine” a major European conference in the field, in 2017. Additionally, 6 ThymiStem members attended 2 workshops run by the FP7 consortium PluriMes.
To ensure wide-reaching dissemination of the project outcomes, and continued public engagement with up-to-date developments in human stem cell research, ThymiStem also developed an ambitious programme of public engagement, that has made a significant contribution to the communications and engagement in the stem cell and regenerative medicine field. The ThymiStem project website has continued to provide dynamic information about the project for peer and public view. This site is actively linked with the leading stem cell and regenerative medicine website eurostemcell.org extending the reach of project news to a wider audience and avoiding duplication of effort. ThymiStem has also collaborated with the FP7/H2020 Coordinating Action EuroStemCell on the re-design of the eurostemcell.org website, to ensure that ThymiStem created material can continue to be discovered, accessed and viewed responsively across digital platforms and devices, thereby creating a public-facing legacy for the project. Based on evaluation evidence the new site, launched in mid-December 2016, will greatly increase access to the high-quality public facing stem cell biology information and resources. We have also collaborated in the translation of the website content into Czech, one of our partner languages. ThymiStem has also developed a short animation to explain thymus biology to a lay audience, as well as a fact sheet “Regenerating the Thymus”, and has translated the book “Your amazing immune system” into Croatian, with launch and release of these resources on the Day of Immunology in 2016 and 2017.
The animation shows how T cells go through a complex journey in the thymus to become mature immune cells ready to fight infection. It was disseminated to all ThymiStem scientists for use in outreach, teaching and websites. Since its launch in April 2016 the animation has been viewed fully 2062 times on the Eurostemcell YouTube channel (see https://youtu.be/FW2Jat00lEs). The ‘Regenerating the Thymus’ factsheet provides an overview of the current scientific field of thymus regeneration as well as the applications of the science, describing the role of the thymus in the immune system and the research avenues currently being investigated to regenerate the organ (see http://www.eurostemcell.org/regenerating-thymus). It reached 689 people on Facebook and received 2453 impressions on Twitter on the Day of Immunology 2017, and has been translated into the six EuroStemCell languages: English, Spanish, German, French, Italian and Polish. Taken together the animation and factsheet represent valuable public engagement resources to explain the function of the thymus, an often poorly understood organ. Additionally, we developed an interactive science game, Thymaze, designed for a family audience, that introduces the player to the thymus and the different cells of the organ. Integral to the game are multiple science messages that can be understood at different levels of scientific literacy. This game is suitable for use at science festivals, open days and other public engagement events. It is accompanied by resources designed to support the scientists when using the game, including game play directions, scientific images and conversation topics for a range of audiences. The game has been distributed to ThymiStem partners for use in future public engagement activities. The highly popular and accessible ‘Your Amazing Immune System’, which we collaborated with the Federation of Immunological Societies to translate, describes the role of the thymus in the context of the immune system in a fun, friendly, illustrated book. With its curriculum relevance, translated copies of the book are being prepared for distribution to schools across Croatia, supported by ThymiStem and the Croatian Ministry for Education.
ThymiStem members have also been active in many international, national and local public engagement events and festivals, and have participated in ‘Wikithons’ to help improve the accuracy of relevant pages on Wikipedia. Finally, to contribute to capacity building in public engagement, we have held two workshops to provide training in public engagement for ThymiStem researchers, and also participated in the ‘Sharing best practice in public engagement’ workshop held by EuroStemCell in 2016. Through these activities, ThymiStem fully met the aims of WP6.
Potential Impact:
Impact on Science
The project has generated advanced knowledge related to controlling differentiation and proliferation of human thymic epithelial stem and progenitor cells and to generation and propagation of thymic epithelial cells in vitro, and hence, increased understanding of thymus biology (including endogenous thymus regeneration). Additionally, it has evaluated preclinical models for stem-cell based human thymus transplantation, and has advanced knowledge in the development of tissue engineering technologies suitable for development of human thymic organoids. The conclusions of this work have been reported in seventeen peer-reviewed publications to date including several in top line journals, with further manuscripts currently in preparation, and have been presented at numerous international conferences. They are therefore expected to impact the direct field of thymus biology beyond the lifetime of this project. Furthermore, since these data can be compared to those obtained in other, non-thymus tissues, they will also contribute to understanding of stem cell and regenerative biology in the broader context. The project additionally aimed to evaluate the effect of a novel treatment, currently in clinical trial, on thymus regeneration and adaptive immune system function in patients, and to evaluate a novel, non-invasive approach for measuring thymus size in patients. The outcome of this research, once known, is anticipated to significantly advance clinical research capacity in the field of thymus-based immune reconstitution. Further, our unexpected findings revealing a role of lutenizing hormone in regulating HSC function may offer a new therapeutic approach for hematopoietic regeneration after hematopoietic injury.
Economic and societal impacts
In the longer term, our research outputs are anticipated to contribute to development of new therapies that enhance immune system function, for instance via safe and effective thymus transplantation. If this goal can be reached, the impact on quality of life, and in some instances, life expectancy, will be substantial. The potential impact on healthy ageing is important in the context of the ageing European demographic, with the potential to contribute to reducing the economic burden related to frailty in old age. Additional beneficiaries would be: bone marrow transplant patients, patients with some classes of congenital primary immunodeficiencies (eg DiGeorge syndrome, Nezelof syndrome, Louis-Bar syndrome, Swiss syndrome, some human SCID patients), and potentially autoimmune, solid organ transplant and cancer patients, patients who have undergone surgical thymectomy, and the elderly population. The market for thymus transplantation approaches is extensive. There are 25,000 allo bone marrow transplant patients annually world-wide, who are potential candidates for thymus transplantation therapy, and this market is currently growing, as the use of allo-BMT is extended.
Beyond its research outputs, ThymiStem has provided specialist training to participating researchers at all career stages. This is expected to enhance their employability in a competitive jobs market, and also to contribute to raising the overall skill-base among European stem cell and regenerative medicine researchers. Additionally, we have linked with other consortia to maintain the highly-valued European Summer School on Stem Cells and Regenerative Medicine and the bi-annual Advances in Stem Cell Research conference. One of the impacts of the project will therefore its contribution to an enhanced European stem cell and regenerative medicine research sector in academia, industry and related fields. Finally, ThymiStem contributed to public engagement with stem cell research and regenerative medicine, including through collaboration with the high-impact H2020 coordinating action EuroStemCell (www.eurostemcell.org) with anticipated positive impacts on public awareness of European science and, importantly, on informed public engagement with this field at a time when new treatments are under active development.
List of Websites:
Website address: http://www.thymistem.org/
Contact details:
Prof Clare Blackburn, MRC Centre for Regenerative Medicine, University of Edinburgh, 5, Little France Drive, EH26 9NL. Email: c.blackburn@ed.ac.uk
ThymiStem was the European Consortium for Development of Stem Cell-Based Therapy for Thymic Regeneration. Throughout the project, our research teams worked together to lay the foundations required for improving immune reconstitution or boosting immune system function in patients, by replacing or regenerating the epithelial component of the human thymus using stem cell-based approaches. ThymiStem’s work therefore addressed the need to control differentiation and proliferation of human thymic epithelial stem cells, in order to achieve their generation or long-term expansion in culture or to stimulate their proliferation and differentiation in vivo to cause endogenous thymus regeneration. Our research aimed to develop robust protocols for the long-term in vitro culture of human thymic epithelial stem cells, including standardized quality controls, and for generating thymic epithelial stem cells from human pluripotent stem cells in vitro.
ThymiStem’s work plan was organised into 5 scientific work packages, plus two work packages dedicated specifically to training and public outreach, and project management. Our main achievements and outputs have been presented in 36 Deliverables, and have led to 17 publications in international peer-reviewed journals and many presentations at international conferences and meetings, with further peer-reviewed publications under review and in preparation. Overall, ThymiStem made substantial progress in all project areas, that has collectively advanced the field towards our specific and overarching project goals. Highlights published to date include:
• Demonstration that thymic epithelial cells can be created in culture, by molecular reprogramming of unrelated cell-types using a single transcription factor (Bredenkamp et al Nature Cell Biology 2014).
• Identification of a multipotent thymic epithelial progenitor/stem cell population in the adult thymus (Ulyanchenko et al., Cell Reports 2016).
• Elucidation of key mechanisms controlling proliferation and differentiation of thymic epithelial progenitor/stem cells during thymus development and thymus regeneration in vivo, based on modelling from bioinformatics data and genetic analyses (Verlardi et al, Blood 2014; O’Neill et al PLoS One 2016; Martin-Gayo et al, J. Exp. Med 2017; Wertheimer et al, Science Immunology 2018).
• Increased insight into mechanisms promoting and limiting thymus-dependent immune reconstitution in patients (Tuckett et al Blood 2014; Velardi et al Nature Medicine 2017; Wertheimer et al, Science Immunology 2018).
The ThymiStem Partners were: Partner 1, The University of Edinburgh (Coordinator and Principal Investigator, Professor Clare Blackburn, Principal Investigators Dr Simon Tomlinson, Dr Andrew JH Smith); Partner 2, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Spain (Principal Investigator Professor Maria Toribio); Partner 3, Rudjer Boskovic Institute, Croatia (Principal Investigator Professor Mariastefania Antica); Partner 4, Open International University of Human Development “Ukraine”, Kyiv, Ukraine (Principal Investigator Professor Valentin Shichkin); Partner 6, Nanopharma, a.s. Czech Republic (Principal Investigator Dr Katerina Vodseďálková); Partner 7, the Memorial Sloan Kettering Cancer Center (MSKCC), New York, USA (Principal Investigator, Professor Marcel van den Brink).
Project Context and Objectives:
Project Context
The European Consortium for Development of Stem Cell-Based Therapy for Thymic Regeneration, ThymiStem, was established in 2013 and brought together 8 academic research teams and 1 SME from 7 countries: UK, Spain, Croatia, Czech Republic, Turkey, Ukraine, and the USA. Collectively, the project comprised scientists and clinicians at the forefront of thymus biology, stem cell biology, and immune reconstitution, plus leading experts in bioinformatics, genetic and tissue engineering, and cell banking, working together in a single team. Our common goal was to lay the foundations for development of an improved therapy for reconstituting or boosting immune system function in patients, by replacing or regenerating the epithelial component of the human thymus using stem cell-based approaches.
Our Objectives
Responding to call HEALTH.2013.1.4-1 Controlling differentiation and proliferation in human stem cells intended for therapeutic use, ThymiStem’s research focus was on the human thymus. Specifically, our work addressed the need to control differentiation and proliferation of human thymic epithelial stem cells, in order to achieve their generation or long-term expansion in culture or to stimulate their proliferation and differentiation in vivo to cause endogenous thymus regeneration. We further aimed to use in vitro human thymic epithelial stem cells to generate thymic organoids that contain all of the differentiated epithelial cell types required for thymus function, as a step towards use of such cells for thymus transplantation.
Our research therefore aimed to develop robust protocols for the long-term in vitro culture of functional human thymic epithelial stem cells, including standardized quality controls, and for generating thymic epithelial stem cells from human pluripotent stem cells in vitro. In addition, we aimed to establish an optimized means of delivering these cells to immunocompromised recipients such that thymus function was fully restored, and to assess risks associated with this protocol. Finally, we aimed to develop optimized procedures for cryopreservation of human thymic epithelial cells.
Beyond our scientific research, ThymiStem aimed to contribute to development of the stem cell sector in Europe and to public engagement across Europe with stem cell research and regenerative medicine. For this, we aimed to provide a programme of training and exchange opportunities for researchers within the consortium, and to engage with other EU-funded research and communications projects – including by supporting the European Summer School in Stem Cells and Regenerative Medicine and the multi-lingual website www.eurostemcell.org.
Our specific measurable goals, over the four years of the project, were to develop:
• A model of the key mechanisms controlling proliferation and differentiation of thymic epithelial
progenitor/stem cells during development and homeostasis in vivo.
• An optimized method for establishment of long-term thymic epithelial stem cell cultures from ex
vivo human thymic epithelial cells and/or human pluripotent cells.
• An optimized transplantation strategy for in vitro thymic epithelial stem cell-based thymic organoids, and
assessment of the impact of the transplanted thymi on immune system function.
• Artificial matrices suitable for generating human thymus organoids in vitro, and for transplantation.
• Optimized and validated SOPs for cryopreservation and thawing of thymic stromal/epithelial cells.
• An integrated network of European human stem cell researchers with training and experience in advanced technologies and high-impact public engagement.
Project Results:
ThymiStem was structured into 5 scientific work packages, plus a work package dedicated to outreach and public engagement. Each of these work packages addressed one of the specific objectives set out above. The final work package was dedicated to project management. The main outcomes of the work performed in each of these work packages over the course of the project are summarised below.
Work Package 1 - Identity and regulation of human thymic epithelial stem cells in vivo.
ThymiStem’s overarching aim in Work Package (WP) 1 was to generate a model of the central mechanisms controlling proliferation and differentiation of thymic epithelial progenitor/stem cells (TEP/SC) during development and homeostasis in vivo, that was at least partially validated experimentally. WP1 thus had two objectives: to identify phenotypically and functionally defined epithelial stem cells within the human thymus, and to establish the molecular pathways through which human TEP/SC are maintained and expanded in vivo. The work of WP1 progressed well throughout the project, with all of the WP1 deliverable reports having been submitted.
The work related to WP1 Objective 1 was undertaken principally by Partners 1 and 2. We initially focussed on characterisation of the human fetal thymus, in order to identify human thymic epithelial cell (TEC) subpopulations equivalent to the mouse thymic epithelial progenitor/stem cells (TEP/SC) that we had previously identified. This work also entailed further characterisation of a bi-potent TEP/SC population we identified in the adult mouse thymus, that can generate both cortical and medullary TEC (the two main types of TEC in the adult thymus), in order that human cell populations taken forward for functional testing should as closely as possible resemble the most enriched mouse TEP/SC population available. We found that 2nd trimester human fetal thymi closely resembled 1 month old mouse thymi in terms of developmental stage and architecture, and were able to identify a candidate TEP/SC in the human fetal thymus at this stage that shared many similarities with the bipotent adult mouse TEP/SC we had identified. Finally, we focussed on development of an assay suitable for functional testing of candidate human TEP/SC populations. The work describing our identification of a bi-potent TEP/SC population in the adult mouse thymus has been published (Ulyanchenko et al., 2016). Deliverable 1.1 (Report on identification and functional characterisation of phenotypically defined human thymic epithelial stem/progenitor cells), which described our work on identification and characterisation of human and mouse TEP/SC, was submitted on time. Our subsequent work in WP1 Objective 1 focussed mainly on two issues: first, we further characterised the human TEC population that appeared equivalent to the bipotent mouse TEP/SC described above, showing that it had equivalent intrathymic localisation. Functional testing of this population was attempted, but so far has been hampered by the low numbers of cells from this human TEC population that can be purified for further testing. This work is on-going. Second, we performed an investigation into potential TEP/SC niche components, in order to gain insight into how human TEP/SC may be regulated. This work was reported in Deliverable 1.2 (Report on characterisation of the in vivo human TEP/SC niche.) which was also submitted on time.
The work related to WP1 Objective 2 was again performed predominantly by Partners 1 and 2, and aimed to establish the molecular pathways through which human TEP/SC are maintained and expanded in vivo. Therefore, we initially focused on collecting global transcription data, which captured the entire gene expression profile of a precisely defined TEPC population at several different stages of development. We collected three replicate datasets at each of three timepoints, and augmented these datasets with equivalent data from a series of mouse mutants that have perturbed thymus development. We also collected datasets detailing the chromatin landscape (using the approach of global chromatin immunoprecipitation and sequencing (ChIPseq) with antibodies that bind three different chromatin modifications) in cells at two of the three timepoints. Deliverable 1.3 (Report on collection of transcriptome datasets), which described this work, was delivered on time. We then performed a preliminary analysis of these ‘RNAseq’ and ‘ChIPseq’ datasets, and related to this, also performed a series of validation (or quality control) experiments which indicated the high quality of the RNAseq and ChIPseq data obtained from fetal thymus, whilst revealing problems with the RNAseq datasets obtained from adult thymus. Therefore, we principally used our fetal data, together with adult thymic epithelial cell (TEC) datasets available in the public domain, for further analyses.
In brief, we collected together into one database, called “ThymiBase” a set of TEC-derived RNAseq and ChIPseq data from public repositories as well as the above data and additional data from Thymistem groups and collaborators. We assessed and reanalyzed each of these datasets in order to provide a data resource for discovery of signalling mechanisms controlling TEC biology. This database has been continuously updated with proprietary and public domain RNAseq data, and will be made publically available once published. Partner 1 then analysed these data to identify signalling pathways implicated in maintaining mouse and human TEP/SC. These analyses were reported in Deliverable 1.4 which were delivered on time, and in later milestone reports. Subsequently, Partner 1 extended this work to form a model of signalling regulation of undifferentiated and differentiating TEPC. Overall, this analysis identified six signalling pathways as active in undifferentiated fetal TEPC. It also generated a model of TEPC differentiation, in which direct and indirect target genes regulated by the major transcription factor responsible for thymic epithelial cell differentiation were also identified, and which incorporated genetic data, obtained in WP1 by Partners 1 and 2 via extensive in vitro and in vivo analysis, regarding the cellular mechanism that may operate to control differentiation of TEP/SC and allow generation of medullary TEC during the earliest stages of thymus organogenesis. These findings were reported in Deliverables 1.5 and D1.6 which were submitted on time. In additional work in WP1, Partner 7 also tested the role of the BMP pathway in regulating TEP/SC via an extensive functional analysis. This revealed a crucial role for BMP4 during thymic regeneration after injury caused by irradiation and suggested that BMP4 potentially regulates this thymic regeneration by stimulating TEPCs.
Overall, the work of WP1 progressed well throughout the project and has provided considerable insight into fetal and adult TEP/SC regulation, meeting most if not all of the outcomes anticipated at the start of the project. The work performed in WP1 resulted in a number of peer-reviewed publications, with the major outputs and findings being: Identification of a multipotent TEP/SC in the adult mouse thymus (Ulyanchenko et al., Cell Reports 2016) and of a candidate equivalent cell population in human fetal and paediatric thymi (Blackburn et al unpublished); Identification of the Notch pathway as a key regulator of TEP/SC self-renewal and differentiation (Martin-Gayo et al, J. Exp. Med 2017; Liu...Blackburn in revision; García-León et al in preparation); Identification of the BMP pathway as a regulator of endogenous thymus regeneration (Wertheimer et al, Science Immunology 2018); Development of an ‘omics database of thymic epithelial cell datasets, with analytical capacity (ThymiBase, Kousa, Tomlinson, in preparation); and generation, via bioinformatics analysis, of a model predicting the key mechanisms controlling proliferation and differentiation of thymic epithelial progenitor/stem cells (Kousa et al, in preparation). Thus, collectively this work has provided considerable new insight into fetal and adult TEP/SC regulation, meeting most if not all the anticipated outcomes.
Work Package 2 - Generation and propagation of human thymic epithelial stem cell lines.
Our goal in WP2 was to develop an optimized and scalable method for establishment of long-term thymic epithelial stem cell (TESC) cultures from ex vivo human TEC and/or human induced pluripotent stem (iPS) cells. To achieve this, we set out to determine in vitro growth conditions that support proliferation of functionally validated human TESC lines and to generate TESC from human iPS cells, or by direct reprogramming. In the original ThymiStem proposal, we proposed to take as our starting point for culturing human TESC the method described by Bonfanti and colleagues (Nature 2010; on which Partner 1 was an author). However, Partner 1 then developed a novel method for generating TEC in the laboratory by converting (or ‘reprogramming’) into TEC an unrelated cell-type called primary embryonic fibroblasts. This work was partly supported by ThymiStem. This appeared to be a better method for producing large quantities of TEC in vitro, since in contrast to the cells cultured in the previous paper, the TEC generated by reprogramming (termed induced TEC, or iTEC), could generate a fully organized, functional thymus when transplanted (Bredenkamp et al., 2014). In addition to this advance, a subsequent report demonstrated that ex vivo mouse TEC could be propagated in vitro as spheroid cultures, termed thymospheres, and that these spheroids appear to be initiated by, and to maintain TESC (Ucar et al., 2014). Therefore, we focused the efforts of WP2 Objective 1 on development and evaluation of human iTEC and on development of human thymospheres, rather than the approach originally proposed. The work conducted progressed well in both areas.
In brief, after extensive screening of different growth factors and attachment matrices for ability to support TEPC growth in vitro (Deliverables 2.1-2.3) Partner 1 established conditions permissive for short to medium term, but not indefinite, growth of TEPC in monolayer cultures, with some cultured cells retaining capacity to contribute to thymic epithelial cell (TEC) networks for at least 3 weeks in culture. Additionally, we identified further modifications that should promote expansion of undifferentiated TEPC - and thus of an in vitro induced TESC cell type, and these remain to be tested. In parallel, Partner 3 showed that human thymic sphere cultures, that appear to be TEPC-derived, could readily be generated and propagated in vitro under conditions modified from the method described by Ucar and colleagues for mouse cells. The human thymic spheres developed by Partner 3 expressed markers of functional TEC, and at least some could support human T cell development in vitro. The above findings were reported in Deliverables 2.1 (Report on expression and functional analysis of TESC cultured on 3T3/J feeder cells), D2.2 (Report on optimization of attachment factors for maintenance of proliferating TESC in vitro), D2.3 (Report on optimization of growth factors and inhibitors for maintenance of proliferating TESC in vitro) and D2.6 (Report on generation and propagation of high efficiency human thymic epithelial stem cell lines) which were all submitted. Collectively, these data represent solid progress towards the goal of maintaining proliferating TESC in vitro. Our currently optimized conditions for the in vitro growth of TEP/SC as monolayers have been tested and validated for short term culture of mouse TEPC and, based on our evaluation of a highly similar medium (as part of WP2), are expected to apply to human. Our recent bioinformatics and functional data suggest modifications to these conditions that should specifically promote expansion of undifferentiated TEPC - and thus of an in vitro induced TESC cell type - and these conditions will be tested in the near future. Additionally, our work on human thymic spheroids indicates that the thymic spheres can readily be generated in vitro and can be propagated through at least six passages, suggesting they are based on TEPC. Importantly, the thymic spheres retain expression of functional TEC markers, and at least some can support human T cell development in vitro, indicating their promise for further development. Protection of intellectual property around this methodology is currently under discussion.
The work related to WP2 Objective 2, on generation of TEC in vitro from other cell types, was more closely related to that in Objective 1 than originally envisaged. We (Partner 1) extended our preliminary findings to show that a cell type called primary mouse embryonic fibroblasts (MEFs) can be converted to TEC by enforced expression of a single transcription factor, FOXN1 (Bredenkamp et al Nature Cell Biology 2014), and subsequently extended this finding to demonstrate that such ‘induced TEC’ could also be generated from mouse pluripotent stem cells (PS) cells. Subsequently, we focused on determining whether human PS cells could be reprogrammed into TEC using a similar strategy. For this purpose, we developed a reporter transgene suitable for introduction into human iPS cell lines, and on development of genetic constructs (transgene vectors) predicted to enable reprogramming of human iPS cells to iTEC. To this end, Partner 1 generated a bacterial artificial chromosome (BAC) carrying a human FOXN1-Venus reporter transgene suitable for introduction into human iPS cell lines, as detailed in Deliverable 2.5. However, this avenue was not pursued since, unexpectedly, we were unable to convincingly detect expression of the Venus reporter protein. In addition, Partner 1 focused on construction of vectors for promoting conversion of human iPS cell lines into iTEC, by human FOXN1 overexpression. This was reported in Deliverable 2.5. Preliminary studies indicated some difficulties with this approach. Therefore, we are now implementing a strategy to introduce the transgenes into a ‘safe harbour’ locus. The expectation is that this will enable robust transgene expression.
Overall, therefore, the work performed generated solid progress towards the two WP2 goals, and all of the WP2 deliverables were submitted. To date WP2 has resulted in one major publication (Bredenkamp et al Nature Cell Biology 2014), with additional several arising publications currently in preparation and discussion on-going around protection of the potential intellectual property arising.
Work Package 3 - Preclinical analysis of human thymic epithelial stem cell transplantation in humanized mice, and comparison to endogenous stimulation in clinical trials.
The work in WP3 aimed to assess the functional outcome of transplanting human TEP/SC-based organoids, using a preclinical humanized murine model, and also to analyse thymus data from a phase I clinical trial evaluating the effect of activating endogenous TESC. To achieve this, we addressed four specific goals. Goals 1-3 evaluated: the outcome of transplanting primary human TEP/SC in a humanized mouse model; the functional outcome of transplanting human TEP/SC-based organoids, including the longevity of the functioning grafts and the tumorigenic potential of the grafted material; and the thymus architecture and distribution of intrathymic cell types in transplanted mice; while goal 4 aimed to assess the functional outcome of activating endogenous TESC in patients.
The work performed on Aims 1-3 was performed principally by Partners 2, 3, and 6, and evaluated two alternate humanized mouse models for in vivo human T-cell reconstitution from human cord blood cells and human early thymocyte progenitors (García-Peydró et al, J Clin Inv final revision); established reproducible methods for isolation of human TEC and for generation from these cells and from human thymic sphere cultures (WP2) of reaggregate thymic organ cultures (RTOCs); and investigated the potential use of human thymic spheres for generating transplantable bioengineered thymus organoids (WP4). The results obtained indicate that implanted human thymic sphere-based RTOCs provide the appropriate thymus microenvironment for human HSPC expansion and early T-cell development, and lack tumorigenic potential. However, under current conditions, full T-cell development is precluded due to loss of architecture and collapse of the grafted thymic sphere-based RTOCs at 2-3 weeks post-implantation. These data emphasised the importance of the thymus architecture of the transplanted organoids for supporting full T-cell development, and in this regard, we have identified specific functional microenvironments for T and non-T cell development in the human thymus (Martín-Gayo et al., J Exp. Med 2017; García-León et al, in preparation). Additionally, work integrating the first-generation scaffolds produced in WP4 with human thymic-sphere-derived cells showed that the scaffolds provided an appropriate 3D structure for TEC infiltration and growth, but did not support full T cell development in vitro, suggesting that further scaffold development was required; such development was therefore undertaken in WP4. The above work was reported in Deliverables 3.1 (Report on evaluation of human TESC transplantation in humanised mice), 3.2 (Report on evaluation of thymus architecture and distribution of intrathymic cell types in the TESC-based transplanted organoids) and 3.5 (Report on the functional outcome of transplanting in vitro-expanded human TEP/SC and TESC-based organoids in humanized mice, as proof-of-principle for development of thymic regenerative immunorehabilitation therapy), all of which were submitted on time.
With respect to the fourth aim of this WP, Partner 7 set out to evaluate a new non-invasive protocol for assessing changes in thymus size and functional volume in patients after total body irradiation (TBI-based) CD34-selected allogeneic hematopoietic stem cell transplantation (allo-HSCT). This protocol, if proven useful, will be invaluable for measuring thymus regeneration in patients after activation of endogenous TESC. The first patients have been recruited to this study, which is on-going. Additionally, we have characterized the pattern of immune reconstitution in the first year after transplant, and its effects on survival and relapse, for a large retrospective study of patients who received myeloablative T cell depleted (TCD) allo-HSCT for hematologic malignancies. The data have shown that T cell recovery, which is an indirect measure of thymus regeneration and therefore most probably activation of endogenous TESC, is a predictor of outcome after TCD allo-HSCT. This study will serve as a baseline to assess effects of interventions such as KGF, Lupron and other combinations as therapeutic approaches to enhance immune recovery of patients receiving allogeneic HSCT. In this regard, we have also investigated how inhibition of sex-steroid signaling affects the outcome of HSCT after lethal TBI in mice; unexpectedly, this work uncovered a direct, sex-steroid- and thymus-independent, regulatory role for lutenizing hormone on haematopoietic stem cells (Velardi et al Nature Medicine 2018).
The work performed in WP3 has resulted in two peer-reviewed publications, with the major outputs being: Martín-Gayo et al., J Exp. Med 2017 and Velardi et al Nature Medicine 2018. A further manuscript is currently under revision (García-Peydró et al, J Clin Inv final revision) and several additional manuscripts are in preparation. All of the WP3 deliverables have been submitted.
Work Package 4 - Development of artificial matrices for generating human thymus organoids in vitro and for transplantation.
Our focus in WP4 was on the integration of tissue engineering with thymus biology, to facilitate the production of thymic organoids at the scale required for transplantation into human patients. Specifically, WP4 aimed to develop scaffold materials suitable for growth of human thymus organoids in vitro, and for transplantation. The work performed required extensive selection, fabrication and functional testing of scaffold materials and types, which was performed by Partner 6 working in conjunction with Partner 7. Overall, the focus was on development of electrospun materials. In brief, Partner 6 initially identified biocompatible polymer materials able to enhance cell attachment and proliferation, based on library searching, and then used these selected polymer materials for production of 2D scaffolds using electrospinning methods including classic, coaxial, and force spinning. Different concentrations of polymer solutions, different solvent systems, and different electrospinning conditions were tested. More than 150 electrospinning experiments were conducted to produce a range of 2D structures. Based on the electrospinning process and the quality of produced nanofibrous layers 15 samples were selected as the most promising scaffold prototypes. These were subjected to biological testing by Partner 7, were to optimization and reproducible production by Partner 6, which identified two scaffold materials as suitable for further development, as reported in Deliverable 4.1 (Report on the feasibility of enhancing TESC growth in vitro using biopolymer-based attachment matrices).
To prepare 3D tissue engineering scaffolds for testing for capacity to support TESC attachment, survival and differentiation, Partner 6 then used three different electrospinning techniques. Firstly, the technique of AC spinning was used for preparation of 3D scaffolds based on the combination of two materials. Secondly, a combination of electrospinning and linearized air flow was employed to obtain voluminous structure with the required pore diameter. Thirdly, a combination of electrospinning and melt blown technologies was used, enabling the formation of a layer containing both microfibers and nanofibers while guaranteeing good mechanical properties of the 3D scaffolds. Partner 7 assessed the adhesive properties of these biomaterials as well as cell morphology and growth of stromal cells, which Partner 6 further analysed the scaffold properties. The results of these structural studies showed that the produced tissue 3D scaffolds imitated the structure of the extracellular matrix through fibre diameters in the nanometre and micrometre dimensions and sizes of continuous pores in tens of micrometres. Thus, this structure may help recapitulate the structures formed by TEC during normal development and homeostasis in vivo. Biological tests carried out by Partner 7, demonstrated the biocompatibility of these scaffolds in vitro and in vivo and also demonstrated that the test cells (TEC) penetrated into the scaffolds and also proliferated. Partner 7 found that the resulting thymic organoids supported growth of stromal cells, with preliminary data suggesting that subcutaneous implantation of fetal thymus-derived thymic organoids resulted in generation of CD8+ and CD4+ T cells in nude mice. This work was reported in Deliverables 4.2 (Report on the use of biopolymer-based scaffolds to support generation of thymic organoids in vitro) and 4.3 (Report on the development of optimised biopolymer-based scaffolds to support generation of human thymic organoids in vitro).
Based on these results, one microfiber/nanofiber composite was identified as suitable for tissue engineering. This underwent functional testing in several partner labs, and based on this further functionalization was attempted. Technological complications were encountered with some approaches. However, promising results were obtained with two approaches: the combination of meltblown electrospun scaffolds with hydrogels, such that the hydrogels provided a superabsorbent reservoir of bioactive factors; and modification of the scaffold with polyfunctional coatings, deposited on the surface scaffold fibres, that could act as carriers of bioactive molecules. Both approaches have yielded encouraging data, with the polyfunctional coating approach appearing to provide a universal, simple and efficient method of scaffold functionalization. Of note is that meltblown technology allows for highly productive manufacturing of the scaffold, and when suitable equipment is used, also its almost unlimited size in all three axes, making this approach highly appropriate for scaling of scaffold production to the size required for clinical use. These results were reported in Deliverables 4.3 (Report on the development of optimised biopolymer-based scaffolds to support generation of human thymic organoids in vitro), 4.4 (Report on scaling of the optimized thymus organoids to a size compatible with clinical use) and 4.5 (Report on development of artificial matrices for generating human thymus organoids in vitro, and for transplantation), which have all been submitted.
Overall, the work of WP4 has provided insight into the use of tissue engineering approaches for thymic organoid production including upscaling to a size compatible with clinical use. However, some of the approaches initially identified as promising were shown through further testing to be unsuitable for production of human thymic organoids in vitro. In particular, although some evidence suggested that the MF/NF scaffolds might be useful with further optimization, we concluded that they did not represent the best option for generating human thymus organoids and that alternative bioengineered materials should be analyzed for their potential to support long-term human T-cell development, based on the outcome of suitable functional assays established, validated and then used in WP3. Both the functionalised hydrogel modified scaffolds and the coated scaffolds appear promising for this purpose. Additionally, the use of meltblown electrospinning appears suitable for generating scaffold materials at the scale required for development of transplantable human thymic organoids. Overall, substantial work was undertaken and delivered related to each of the objectives of this workpackage and, taken together, our systematic testing of molecularly tailored biomaterials and innovative scaffold designs resulted in the identification of a promising implantable platform for thymic organoid engineering. However, further development work is required to develop matrices, including those identified herein, such that they support development of a functional human thymus organoid in vitro or for transplantation.
All of the deliverables for this WP were submitted, the work has been presented at several international scientific meetings, and we anticipate that manuscripts describing the main findings will be submitted for publication in the near future.
Work Package 5 - Development of clinical grade protocols for cryopreservation and quality control of primary thymic stromal cells and cultured thymic epithelial stem cell lines.
The goal of WP5 was to enable use of TEC/TESC for research and clinical purposes, by developing standard operating protocols (SOPs) for cryopreservation and quality control of primary thymic stromal cells and cultured TESC lines. The work in WP5 was predominantly conducted by Partner 4, who performed extensive comparative testing of different conditions for tissue dissociation, freezing and thawing of human paediatric thymus tissue, and the subsequent development from these of SOPs for: thymic tissue enzymatic digestion; in vitro culture of human thymic cells; cryopreservation of human thymic cells in liquid nitrogen; thawing of human thymic cells after storage in liquid nitrogen; evaluation of human thymic cell viability by trypan blue staining; evaluation of human thymic cell viability by 7-AAD staining; evaluation of human thymic cell phenotype by flow cytometry; evaluation of the functionality of human frozen and thawed TEC by co-culturing with authologous thymocytes; and evaluation of mRNA expression in human thymic cell subpopulations. Over the course of the project, 93 paediatric thymi were collected and stored in liquid nitrogen, and used for these experiments. All of the deliverables for this WP were submitted, some has of the outcomes have been published in a peer-reviewed journal (Shichkin et al., Cryobiology 2017; Shichkin et al Trends in Transplantation 2018), with four further manuscripts currently in preparation.
Work Package 6 - Training and Dissemination.
In addition to its research programme, ThymiStem also aimed to provide training to project participants at all career stages; and to stimulate public engagement with the project aims and the wider stem cell field, since contribution in these areas is required to develop the field of human stem cell research, and also to fulfil our responsibility to European publics to enable and participate in discussion and wider consideration of our research field and related ethical and societal issues. Extensive work was therefore performed related to each of these objectives, and all of the deliverable reports from this WP were submitted. In particular, ThymiStem facilitated 9 exchange visits in which early career stage researchers visited the labs of other partners, or other internationally respected scientists in the thymus field, in order to learn new techniques and exchange knowledge. We also held two training workshops for consortium members. We also contributed to the 2014, 2015, 2016 and 2017 European Summer Schools on Stem Cells and Regenerative Medicine with a total of 19 ThymiStem researchers having attended this School, and to organising “Advances in Stem Cell Research and Regenerative Medicine” a major European conference in the field, in 2017. Additionally, 6 ThymiStem members attended 2 workshops run by the FP7 consortium PluriMes.
To ensure wide-reaching dissemination of the project outcomes, and continued public engagement with up-to-date developments in human stem cell research, ThymiStem also developed an ambitious programme of public engagement, that has made a significant contribution to the communications and engagement in the stem cell and regenerative medicine field. The ThymiStem project website has continued to provide dynamic information about the project for peer and public view. This site is actively linked with the leading stem cell and regenerative medicine website eurostemcell.org extending the reach of project news to a wider audience and avoiding duplication of effort. ThymiStem has also collaborated with the FP7/H2020 Coordinating Action EuroStemCell on the re-design of the eurostemcell.org website, to ensure that ThymiStem created material can continue to be discovered, accessed and viewed responsively across digital platforms and devices, thereby creating a public-facing legacy for the project. Based on evaluation evidence the new site, launched in mid-December 2016, will greatly increase access to the high-quality public facing stem cell biology information and resources. We have also collaborated in the translation of the website content into Czech, one of our partner languages. ThymiStem has also developed a short animation to explain thymus biology to a lay audience, as well as a fact sheet “Regenerating the Thymus”, and has translated the book “Your amazing immune system” into Croatian, with launch and release of these resources on the Day of Immunology in 2016 and 2017.
The animation shows how T cells go through a complex journey in the thymus to become mature immune cells ready to fight infection. It was disseminated to all ThymiStem scientists for use in outreach, teaching and websites. Since its launch in April 2016 the animation has been viewed fully 2062 times on the Eurostemcell YouTube channel (see https://youtu.be/FW2Jat00lEs). The ‘Regenerating the Thymus’ factsheet provides an overview of the current scientific field of thymus regeneration as well as the applications of the science, describing the role of the thymus in the immune system and the research avenues currently being investigated to regenerate the organ (see http://www.eurostemcell.org/regenerating-thymus). It reached 689 people on Facebook and received 2453 impressions on Twitter on the Day of Immunology 2017, and has been translated into the six EuroStemCell languages: English, Spanish, German, French, Italian and Polish. Taken together the animation and factsheet represent valuable public engagement resources to explain the function of the thymus, an often poorly understood organ. Additionally, we developed an interactive science game, Thymaze, designed for a family audience, that introduces the player to the thymus and the different cells of the organ. Integral to the game are multiple science messages that can be understood at different levels of scientific literacy. This game is suitable for use at science festivals, open days and other public engagement events. It is accompanied by resources designed to support the scientists when using the game, including game play directions, scientific images and conversation topics for a range of audiences. The game has been distributed to ThymiStem partners for use in future public engagement activities. The highly popular and accessible ‘Your Amazing Immune System’, which we collaborated with the Federation of Immunological Societies to translate, describes the role of the thymus in the context of the immune system in a fun, friendly, illustrated book. With its curriculum relevance, translated copies of the book are being prepared for distribution to schools across Croatia, supported by ThymiStem and the Croatian Ministry for Education.
ThymiStem members have also been active in many international, national and local public engagement events and festivals, and have participated in ‘Wikithons’ to help improve the accuracy of relevant pages on Wikipedia. Finally, to contribute to capacity building in public engagement, we have held two workshops to provide training in public engagement for ThymiStem researchers, and also participated in the ‘Sharing best practice in public engagement’ workshop held by EuroStemCell in 2016. Through these activities, ThymiStem fully met the aims of WP6.
Potential Impact:
Impact on Science
The project has generated advanced knowledge related to controlling differentiation and proliferation of human thymic epithelial stem and progenitor cells and to generation and propagation of thymic epithelial cells in vitro, and hence, increased understanding of thymus biology (including endogenous thymus regeneration). Additionally, it has evaluated preclinical models for stem-cell based human thymus transplantation, and has advanced knowledge in the development of tissue engineering technologies suitable for development of human thymic organoids. The conclusions of this work have been reported in seventeen peer-reviewed publications to date including several in top line journals, with further manuscripts currently in preparation, and have been presented at numerous international conferences. They are therefore expected to impact the direct field of thymus biology beyond the lifetime of this project. Furthermore, since these data can be compared to those obtained in other, non-thymus tissues, they will also contribute to understanding of stem cell and regenerative biology in the broader context. The project additionally aimed to evaluate the effect of a novel treatment, currently in clinical trial, on thymus regeneration and adaptive immune system function in patients, and to evaluate a novel, non-invasive approach for measuring thymus size in patients. The outcome of this research, once known, is anticipated to significantly advance clinical research capacity in the field of thymus-based immune reconstitution. Further, our unexpected findings revealing a role of lutenizing hormone in regulating HSC function may offer a new therapeutic approach for hematopoietic regeneration after hematopoietic injury.
Economic and societal impacts
In the longer term, our research outputs are anticipated to contribute to development of new therapies that enhance immune system function, for instance via safe and effective thymus transplantation. If this goal can be reached, the impact on quality of life, and in some instances, life expectancy, will be substantial. The potential impact on healthy ageing is important in the context of the ageing European demographic, with the potential to contribute to reducing the economic burden related to frailty in old age. Additional beneficiaries would be: bone marrow transplant patients, patients with some classes of congenital primary immunodeficiencies (eg DiGeorge syndrome, Nezelof syndrome, Louis-Bar syndrome, Swiss syndrome, some human SCID patients), and potentially autoimmune, solid organ transplant and cancer patients, patients who have undergone surgical thymectomy, and the elderly population. The market for thymus transplantation approaches is extensive. There are 25,000 allo bone marrow transplant patients annually world-wide, who are potential candidates for thymus transplantation therapy, and this market is currently growing, as the use of allo-BMT is extended.
Beyond its research outputs, ThymiStem has provided specialist training to participating researchers at all career stages. This is expected to enhance their employability in a competitive jobs market, and also to contribute to raising the overall skill-base among European stem cell and regenerative medicine researchers. Additionally, we have linked with other consortia to maintain the highly-valued European Summer School on Stem Cells and Regenerative Medicine and the bi-annual Advances in Stem Cell Research conference. One of the impacts of the project will therefore its contribution to an enhanced European stem cell and regenerative medicine research sector in academia, industry and related fields. Finally, ThymiStem contributed to public engagement with stem cell research and regenerative medicine, including through collaboration with the high-impact H2020 coordinating action EuroStemCell (www.eurostemcell.org) with anticipated positive impacts on public awareness of European science and, importantly, on informed public engagement with this field at a time when new treatments are under active development.
List of Websites:
Website address: http://www.thymistem.org/
Contact details:
Prof Clare Blackburn, MRC Centre for Regenerative Medicine, University of Edinburgh, 5, Little France Drive, EH26 9NL. Email: c.blackburn@ed.ac.uk