Community Research and Development Information Service - CORDIS

FP7

THYMISTEM Report Summary

Project ID: 602587
Funded under: FP7-HEALTH
Country: United Kingdom

Periodic Report Summary 2 - THYMISTEM (Development of Stem Cell Based Therapy for Thymic Regeneration)

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 brings together 7 academic research teams and 1 SME from 6 countries: UK, Spain, Croatia, Czech Republic, Ukraine, and the USA. Collectively, the project comprises 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 is to develop 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 is on the human thymus. Specifically, our work addresses 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 aim to use in vitro human thymic epithelial stem cells to generate thymic organoids - suitable for transplantation - that contain all of the differentiated epithelial cell types required for thymus function.
Our research therefore aims to develop robust protocols for the long-term in vitro culture of functionally validated 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 aim to establish an optimized means of delivering these cells to immunocompromised recipients such that thymus function is fully restored, and to assess risks associated with this protocol. Finally, we aim to develop optimized procedures for cryopreservation of human thymic epithelial cells.
Beyond our scientific research, ThymiStem aims 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 aim 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, are to develop:
• A validated model of the key mechanisms controlling proliferation and differentiation of thymic epithelial progenitor/stem cells during development and homeostasis in vivo.
• An optimized, scalable 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.

ThymiStem is structured into 5 scientific work packages, plus a work package dedicated to outreach and public engagement. Each of these workpackages addresses one of the specific objectives set out above. The final workpackage is dedicated to project management.

Project Results:
• WP1: Partner 1 has identified a multipotent epithelial stem/progenitor cell in the adult mouse thymus (Ulyanchenko, O’Neill, ...Blackburn, Cell Reports 2016). Partners 1, 2 and 3 have extensively characterised the human fetal and paediatric thymus, and have identified a cell population that shares phenotypic characteristics with these mouse thymic epithelial stem/progenitor cells (TEP/SC). Partner 1 has developed an assay of the potency of defined human thymic epithelial cell populations. Additionally, Partners 1 and 2 have identified the Notch pathway as a key regulator of thymic epithelial stem/progenitor cell differentiation. Partner 1 has generated RNAseq data for 3 defined mouse fetal thymus populations, and is currently generating RNAseq datasets from defined adult mouse and postnatal human thymic epithelial cell populations. Through bioinformatics analysis of these and public domain TEC RNAseq and ChIPseq datasets, Partner 1 has identified signaling pathways that are implicated in regulation of undifferentiated fetal TEPC. Members of these pathways these are therefore candidate self-renewal factors for TEP/SC and, by extension, may be components of the adult TEP/SC niche. Partner 1 has investigated the nature of the in vivo adult TEP/SC niche directly by screening candidate proteins. From this analysis, Partner 1 has identified a number of proteins as candidate niche components. These factors have all been identified as stem cell niche components in other lineages. The above results are described in detail in deliverables D1.1-D1.4.

• WP2: Partner 1 has developed a novel method for generating functional thymic epithelial cells (induced thymic epithelial cells, iTEC) in vitro by reprogramming (Bredenkamp Nature Cell Biology 2014). This breakthrough is highly relevant to ThymiStem’s goals and therefore we refocused WP2 around transposing this system to human (see first periodic report). Additionally, Partner 3 has modified a recently described protocol for growing mouse thymic epithelial cell-based feeder-free cultures, as thymospheres, to enable growth of human thymospheres. The remodeled WP2 thus includes significant work aimed at the thymosphere culture approach as a means of growing TEP/CS in vitro (see first periodic report). In order to identify synthetic polymers for use as attachment factors for growing TEP/SC or iTEC in vitro, and for use as the basis for 3D scaffold formation, Partner 1 has now screened a library of synthetic polymers for capacity to bind iTEC. Partner 1 has also tested whether the adhesion, proliferation and function of iTEC grown on polycaprolactone (PCL) scaffolds generated by Partner 6 in collaboration with Partner 7 (in WP4), can be improved by functionalization of the PCL MF/NF scaffolds. Partner 3 has shown that human thymospheres can be readily established from dissociated thymus tissue, and has worked extensively with Partner 2 to characterize these cells. Partner 3 also investigated optimal culture conditions for thymosphere expansion. Functional testing of the thymospheres is reported in WP3. These results are described in D2.1 and D2.2. We have also progressed our work aimed at generating TEC from human pluripotent stem cells, Partner 1 has investigated differentiation of pluripotent stem cells (PSCs) into iTEC, and has generated a human pluripotent stem cell line carrying a FOXN1 reporter allele. Partner 1 has also constructed advanced vectors for introducing relevant reprogramming factors into human PSC, and has generated conventional and ‘reset’ human ES cell lines that carry these vectors. Partner 1 has conducted preliminary investigation of transgene expression in these cell lines. The data on human PSC programming will be reported in D2.5.

• WP3: Several experimental approaches have been undertaken as part of WP3, to evaluate the outcome of transplanting thymic organ reaggregates (RTOCs) generated from ex vivo human thymic epithelial progenitor/stem cells (TEP/SC). Partner 2 has described optimal conditions for in vivo human T-cell reconstitution from either cord blood hematopoietic stem/progenitor cells (CB HSPCs) or early thymic T-cell progenitors (ETPs) in two xenotransplantation models validated for this work by Partner 2 (‘humanized’ NSG and RAGγc-/- mice). In collaboration with Partner 1, Partner 2 has also established reproducible methods for isolation of human postnatal thymus TEC components, including TEP/SC, and has reported their suitability to generate reaggregate thymic organ cultures (RTOCs). Partners 2 and 3 have tested the functionality of human thymosphere-based reaggregates, and Partner 1 the outcome of transplanting human thymus fragments into immunocompromised mice. To refine this work, Partner 2 has now characterized the minimal cell composition required to generate functional RTOCs based on ex vivo-isolated human TEC components, and has identified the particular components required to selectively support human T- and non-T-cell intrathymic development. Comparison of functional properties of fetal vs postnatal primary TECs has also been evaluated. Partner 3 has generated thymic spheres (thymospheres) in culture from human postnatal TECs (see WP2). The functional attributes of these cells have been tested in WP3. Partner 2 in collaboration with Partner 3 have developed assays for evaluating the potential of in vitro-derived thymospheres to support the differentiation of human haematopoietic progenitors into T- and non-T intrathymic cell subtypes both in vitro and in vivo. Partners 2 and 3, together with Partner 6, have begun to investigate the potential use of human thymospheres for generating transplantable bioengineered thymus organoids supporting human T-cell development. Additionally, Partner 2 has investigated the tumorigeneicity of transplanted thymospheres. Collectively, the above data are reported in D3.1, D3.2, and D3.5. To meet the third aim of WP3, Evaluation of the outcome of activating endogenous thymic epithelial stem cells in patients, Partner 7 is performing a phase 2 trial to assess the effects of two drugs on thymic recovery in patients with advanced hematologic malignancies). Partner 7 has also developed an FDG PET/ CT scanning protocol for use in an open-label phase 2 trial to assess a protocol for enhancing thymic recovery in patients with advanced hematologic malignancies. These data will be reported in D3.3 and D3.4.

• WP4: Partner 6 has used electrospinning technology to develop a large number of nanofibre scaffolds for testing for capacity to support thymic epithelial stem cell growth in vitro, and development of 3-dimensional thymic organoids. Fifteen scaffolds were initially selected for testing for reproducibility of manufacture by Partner 6, and for biocompatibility by Partner 7. This identified two nanofibre preparations as promising materials for further development as 2D matrices for supporting human thymic epithelial stem cell growth. These results are described in detail in D4.1 and D4.2 (see also D2.2). Partner 6, working with Partner 7, has also developed PCL MF/NF scaffolds for use to support 3D thymic organoids. Partner 6 tested the effect of the varying the ratio of nanofibers (NF) to microfibers (MF) in the composite, using a combination of melt blown and electrospinning technology, on cell proliferation. On the basis of these results from in-vitro testing, Partner 6 proposed one composite as most suitable for tissue engineering. This has now undergone preliminary testing by Partner 7. Partner 6 has now extended this work by developing functionalised PCL-based scaffolds, using a variety of approaches centred on core-shell technology. Partner 7 has tested the kinetics of factor release from these scaffold preparations. Results from the above studies are reported in D4.1, D4.2 and D4.3. The results on the functionalized scaffolds will be reported in D4.4 and D4.5.

• WP5: Partner 4 has tested a number of protocols for freezing human paediatric thymus samples. Work performed by Partner 4 together with Partner 1 has shown that one of these is suitable for freezing of human thymus samples that can then be recovered for use in further experiments. Partners 4 and 5 also developed a draft manual for QC and QA. Partner 4 developed a set of SOPs and protocols for receipt and storage of human thymus fragments, primary thymic cell suspensions, thymic cell suspensions enriched by TEC and stromal-epithelial cultures. Partner 4 has now developed a standardized method for preparation and freezing of dissociated cell preparations generated from thymus samples, based on cell viability and efficacy of stromal-epithelial cells monolayer formation after thawing. Partner 4 has also shown that cryopreservation can be used for long-term storage of thymic samples and has investigated the viability of human TEC after long term storage in a variety of different conditions. Partner 4 has also done a substantial amount of work growing thymic cells under different culture conditions. These results are presented in D5.1 and D5.2, and related milestones. Partner 4 has started experiments in additional evaluation and identification of TEC in human thymic samples and is also is testing a set of scaffolds provided by Partner 6 on human thymic samples.

• WP6: Partner 1 has held two training workshops for consortium members. ThymiStem contributed to the 2014, 2015 and 2016 European Summer Schools on Stem Cells and Regenerative Medicine; six ThymiStem students attended this School in 2014, two in 2015 and eight in 2016. ThymiStem members attended the workshop ‘Tissue engineering through stem cell-based self-organisation’ run by the FP7 consortia PluriMes in September 2015. Partner 1 developed the project website www.thymistem.org. ThymiStem has contributed to eurostemcell.org through fact sheet development; we have also developed a short animation to explain thymus biology to a lay audience. Thymsitem scientists in Edinburgh have undergone training with an experienced Wikipedian to equip them with editing skills and work has begun on editing pages related to Thymistem’s work and the wider stem cell field. In collaboration with Eurostemcell and other FP7 stem cell consortia, Thymistem has taken a lead role in the conception and development of two 2-day training events aimed at sharing best practice, for stem cell communicators across the Eurostemcell partnership including Thymistem members. Additionally, Thymistem has been active in many international, national and local public engagement events, including European Researchers night 2014, ISSCR public symposium (Stockholm June 2015), Day of Immunology (April 2016, Croatia and Ukraine), The Edinburgh International Science Festival (April 2016), Nanoden (May 2016, Prague, Czech Republic), MRC Festival of Science (June 2016, Edinburgh).

Potential Impact:
Impact on Science

• The project will generate advanced knowledge related to controlling differentiation and proliferation of human thymic epithelial stem and progenitor cells. This will lead to increased understanding of thymus biology, including the cellular interactions needed to maintain the thymus and how these are affected by age-related thymus involution. The work will therefore impact our direct field beyond the lifetime of this project. Furthermore, since our results can be compared to those obtained in other tissues, they will contribute to understanding of stem cell and regenerative biology in the broader context.
• The project is also developing stem-cell based preclinical models for stem-cell based human thymus transplantation. This includes development of relevant tissue engineering technologies; and evaluation of thymic epithelial stem cell-based organoid transplantation into humanized mice including to test the function and longevity of thymic epithelial stem cell-based organoids.
• The project will additionally evaluate the effect of a novel clinical treatment, currently in clinical trial, on thymus regeneration and adaptive immune system function in patients. For this, we will develop a novel FDG PET/CT scanning protocol for non-invasive evaluation of thymus size in patients: this technology is anticipated to significant advance clinical research capacity in the field of thymus-based immune reconstitution.
Overall, the questions addressed and methods and approaches used throughout this project are at the cutting edge of human stem cell biology and of preclinical research on the application of thymic epithelial stem cells for immune reconstitution. Our research outcomes are therefore anticipated to have a significant impact on the broad field of stem cell research and regenerative medicine, as well as on basic and preclinical thymus research, in Europe and internationally.

Economic and societal impacts

Our research outputs are anticipated to pave the way for new therapies that could benefit several patient groups, including: bone marrow transplant patients, patients with some classes of congenital primary immunodeficiencies (such as DiGeorge syndrome, Nezelof syndrome, Louis-Bar syndrome, Swiss syndrome, some human SCID patients), and may also benefit autoimmune, solid organ transplant and cancer patients, patients who have undergone surgical thymectomy, and the elderly population. The envisaged therapies are anticipated to enhance immune system function. Therefore, 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.

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. This market is currently growing, as allo-BMT is increasing being used as therapy for malignant and non-malignant diseases of the hematopoietic system, including to ‘reprogramme’ the immune system in autoimmune patients.

Beyond its research outputs, ThymiStem is providing specialist training to participating researchers at all career stages. 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 be to contribute to an enhanced European stem cell and regenerative medicine research sector in academia, industry and related fields.

Finally, ThymiStem will contribute to public engagement with stem cell research and regenerative medicine, including through collaboration with the high-impact H2020 coordinating action EuroStemCell (www.eurostemcell.org).

List of Websites:
www.thymistem.org

Contact

Angela Noble, (Senior European Research Advisor)
Tel.: +44 1316509024
E-mail

Subjects

Life Sciences
Record Number: 195804 / Last updated on: 2017-03-14