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Periodic Report Summary 2 - PLURIMES (Pluripotent stem cell resources for mesodermal medicine)

Project Context and Objectives:
Project Context
The concept of PluriMes is to exploit the developmental programme of human pluripotent stem cell differentiation in order to capture mesodermal progenitor cells with therapeutic potency for diseases of skeletal muscle, bone and cartilage.

Reconstructive cell replacement therapies offer a prospect for alleviating the enormous medical and economic burdens presented by injury and degenerative disease of muscles, bone and cartilage. Mesenchymal stromal cells (MSCs) harvested from post-natal tissues are a possible source for such therapies. Indeed, MSCs currently dominate clinical trials in cell therapy and are frequently portrayed as a panacea for conditions ranging from heart failure and ischemic limb conditions to colitis and graft versus host disease. To date, however, evidence of clinical efficacy is inconsistent and controversial (Bianco et al., Nature Medicine, 2013). Most importantly, the putative therapeutic entity, the MSC, is arbitrarily defined by individual practitioners (Bianco et al., EMBO J, 2013). Consequently critical parameters such as cell purity, self-renewal capacity, differentiation stability, biological potency and clinical mode(s) of action are all indeterminate.

The central idea of the PluriMes consortium is to provide a rigorous scientific foundation for prospective cell therapies by defining the cell state(s) and potency of MSC in the framework of normal tissue development. This requires isolating and defining human mesenchymal progenitors at progressive steps in tissue differentiation. PluriMes will achieve this by harnessing the capacity of pluripotent stem cells to recapitulate ontogeny in the laboratory environment. The ultimate cell therapy delivery vehicles may be MSC derived from PSC and/or post-natal tissue sources. The strategy requires integrating knowledge in fields of stem cell biology, mammalian development and mesenchymal tissue biology to address basic science challenges, combined with expertise in cell culture, bioengineering, pre-clinical models and cell therapy for extension to clinical applicability. Accordingly the consortium comprises world-leading academic expertise in each of these areas complemented with three bioindustry SMEs providing development for commercial exploitation (see figure 1).

Project Objectives
PluriMes has three major objectives:
(i) Provision of generic platforms for human pluripotent stem cell self-renewal and directed differentiation based on stratification into ground state and lineage-biased sub-states.
(ii) Delineation of the molecular identities and biological potencies of defined classes of mesodermal progenitor generated during developmental progression from pluripotent stem cells, and comparison with cells harvested from post-natal human tissues.
(iii) Scaleable expansion of mesodermal progenitors in fully defined culture conditions suitable for pre-clinical manufacturing with retention of genetic integrity, identity, and engraftment potency

Overall, PluriMes will make significant advances in understanding and controlling two of the most potent and promising stem cell types for regenerative medicine, pluripotent stem cells (PSC) and mesodermal progenitors.
Project Results:
Following a productive kick-off meeting in February 2014, the consortium funds were distributed and all beneficiaries commenced activities within 3 months. The consortium recruited an experienced Project Management team and established the project website ( Quarterly web conferencing was implemented for each Workpackage to facilitate continuous communication and interaction between beneficiaries and for oversight of the strategic operation and integration of the project.

In the first period 1 Partner 1 demonstrated that conventional human PSC can be reset to a naïve state. This was achieved by short-term expression of NANOG and KLF2 transgenes and subsequent culture in conditions based on those for ground state mouse embryonic stem cells with addition of a protein kinase C inhibitor. These reset PSC have gene expression and epigenetic features expected for naive cells and distinct from conventional PSC. The findings were published in the leading international journal CELL (see figure 2).

Partner 1 subsequently devised a method for generating human naïve cells by resetting without transgenes. Based on these advances Partner 11 is developing commercial culture media for transgene-free resetting and for expansion of naïve PSC. Partners 7 and 2b furnished evidence for the existence of lineage-biased sub-states in conventional PSC and have generated single cell RNA transcriptomes for in-depth characterisation.

Partner 6 and Partner 8 have created PSC carrying lineage-specific fluorescent protein reporters by BAC transgenesis and homologous recombination. In addition CRISPR/Cas 9 technology was employed to correct the mutated α-sarcoglycan gene in iPSC derived from patients with Limb-Girdle Muscular Dystrophy 2D.

Partner 10 described for the first time robust generation of skeletal muscle from PSC, a major finding published in Nature Biotechnology. Partner 8 published defined protocols for the differentiation of lateral plate mesoderm derivatives; cardiac muscle and endothelial cells (Nature Protocols, Nature Biotechnology, Development). This technology has been licensed to Partner 4 who is developing robust processes for upscaling and manufacturing.

Partner 3 developed stringent in vivo assays to measure differentiation properties of mesoderm progenitors. Assays for osteogenesis, chondrogenesis, angiopoiesis, myogenesis, and establishment of hematopoietic niches revealed strikingly divergent differentiation capabilities of peri- and postnatal progenitors isolated from bone, muscle, fetal blood, and other tissues. The findings have been discussed in two major review articles. The diversity of postnatal progenitors based on tissue source has been confirmed by transcriptome analysis. These results, published in Stem Cell Reports, disprove the notion of a common mesenchymal stem cell (MSC).

Partners 5 and 11 collaborated with Partners 2a and 4 to optimize cryopreservation media and methods for mesodermal progenitors. Partner 11 has worked with Partners 2a and 4 to demonstrate animal-component free media expansion of mesodermal progenitors. Partner 9 has designed and tested a synthetic microcarrier system for bioreactor expansion of mesodermal progenitors.

PluriMes appointed a Scientific Advisory Panel (SAP) of leading experts who attended the consortium meetings in February 2015 and 2016 and provided reports with advice on our programme and progress to date.

Over the last 36 months PluriMes has:
• Funded six exchanges through the PluriMes mobility programme
• Co-hosted three European Summer Schools on Stem Cells & Regenerative Medicine
• Run four training workshops plus two short public engagement training sessions
• Run two full consortium meetings and two mini-consortium meetings
• Worked cooperatively with 4 other stem cell consortia; HuMen, Thymistem, Neurostemcellrepair, REVIVE, to run two major conferences and include them in PluriMes training workshops
• Collaborated with EuroStemCell on various public engagement activities

PluriMes’ main results are presented in 19 Deliverables and 12 Milestones. Research discoveries are presented in 26 publications in international journals and 1 patent filing. Additional manuscripts are currently submitted or in preparation. The majority of publications are Open Access and ’omic datasets have been deposited in public databases.
Potential Impact:
By bringing together leading European research teams in an integrated cross-disciplinary programme, PluriMes expects to have a major impact on stem cell research in the dual fields of pluripotency and mesoderm. The outcomes will be new and advanced knowledge, improved technology platforms, standardised protocols, and a coherent body of highly skilled stem cell investigators.

The most important impact of PluriMes will be in defining cellular parameters for therapeutic relevance and thereby setting the stage for future clinical implementation. This will be achieved by addressing key scientific and translational challenges:
• defining the mesodermal differentiation potential of naïve ground state and lineage-biased substates of pluripotent stem cells
• establishing a platform for directed differentiation and expansion of mesodermal stem/progenitor cells from renewable PSC resources;
• clarifying the origin, identity and culture requirements of different classes of mesodermal progenitors;
• validating differentiation potency in animal models.
• defining culture conditions and high quality reagents suitable for future scale up and GMP cell production

Expected impacts of the project include:
(a) Therapeutic Purpose
Elucidating the identity and properties of defined classes of mesoderm progenitors will be enabling in two fields: (i) development of direct strategies for tissue replacement or regeneration, which would meet a growing market in Europe (43% of the global market for a value of >$25 billion, expected to grow to >$36 billion by 2016); (ii) dissection of mechanisms underpinning self-renewal and differentiation of progenitors in vivo, which will be key to targeting dysfunction across a broad range of diseases.
(b) Knowledge to Harness the Potential of Human Pluripotent Stem cells
PluriMes addresses general hurdles associated with potential clinical application of PSC. Deeper knowledge of the human ground state and of pluripotent sub-states will provide a rational framework for much-needed harmonisation and standardisation in the field.
(c) New Technologies
The technologies developed and exemplified in this project will make available new ways to analyse and control stem cells, providing for example bioengineered stem cell culture methods to enable precision application of extrinsic factors and determine how they influence cell fate, or techniques that facilitate scale-up of ground state or lineage-biased PSC while minimising genetic change. Such technologies offer new opportunities for commercialisation. Thus the naïve human pluripotent stem cell medium developed by Partner 1 has been licensed by Partner 11 for development into a commercial product.
(d) Regulatory Standards
PluriMes addresses key regulatory issues, meeting or surpassing current standards in order to facilitate approval by relevant EU Regulatory authorities (EMA). A workshop on this topic was jointly organised with the UK Regenerative Medicine Platform.
(e) Bioindustry
PluriMes includes two SME partners. Their involvement realises impacts for employment, competitiveness of the technological R&D base, and wealth creation in Europe. Specifically, SCT UK has located research activity related to PluriMes in a new facility in Cambridge.
(f) Skills and Training
PluriMes places a major emphasis on developing skills of junior researchers and promotes this through laboratory exchanges and specialist workshops.
(g) Consolidation of the European Regenerative Medicine Community
A broad impact aimed at by PluriMes is to promote and strengthen the general field of stem cell research and regenerative medicine in Europe. This is achieved most effectively through coordinated activities with complementary projects, such as the Hydra Summer School.
(h) Public Communication and Engagement
PluriMes works with the FP7 coordination action EuroStemCell ( to ensure dissemination of accurate information in accessible formats to a diversity of public audiences.
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