Final Report Summary - PLURIMES (Pluripotent stem cell resources for mesodermal medicine)
Executive Summary:
The ambition of PluriMes is to advance understanding and control of human pluripotent stem cell differentiation in order to produce mesodermal progenitor cells for use in the study and treatment of diseases of muscle, bone and cartilage.
Cell replacement therapies offer a prospect for alleviating the enormous medical and economic burdens presented by injury and degenerative disease. Human pluripotent stem cells offer a renewable source of cells for cell therapy because they can, in principle, be converted into any cell type. In particular progenitor cells for mesodermal tissues such as muscle, bone and cartilage, may be produced through controlled differentiation in the laboratory. In contrast, current clinical trials in cell therapy are dominated by mesenchymal stromal cells (MSCs) harvested from post-natal tissues. MSCs are typically portrayed as universal stem cells and as a panacea for conditions ranging from heart failure and ischemic limb conditions to colitis and graft versus host disease. However, evidence of clinical efficacy is inconsistent and controversial (Bianco et al., Nature Medicine, 2013). Crucially, the MSC entity does not fulfil scientific criteria for an authentic stem cell but is arbitrarily defined by individual practitioners (Bianco et al., EMBO J, 2013). Critical properties such as cell purity, self-renewal capacity, differentiation stability, biological potency and clinical mode(s) of action are all indeterminate.
PluriMes aimed to provide a rigorous scientific foundation for prospective cell therapies by defining the cell state(s) and potency of MSCs compared to mesodermal progenitors generated from pluripotent stem cells. To reach this goal the project had to meet three key challenges:
(i) Provision of common platforms for human pluripotent stem cell self-renewal and directed differentiation based on isolation of naive and lineage-biased classes of stem cell.
(ii) Detailed characterisation of mesodermal progenitors generated during differentiation from pluripotent stem cells and comparison with MSC harvested from post-natal human tissues.
(iii) Growth of mesodermal progenitors with therapeutic potential in controlled environments suitable for pre-clinical manufacturing.
PluriMes combined the know-how of 10 academic and 3 bioindustry groups to reach each of these objectives, as summarised below:
(i) The project defined two new classes of human pluripotent stem cell; naïve cells representing the earliest embryonic founder cells with unlimited potential, and lineage-biased substates that are poised to develop along a particular developmental pathway. These fundamental advances in knowledge of pluripotency will guide efforts to further improve control of differentiation.
(ii) PluriMes teams developed several reagents and protocols for generating various mesodermal cell types, notably including functional skeletal muscle, by differentiation of pluripotent stem cells. In a parallel and comprehensive series of investigations, MSCs were obtained from different tissues but found to share no common molecular identity nor to exhibit regenerative properties in the body. These critical findings confirm other scientific data that MSCs are not a common cell type present in all tissues, do not contribute directly to tissue regeneration, and should not be termed stem cells.
(iii) Close collaborations between academic and industry partners resulted in new and improved media and devices for preserving and expanding stem cells. Selected formulations have been further refined to meet the high quality Good Manufacturing Practice regulations required to produce cells for therapeutic applications in humans.
PluriMes research results have been presented at numerous scientific conferences and are available in 41 publications in international peer-reviewed journals and books, with a further 16 manuscripts in preparation. The publications are open access and 11 high content datasets have been deposited in public databases. Two patents were filed and are in the process of being commercialised and one patent will be filed shortly. As a result of the project 11 new culture media and other products for stem cell research are being produced and released. Finally, in addition to the scientific activities, the PluriMes project engaged in an active programme of training and dissemination activities, promoting the field of stem cell research both within the consortium and externally to specialist and public audiences.
Project Context and Objectives:
Context
The PluriMes Project bought together leading European research teams and SMEs to create an integrated cross-disciplinary programme that would have a major impact on stem cell research in the dual fields of pluripotency and mesodermal progenitors for muscle, bone and cartilage.
Mesoderm derivatives comprise the largest proportion of the body mass and are central to a vast range of both system specific or organism-wide physiological functions. They are among the prime targets of aging-related involutional or degenerative changes. Diseases for which mesoderm derivatives represent the direct sites of morbidity include highly prevalent conditions, as well as rare, crippling or lethal diseases appearing in childhood, adolescence or adult life. Due to their role in growth, energy metabolism, hormonal integration of organ systems, reproductive function, control of hematopoiesis, circulation, and many other pivotal physiological functions, mesoderm-derived tissues are also central to a number of organism derangements such as obesity, menopause-related morbidity and vascular disorders.
Highly prevalent conditions such as osteoarthritis, osteoporosis, sarcopenia, frailty, and obesity represent some of the most significant determinants of morbidity that worsen quality of life in aging populations. These conditions arise from derangement in post-natal life of the function of connective tissue cells and their progenitors. They generate high social, medical and human costs. For example, the estimated direct healthcare cost attributable to sarcopenia in the United States in 2000 was $18.5 billion ($10.8 billion in men, $7.7 billion in women), which represented about 1.5% of total healthcare expenditures for that year. A 10% reduction in sarcopenia prevalence would result in savings of $1.1 billion (dollars adjusted to 2000 rate) per year in U.S. healthcare costs. In the United Kingdom direct cost of treatment for osteoarthritis exceeds £870 million per year, while cost of social and community services and loss of economic production exceeds £250 million. On the other hand, less prevalent or rare diseases such as muscular dystrophies and skeletal dysplasias typically emanate from genetic abnormalities that affect the morphogenesis, differentiation and growth of specialized cell types of mesodermal origin. Consequences for the affected individuals and their relatives are devastating.
Success of any therapy targeting postnatal mesoderm-derived tissues postulates a clear appreciation of the cellular tools at hand. This can only be based on a rigorous definition of their identity, biology, mode of culture, and functional properties. Major uncertainties have plagued the field in all these areas (reviewed in Bianco et al, Nature Med, 2013).
An important goal for PluriMes, therefore, was to define cellular parameters for therapeutic efficacy and thereby set the stage for future clinical implementation.
Key Challenges
At the outset, PluriMes needed to resolve state of the art challenges in pluripotent stem cell (PSC) biology in order to provide optimal platforms for production of efficacious differentiated progeny. Foremost among these issues was the fundamental nature of pluripotency in human. Studies in rodents had revealed that pluripotency is not a unitary identity but comprises at least two distinct stable states, naive ES cells and primed EpiSCs (Tesar et al, Nature, 2007; Brons et al, Nature, 2007; Nichols and Smith, Cell Stem Cell, 2009), together with heterogeneous, or metastable, sub-states (Chambers et al, Nature, 2007; Toyooka et al, Development, 2007; Hayashi et al, Cell Stem Cell, 2008; Hanna et al, Cell Stem Cell, 2009; Bao et al, Nature, 2009; Canham et al, PLoS Biology, 2010; Greber et al, Cell Stem Cell, 2011; Bernemann et al, Stem Cells, 2011). Importantly, however, a “ground state” of naïve pluripotency had been captured and stabilised by inhibition of the mitogen activated protein kinase (Erk) signalling pathway (Ying et al, Nature, 2008). Ground state PSC share common features across different strains of mice and rats. These characteristics include: high clonogenic efficiency from dissociated cells, near-homogenous expression of key pluripotency factors (Wray et al, Biochem Soc Trans, 2010); suppression through transcriptional pausing of many developmental genes (Marks et al, Cell, 2012); reduced levels of epigenetic histone modifications associated with gene silencing (Marks et al, Cell, 2012); global hypomethylation of DNA (Leitch et al, Nature Strut Mol Biol, 2014); and a distinct miRNA profile (Jouneau et al, RNA, 2012). These transcriptional and epigenetic features were postulated to underlie unbiased access to definitive germ layer differentiation (Smith and Nichols, Cold Spring Harbor Perspectives, 2012). Isolating human PSC that recapitulate all of these properties was therefore a key target for PluriMes. On the other hand, the heterogeneity of conventional human PSC was postulated to reflect differential lineage priming (Enver et al, Cell Stem Cell, 2009). An important goal for PluriMes therefore was to explore the existence of PSC sub-states, in particular with a lineage bias towards mesoderm. These two complementary approaches were anticipated to lead to definitive advances in understanding of the pluripotent constellation in human which could in turn shape downstream approaches to control differentiation.
Directed differentiation of PSC, particularly into skeletal mesoderm, was a further barrier in the field and a major focus for the collaborative research programme in PluriMes. This would entail the creation of a set of reporter lines, the development of new culture systems and functional assays, and detailed transcriptomic characterisation. In addition, there was a clear need to develop improved cell cryopreservation and expansion methods, compatible with clinical manufacturing (GMP) for cell therapy applications.
Most importantly, prior to PluriMes, and still today, post-natal mesodermal progenitors, variously described as mesenchymal stromal cells (MSC), mesenchymal stem cells (MSC), mesoangioblasts or pericytes, are proffered for a range of cell therapy applications. The different names alone, however, testify to the ambiguities and controversies in this field. In reality there is a dearth of reliable information on the origin and relatedness of these cell populations or their fate after transplantation (reviewed by Bianco et al, Nature Med, 2013). PluriMes therefore proposed a radical new approach: directed differentiation of human PSC would be exploited to establish developmental hierarchy, define cell identities, and determine biological activity and prospective therapeutic potency of specific mesodermal progenitors. PluriMes would then compare for the first time developmentally discrete mesodermal progenitors derived through PSC differentiation with cell preparations from post-natal human tissue that are presently considered for clinical use (so-called MSC). This exercise is designed in an unbiased way to provide new knowledge and advance understanding of mesodermal progenitor cell ontogeny, biology and function. PluriMes thereby aimed to provide defined cell classes and associated culture protocols to facilitate translational applications using both PSC-generated and post-natal tissue derived mesodermal progenitor cells.
The Consortium
Against this background, PluriMes sought to integrate knowledge in fields of stem cell biology, mammalian development and mesodermal 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 comprised world-leading academic expertise in each of these areas complemented with two bioindustry SMEs providing research and development for commercial exploitation. The overall multidisciplinary approach comprised a series of collaborative scientific workpackages in which PluriMes investigators addressed: (1) human ground state pluripotency; (2) Lineage biased sub-states (3) Genetic tools; (4) Lineage specification for paraxial and lateral mesoderm; (5) Comparative assessment of biological activity and therapeutic potential; (6) Bio-processing and scale-up. In addition, two work packages (7 and 8) were dedicated to management activities, and to dissemination and training respectively. See Project component schematic (Figure 1 of the attachment).
Overall the consortium aimed to maximise cross-fertilisation and synergy between the participating research teams. Thus the research plan was constructed to maximise collaboration rather than individual contributions. To stimulate and enable new directions and interactions, a contingency fund was set aside specifically to support innovative sub-projects proposed jointly by Principal Investigators.
Finally, to evaluate and advise on progress, PluriMes engaged a Scientific Advisory Panel of leading experts who reviewed the project at each of the annual consortium meetings and made recommendations on funding of new sub-projects.
Objectives
The over-arching goal of PluriMes was to exploit human pluripotent stem cell (PSC) differentiation to generate mesodermal progenitor cells with therapeutic potency for diseases of skeletal muscle, bone and cartilage, and to compare these with mesenchymal stromal cells (MSCs) isolated from various human tissues. To this end PluriMes set three primary 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 mesenchymal progenitor generated during developmental progression from pluripotent stem cells, and comparison with cells harvested from post-natal human tissues.
III. Scaleable expansion of mesenchymal progenitors in fully defined culture conditions suitable for pre-clinical manufacturing with retention of genetic integrity, identity, and engraftment potency
The PluriMes research plan was designed to achieve the objectives within the four years of the project, measured and verified by, amongst others:
• open access peer-review publications in leading international journals;
• data deposition in public archives;
• conference presentations;
• training courses and exchanges;
• patent applications;
• new protocols and commercial products for research;
• regulatory compliant protocols, materials and culture media for human stem and progenitor cells.
PluriMes also aimed to:
• Make Project knowledge/information accessible to both specialist and public audiences via a user-friendly website.
• Increase scientific knowledge, interaction, training and communication within and beyond the consortium.
• Engage effectively with a range of stakeholders and public through outreach activities
Project Results:
Overview
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 (www.plurimes.eu). 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. PluriMes also appointed an international Scientific Advisory Panel (SAP) of leading experts; Professor Margaret Buckingham (Chair), Professor Pam Robey (co-Chair), Professor Peter Zandstra, Professor Shahragim Tajbakhsh and Dr Glyn Stacey. The SAP attended the four annual consortium meetings and provided detailed reports with evaluations on progress and strategic guidance towards final outputs.
Objective (i) concerns the fundamental nature and regulation of pluripotent stem cells, which should be understood fully to achieve optimal control and translational efficacy. PluriMes has advanced the state of the art, firstly by generating and expanding human PSC in a naïve, or ground state, condition (Partner 1), and secondly by trapping a mesoderm-lineage biased sub-state of PSC (Partners 7 and 2b). Both of these new PSC culture systems are based on specific manipulations of the cell signalling environment. The resulting cell states have been validated by global transcriptome analyses and by in vitro differentiation. These findings constitute original advances in basic PSC biology and fulfil our aim to provide new routes to exploitation in biomedical research, whether for disease modelling or cell therapy applications. To this end, Partner 11 has developed commercial media products for naïve PSC and in addition a cGMP compatible media formulation.
Objective (ii) encompasses both directed differentiation of PSC into mesodermal progenitors, and characterisation of in vivo post-natal progenitors, including the so-called MSCs. For the former, a challenging but ultimately productive technical endeavour has been the generation of PSC carrying various lineage-specific fluorescent protein reporters (Partners 6, 8 and 10). In addition, the potential for autologous therapy with gene-corrected cells was established by using CRISPR/Cas 9 technology to correct the mutated α-sarcoglycan gene in iPSC derived from patients with Limb-Girdle Muscular Dystrophy 2D that were then differentiated into mesangioblasts (Partners 6 and 2a). Guided and validated by the reporters, Partner 8 refined protocols for the differentiation of lateral plate mesoderm derivatives (cardiac muscle and endothelial cells), while Partner 10 described for the first time robust generation of skeletal muscle from PSC, a major advance. Partner 9 developed a microfluidic device for fine environmental control over PSC differentiation and derivative mesodermal progenitors, while partner 2a developed skeletal muscle organoids for measuring functional differentiation properties. Finally, as a resource for testing the in vivo regenerative potency of human mesodermal progenitors, Partner 3 created the first genetically modified immunodeficient mouse model of skeletal disease.
In a critical study to examine the biological existence of the MSC entity, Partner 3 employed stringent in vivo assays to measure differentiation properties of cell populations derived from different sources. Assays for osteogenesis, chondrogenesis, angiogenesis, myogenesis, and establishment of hematopoietic niches revealed strikingly divergent differentiation capabilities of peri- and postnatal progenitors isolated from bone, muscle, umbilical cord blood, and other tissues, or produced by differentiation of PSC. Cellular diversity according to source was independently confirmed by transcriptome analysis. These published results demonstrate that only the bone marrow contains a multipotent skeletal stem cell fraction and disprove the notion of common stem cells in different tissues. As discussed in several review articles, the findings confirm that the concept of MSCs as mesenchymal stem cells lacks any solid scientific foundation.
Objective (iii) comprises the translational component of Plurimes. Partner 4 developed robust processes for upscaling and manufacturing of PSC-derived cardiomyocytes and endothelial cells based on directed differentiation methodology developed by Partner 8. Partner 9 designed and successfully tested a synthetic microcarrier system for bioreactor expansion of mesodermal progenitors. GMP-compatible cryopreservation media for mesodermal progenitors have been optimised in a collaborative effort between Partners 5, 11, 2a and 4. Partner 11, working with Partners 2a, 4 and 12, has demonstrated animal-component free media expansion of mesodermal progenitors. Importantly, the requisite high quality raw materials have been identified and documentation put in place for a GMP-compliant Technology Transfer Package to enable GMP production of this media. Finally, Partners 12 and 6 developed a strategy to engineer mesangioblasts to deliver exon-skipping RNA and thereby enhance their potency for cell therapy in muscular dystrophy.
In addition, over 48 months PluriMes has:
• Funded nine exchanges through the PluriMes mobility programme
• Co-organised four European Summer Schools on Stem Cells & Regenerative Medicine
• Run seven training workshops plus two specialist training courses
• Run four full consortium meetings and two mini-consortium meetings
• Worked cooperatively with 6 other stem cell consortia; HuMen, Thymistem, Neurostemcellrepair, Intens, REVIVE and UKRMP, to run two major conferences, and to provide places in PluriMes training workshops
• Collaborated with EuroStemCell on various public engagement activities and developed new outreach activities.
The principal results from PluriMes are presented in 44 Deliverables and 15 Milestones. Research discoveries are disseminated in 37 publications in peer-reviewed journals and 4 book chapters. An additional 16 manuscripts are currently submitted or in preparation. The publications are Open Access and high 11 datasets have been deposited in public databases. Intellectual property is being protected in 3 patent filings and 2 trademarks.
a) Work Package 1: Human ground state pluripotency
Pluripotent stem cell biology is based on the paradigm of mouse embryonic stem cells which consistently show the ability to produce all cell types of the body. These mouse cells can be propagated in a stable uniform condition of naivety, known as the ground state. Human pluripotent stem cells (PSC) by contrast are heterogeneous and show variable propensity for differentiation to particular lineages. In this workpackage we first determined conditions for capturing human PSC in a naïve condition both by conversion of conventional PSC and by reprogramming of somatic cells. We have characterised extensively the molecular properties of human naïve PSC and have examined their genetic stability. We then developed protocols to capacitate the naïve PSC for differentiation and determined their potential for multilineage development in vitro. Finally, in collaboration between academic and industry partners we have developed and refined media, substrates and protocols for generation and maintenance of human naïve PSC. These products are being commercialised for the research community and conditions have also been determined that are compatible with GMP cell manufacturing for future clinical applications.
i) WP1 Objectives
• Objective 1: To generate ground state (naïve) human PSC by conversion of extant ESC and iPSC and directly from somatic cells.
• Objective 2: Characterisation of ground state PSC
• Objective 3: Development of reagents and protocols suitable for pre-clinical application
ii) WP1 Results
For Objective 1 Generation of ground state PSC we demonstrated that conventional human PSC can be reset to a naïve state 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. Partner 1 went on to establish conditions for resetting to the naïve state using small molecules, avoiding the use of transgenes, and evaluated chromosomal stability with Partner 7. Finally, we showed that human somatic cells could be reprogrammed directly to naïve PSC and also identified a unique cell surface marker to discriminate naïve and primed PSC.
For Objective 2 Characterisation of ground state PSC we have shown that the reset PSC described above have metabolic features and global transcriptome and DNA hypomethylation features expected for ground state cells that are distinct from conventional PSC. Multilineage differentiation ability has been demonstrated in vitro and in vivo, for both transgenic and chemically reset naïve cells and compared with the potency of naïve PSC derived from human embryos. In addition, Partner 7 provided information on characterisation of available antibodies for PSC characterisation, and Partner 1 employed CRISPR/Cas9 to achieve targeted gene mutation in naïve PSC. Finally, Partner 11 received cell lines and protocols from Partner 1 and developed improved culture media for resetting and maintaining human naive PSC. This has resulted in new commercial media products.
Towards Objective 3 Development of reagents and protocols suitable for pre-clinical application Partner 9 and Partner 1 collaborated on a preliminary screen to identify substrates and Partner 1 extended this to develop conditions that support feeder-free propagation of reset PSC. Partner 11 identified higher grade, defined and quality controlled raw materials suitable for a GMP-compatible media formulation and produced a GMP-compliant Technology Transfer Package. Partner 1 and Partner 7 evaluated chromosomal stability and reset clinical grade primed PSC to a naïve condition.
In summary, this workpackage has established and validated a new type of human PSC that displays the features anticipated for a ground state stem cell closely related to naïve pre-implantation epiblast, and has laid groundwork for potential future applications in biomedical settings.
iii) WP1 Outputs
This work package has resulted in the following outputs:
• 2 peer-reviewed research publications
• 3 manuscripts are currently in preparation
• 0 reviews
• 2 patents
• 2 other products
• 3 data sets published in public database resources
• 0 trademarks
b) Work Package 2: Lineage biased sub-states
The body is made up of many tissues and cell types. These are generated during human development from stem cells that divide and produce cells of many different types and functions. This process can be recapitulated in the laboratory using cultures of pluripotent stem cells which retain the capacity to produce all cells of the body. These stem cells could be used to produce cells that could then be used clinically to replace tissues lost through disease. However, the challenge is to control the process of stem cell differentiation so that we can efficiently produce the cell types and tissues we require. Our work suggests that cultures of pluripotent stem cells are heterogeneous, so that some cells may be biased to produce one tissue type, while others may tend to differentiate into other tissues types. We think that this is not fixed but that the stem cells may transition between these different preferences with time. To make tissue generation more reproducible and efficient we have identified different stem cell states with different tissue preferences. We have characterised these different states at the molecular level and also the mechanisms that control their behaviour. From this we have now developed a fully defined culture medium which traps the stem cells in one of these states from which they can efficiently be directed to generated mesodermal cell types such as muscle bone and cartilage.
i) WP2 Objectives
• Objective 1: Characterisation of PSC substates
• Objective 2: Identification of mesoderm-biased substates of hPSC
• Objective 3: Trapping mesoderm biased hPSC subsets
ii) WP2 Results
The proposition underlying WP2 is that hPSC can exist in multiple interconvertible substates within the stem cell compartment but when occupying different substates they exhibit different properties with respect to self-renewal and differentiation. In particular, different substates may be associated with a bias in the potential fate of the cells upon differentiation. The work of WP2 was focused upon confirming this hypothesis, identifying substates with a mesoderm bias and elucidating molecular mechanisms that control the dynamics with which stem cells may transition between substates. Our ultimate goal was to identify conditions that may trap cells in a mesoderm biased substate, and assess whether doing so may offer practical advantages for expansion and derivation of mesodermal derivatives for safe clinical applications.
Initially, we carried out an extensive screen of several approaches to identify and isolate hPSC occupying distinct substates within the undifferentiated stem cell compartment, including assessment of surface antigen patterns, metabolic state and reporter genes. Using these tools we confirmed that hPSC can occupy different substates in which the cells exhibit different patterns of gene expression, and propensities for differentiation, while retaining the capacity to proliferate as an undifferentiated stem cells. Building on these results and using a combination of a cell surface antigen (SSEA3) characteristically expressed by undifferentiated hPSC, and a fluorescent reporter gene that indicates expression of MIXL1, a transcription factor turned on during the early phases of mesoderm specification, we found that a small proportion of cells in hPSC cultures expressed both SSEA3 and MIXL1. By isolating cells with these characteristics (i.e. SSEA3+/MIXL1+ cells) we were able to show that they would not only proliferate as undifferentiated cells but also that they would tend to towards mesodermal derivatives when allowed to differentiate. Further, from single cell transcriptomic analysis of SSEA3+/MIXL1+ cells we found that regulatory genes characteristic of both undifferentiated hPSC and of mesodermal pathways of differentiation could be found co-resident in the same cell, consistent with the notion of intermediate metastable states. Similar results were found using a reporter for another mesoderm related gene, BRACHYURY.
Importantly, we then discovered that whereas we could find these mesoderm biased stem cells when hPSC were grown under culture conditions that included inactivated feeder cells, they disappeared when hPSC were grown under fully defined conditions. This allowed us to identify specific signalling molecules that promote the differentiation of hPSC to mesoderm during which the SSEA3+/MIXL1+ appeared transiently. By screening other molecules that tended to inhibit differentiation, we were able to identify a combination of pro-differentiation and anti-differentiation factors that cross-antagonised each other and trapped the cells in the SSEA3+/MIXL1+ state. A transcriptomic analysis of these trapped cells showed that they correspond to a state on the trajectory of hPSC as they differentiate towards mesodermal derivatives. Using these data we were able to develop an optimised, defined medium formulation, called Primo, in which it were able to maintain mesoderm-biased hPSC over 10 passages while the cells retained karyotypic normality. We are currently seeking patent protection for the formulation of Primo medium.
iii) WP2 Outputs
This work package has resulted in the following outputs:
• 3 peer-reviewed research publications
• In addition, 1 manuscript is in press, 2 manuscripts have been submitted and 1 manuscript is in preparation
• 0 reviews
• 0 datasets
• 1 patent is in preparation
• 0 other products
• 0 trademarks
c) Work Package 3: Genetic Tools
Human pluripotent stem cells (hPSCs) have the capacity to produce other cell types, including cells belonging to the mesodermal lineage, such as cardiac and skeletal muscle. Defined protocols that allow a reproducible and efficient differentiation of hPSCs to specialized cell types in the culture dish are still under development. In WP3 we introduced into hPSCs fluorescent markers which make the cells glow green or red when they become a different cell type. Those so-called reporter cell lines carry fluorescent proteins that are under the control of tissue-specific genes and mark distinct cell types that arise during differentiation to mesoderm. Up to date we generated in PluriMes more than 10 reporter lines marking different cell types. In addition, we produced reporter lines carrying two independent colors under the control of specific genes, making possible to visualize the transition between differentiation steps. The reporter lines will facilitate the development of reliable differentiation protocols by other members of the PluriMes Consortium.
Moreover, in WP3 we developed techniques for changing the genetic makeup of the hPSCs. One of the aims is to correct genetic mutations causing diseases such as muscular dystrophies and in collaboration with other PluriMes partners we succeeded in repairing the mutation in patient specific induced PSCs with Limb-girdle-muscular dystrophy. Another reason for changing the genetic makeup is to model diseases such as Duchenne muscular dystrophy in the culture dish. The other PluriMes partners will use the modified cells to perform screens for therapeutic reagents.
i) WP3 Objectives
• Objective 1: Generation of PSC reporter lines
• Objective 2: Forward programming by inducible TFs
• Objective 3: Correction of mutations by gene targeting.
ii) WP3 Results
In Objective 1 we have generated a series of transposition compatible BAC vectors and targeting constructs carrying fluorescent proteins (FP; Venus, Cherry or TagBFP) under the control of mesodermal specific genes using recombineering technology. The reporters will facilitate the detection by live imaging and isolation by flow cytometry of relevant cell states during in vitro differentiation of hPSCs. This approach will be applied to analyse and optimise the mesodermal/mesenchymal trajectories in collaboration with Partners 7, 8 and 10. The reporters are specific for the pluripotent state (OCT4), presomitic mesoderm (MSGN1, BRACHYURY), paraxial mesoderm (MEOX1; PAX3, PAX7 for somatic skeletal muscle and PAX1, PAX9 for axial skeleton), lateral plate mesoderm (COUPTFII, PRRX1) and vascular derivatives (FOXC2). In summary, we generated 8 lines by BAC transposition, 10 single reporter lines by gene targeting and 4 dual reporter lines. Generation of reporter lines by gene targeting using CRISPR technology turned out to be more reliable for the expression of the fluorescent protein as compared to lines generated by BAC-transposition. Finally, the reagents that we have generated in PluriMes (targeting vectors, gRNA-expression vectors, hybridization probes) and protocols for gene-targeting and analysis of the clones can be easily applied to other human cell lines.
In Objective 2 we have generated a transposition compatible vector that allows the easy insertion of transcription factors by Gateway reaction under the control of a tetracycline inducible promoter. We cloned the cDNA of Mesogenin (MSGN1) together with the fluorescent protein mCherry. Induction of MSGN1 expression by Doxycycline administration in stable colonies resulted to changes in cell morphology already after 24 hours. The cells lost their epithelial morphology and acquired a mesenchymal phenotype, downregulated pluripotency markers (OCT4, NANOG) and upregulated epithelia to mesenchymal transition markers (SNAIL1, 2) and other mesodermal genes (MEOX1, PRRX1). The above results prove that it is possible to change cell fate by overexpression of specific transcription factors. For resetting hPSCs to the ground state of pluripotency we generated a vector for inducible expression of NANOG, KLF2 and mCherry. Stable NCRM1 hiPSCs formed dome shaped colonies and proliferated under culture conditions that support the ground state of mESC pluripotency in the presence of Doxycycline, whereas uninduced colonies could not be propagated under those conditions. Altogether, the functionality of the inducible vector was demonstrated in two different applications: a) forward programming and b) resetting to ground state.
In Objective 3 we have generated vectors for correcting mutations in the α-sarcoglycan gene (SGCA) in iPSCs derived from patients with Limb-Girdle Muscular Dystrophy 2D (LGM2D). In one of the two available LGMD2D iPSCs lines characterized by Partner 2a the mutation is a homozygous substitution (C>T) in exon 3, implying that correction in only one allele is sufficient to revert the disease phenotype. For correcting the mutation we used two types of targeting vectors: a) BAC based vectors, with one short and one very long homology arm and b) Cas9 compatible vectors with short homology arms. We successfully targeted the locus in patient iPSCs and corrected the mutation in one allele with a frequency varying between 5.5% and 31% and verified by sequencing. Targeting frequency was higher with CRISPR assisted targeting, however many colonies carried random integrations of the targeting vector in addition to the targeted event. Altogether, this study outlines a step towards autologous cell therapy in LGMD2D patients
iii) WP3 Outputs
This work package has resulted in the following outputs:
• 2 peer-reviewed research publications
• No additional manuscripts are currently under review or in preparation
• 0 datasets
• 0 reviews
• 0 patents
• 0 other products
• 0 trademarks
d) Work Package 4: Lineage specification for paraxial and lateral mesoderm
Human pluripotent stem cells can form any type of cell in the body but for some purposes it is very useful to be able to direct their differentiation to specialized cell types of interest. In Plurimes, the aim is to form cells of the blood vessels and various types of muscle. These are known to originate from different types of mesoderm in normal embryonic development. Our goal in this work package was to generate a set of cells lines that could “report” on the basis of expression of different colours where they are in their differentiation trajectory. To create these so-called transgenic human pluripotent cell lines we used gene editing to express green, red, yellow or blue fluorescent proteins in each different type of mesoderm. This allowed us to form endothelial and smooth muscle cells of the blood vessels very efficiently as well as heart and skeletal muscle. In addition, by separating populations of cells expressing several colours into cells expressing just one we were able to purify these cell types and identify proteins on the cell surface specific for each of the cells. This allowed us later to separate cells from pluripotent cells which did not express the coloured reporters. In this way we were able to refine and improve differentiation protocols so that they now contain no animal reagents (in principle applicable for further development in clinical applications) in a process that is applicable and efficient in multiple different pluripotent stem cell lines. This has been one of the bottlenecks in moving the field forward: how to differentiate all cell lines efficiently using similar protocols.
i) WP4 Objectives
• Objective 1: Defined conditions for differentiation of hPSC to lateral plate (LPM) and paraxial mesoderm (PM)
• Objective 2: Transcriptional profiling to identify surface markers, signalling pathways, transcriptional regulators and micro-environmental stimuli that promote mesoderm specification, patterning and expansion.
• Objective 3: Functional validation of regulatory pathways and surface proteins.
ii) WP4 Results
In this work package we aimed to define human PSC differentiation conditions for efficient derivation of early mesoderm and subsequently specific mesodermal cell types like heart cells, blood vessels and skeletal muscle relevant to tissue repair and regeneration. To make these different cell types, it is important that the early mesoderm that first forms as the hPSCs leave the pluripotent state receives the right signals to programme the later differentiation into the tissue types of interest. The work showed this is best done using defined differentiation conditions for the induction of lateral plate mesoderm (LPM) which gives rise to cells such as cardiomyocytes, endothelial cells, pericytes/vascular smooth muscle cells (vSMCs), and paraxial mesoderm (PM) which gives rise to skeletal and satellite muscle cells, cartilage and bone. Transgenic hPSC lines that become fluorescent through expression of lineage reporters generated in WP3 were used as an easy method to monitor differentiation and to determine exactly which time points were best for growth factor addition to result in the most efficient differentiation. The Partners thus developed culture conditions for the maintenance and expansion of clonal progenitors and the directed differentiation of presomitic mesoderm into skeletal muscle and satellite cells within 30 days. Functionally, these derivatives were examined using in vitro and in vivo assays in WP5.
The transgenic reporter lines were also exploited to obtain gene signatures for isolated cells at different differentiation stages of human mesoderm formation. Partners also identified sets of surface markers that can be used for the enrichment of mesoderm-derived endothelial cells and cardiomyocytes. The most significant findings were the development of methods to form different endothelial cell subtypes from hPSC. Each tissue and organ has its own type of endothelial cells and through the knowledge we developed on mesodermal patterning we were able to generate heart specific endothelial cells. When grown together with cardiomyocytes that had been selected from mixed differentiated cell populations using cell type specific surface markers we had identified in the study, we formed cardiac microtissues that were representative of human myocardium. In a similar way, close mimics of human skeletal muscle were developed from hPSC. The IP was licensed to one of the industrial partners (Pluriomics).
The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of (human) cardiomyocytes and vascular cells. We therefore established conditions for clonal expansion of cardiovascular progenitors (CPCs) (Birket et al. Nat Biotech 2015) In WP4, we used hPSCs, in which CPCs are genetically marked, to show that regulated MYC expression enables their robust expansion. These CPCs could be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes with distinctive physiological characteristics. The cells were clonogenic and could be expanded over 40 population doublings yet still be directed to differentiate to cardiomyocytes as well as vascular cells Together, this tractable model reveals the potential of hPSC-derived CPCs for precisely recreating elements of heart development in vitro.
Finally, we further confirmed functionality of ECs and cardiomyocytes (Giacomelli et al. Development 2017). The data following transplantation of these cells in zebrafish embryos was reported in Orlova et al, Nat Protocols, 2014). Additional transplantation data in chick embryos was also reported i(Guadix et al, Stem Cell Reports 2017). Various (combinations of) cell surface markers from transcriptomics analysis and other data now allow sorting of early mesoderm populations.
iii) WP4 Outputs
This work package has resulted in the following outputs:
• 7 peer-reviewed research publications (plus 1 pre-print)
• In addition 1 manuscript is in press and 1 manuscript is in preparation
• 0 reviews
• 0 patents
• 1 data set has been deposited in a public database resource. 2 other datasets will be deposited in a public database resource.
• 0 other products
• 0 trademarks
e) Work Package 5: Comparative assessment of biological activity and therapeutic potential.
The use of induced pluripotent stem cells (iPSCs) to regenerate damaged human tissues relies on a) complete knowledge of the characteristics and functions of natural stem/progenitor cells included in the human body b) perfect reproduction of those characteristics and functions in progenitor cells derived from iPSCs c) rigorous demonstration that progenitor cells obtained from iPSCs can effectively regenerate tissues in suitable animal models. We have demonstrated that stem/progenitor cells isolated from different human tissues such as, for example, skeletal muscle and bone, are able to regenerate only the tissue in which they reside, in contrast with the popular hypothesis that stem cells with identical functions (so called Mesenchymal Stem Cells) can be isolated from different sites of the human body. We have shown that, in many human tissues, stem/progenitor cells are located in the blood vessel wall and have identified some of the mechanisms regulating their behaviour and function at that site. We have generated and tested different cell types derived from iPSCs, such as endothelial and muscle cells, demonstrating their functionality in appropriate experimental conditions. Furthermore, we have started to explore the possibility of using iPSCs for regenerating specific components of the skeleton. All these results have been obtained through the development and application of multiple experimental models that represent powerful tools for future studies. Finally, we have tested different nutrient media for natural and iPSCs derived progenitor cells that do not contain any factor of animal origin and therefore do not carry any potential risk for human patients.
i) WP5 Objectives
• Objective 1: Differentiation potential of pre-, peri- and postnatal progenitors of mesoderm-derived tissues.
• Objective 2: Transcriptome of preogenitors of mesoderm-derived tissues
• Objective 3: Microvascular niche of mesoderm progenitors.
ii) WP5 Results
Objectives 1 and 2. We have developed tools and knowledge for the assessment of the effective regenerative potency of natural and hPSC-derived mesodermal progenitors.
We have established/refined multiple, rigorous cell differentiation assays based on heterotopic/orthotopic transplantation in the mouse. Our assays have demonstrated that progenitor cells isolated from different human post- and peri-natal mesodermal tissues represent distinct classes of tissue-specific progenitors rather than a uniform class of “mesenchymal stem cells”. The functional diversity observed in vivo based on the tissue origin of the cells, has been further validated by transcriptome analysis. These results are in contrast with the widely held concept of “mesenchymal stem cells” and may have a great impact on translational research focused on mesodermal tissue regeneration.
We have generated different hPSC-derived cell types, including hPSC-derived endothelial cells, hPSC-derived pericytes and hPSC-derived myogenic cells. The functional competence of all these cell populations has been demonstrated by transplantation in the developing Zebrafish and/or in the mouse and the expression of specific genes of interest has been confirmed by transcriptome analysis. We have also developed multiple populations of mesodermal progenitors derived from hPSCs through intermediate “mesenchymal” stages (hPSC-derived mesodermal/”mesenchymal” stem cells). In agreement with the results obtained with post-natal cells, we have observed that the expression of the “MSC” phenotype in hPSC-derived cells in vitro is not predictive of the ability to regenerate tissues in vivo, e.g. to form bone. We have also begun to develop new platforms for the generation of human muscle organoids, by using post-natal and hPSC-derived cells, and isogenic artificial muscle constructs containing myogenic cells, endothelial cells and pericytes derived from the same iPSCs. These constructs provide valuable experimental systems for regenerative medicine and drug development.
The gold standard in assessing the potential of mesodermal progenitors for clinical intervention is in vivo transplantation in animal models reproducing tissue disease/injury. Therefore, we have converted some of our in vivo experimental assays in the mouse into specific models of therapeutic intervention for skeletal and muscle disease. To this aim, we have developed the first model of human skeletal disease in immunocompromised mice (Fibrous dysplasia/SCID mice) thus providing a novel experimental tool in the skeletal field. Finally, since the clinical application of any progenitor cell type relies on the identification of suitable conditions for cells isolation/expansion, we have identified candidate chemically defined, animal-component-free medium formulations that can potentially be used for human natural and hPSC-derived mesodermal progenitors under GMP-compliant protocols.
Objective 3. Evidence has been provided that mesodermal progenitors in post-natal tissues are incorporated as pericytes in the blood vessel wall and that their differentiation/function at that site is regulated through molecular gradients. For this reason, we have developed cutting edge microfluidic systems that allow to reproduce and to investigate the molecular interaction of natural and hPSC-derived pericytes with the microvascular niche.
iii) WP5 Outputs
This work package has resulted in the following outputs:
• 8 peer-reviewed research publications
• 4 reviews
• 2 book chapters
• In addition there are 2 manuscripts in revision, 3 manuscripts in preparation and 4 published conference abstracts.
• 2 data sets have been deposited in a public database resource. 3 other datasets will be deposited in a public database resource.
• 0 patents
• 0 other products
• 0 trademarks
f) Work Package 6: Bio-processing and scale-up
Work Package 6 (WP6) is intended to lay the foundation for bio-processing and scale-up of the cell types relevant for the general aim of the Plurimes consortium. This comprises the production and validation of highly defined culture and cryopreservation conditions, as well as the development of strategies for the larger-scale production of clinically relevant cell types. New cell culture media formulations for the expansion and cryopreservation of several types of mesodermal progenitor cells were successfully developed, showing superior characteristics compared to the state-of-the-art. In parallel, bioengineers devised powerful biomaterials-based strategies for scalable expansion of the cells in bioreactors. To this end, synthetic hydrogels were formulated as micron-sized beads (‘microcarriers’) such as to expand the cells on their surface in suspension.
i) WP6 Objectives
• Objective 1: Development of GMP-compatible cryopreservation protocols
• Objective 2: Development of GMP-compatible expansion protocols
• Objective 3: Development of scale-up processes for large-scale production of cells in bioreactors.
ii) WP6 Results
In this work package, we aimed at developing Good Manufacturing Practice (GMP)-compatible protocols for the efficient and reliable cryopreservation, as well as the large-scale expansion of human mesodermal progenitors and pluripotent stem cells (hPSC) and their derivatives used in the PluriMes project.
Three cryopreservation medium variants containing GMP-compatible components were developed and tested for their effect on post-thaw cell viability, cell recovery and the re-establishment of successful cultures. One of the media, CryoStor® CS10, showed very good results for three sources of mesodermal progenitors (hPSC-derived MPCs, human mesoangioblasts, iPS- and ES cell-derived inducible myogenic progenitors) and human iPSC-derived vascular smooth muscle cells. All cell types stored for at least 6 months in CryoStor® CS10 maintained high cell viability, marker expression and morphology post thaw and culture.
Recent efforts by one of the PluriMes labs (A. Smith) have demonstrated that hPSCs can be modified, via small molecules and/or the exogenous expression of transcription factors, to adopt a state approximating the naïve state of murine pluripotent stem cells. A new commercial medium was developed to induce and maintain these naïve-like, or ‘reset’, hPSCs from primed hPSCs using a transgene-free or chemical-induction method. Human naïve reset PSCs generated and expanded using these media exhibited proper colony morphologies, expression of key naïve-associated genes and tri-lineage differentiation potential. In addition, an hPSC expansion medium containing high-quality components was developed and found to result in improved growth rates of multiple hPSC lines and compatibility with feeder-independent hPSC maintenance cultures.
Further, enhanced manufacturing protocols, relevant quality control and processes documentation, as well as key relationships with Contract Manufacturing Organizations (CMOs) manufacturing under cGMP compliance were successfully established. A “GMP-compliant Technology Transfer Package” detailing all required documentation and instructions to initiate the transfer of MesenCult™-ACF, a commercial research grade medium for the optimal derivation and expansion of Mesenchymal Progenitor Cells (MPCs), to a Contract Manufacturer Organization (CMO) for GMP-manufacturing.
Towards manufacturing cells in large-scale bioreactors, a chemically defined microcarrier system was developed and demonstrated successful expansion and maintenance of mesoangioblasts and iPSC-derived mesodermal progenitors. Compared to the gold standard commercially available microcarrier, the synthetic version showed improved cell detachment after bioreactor culture, overcoming a significant problem in the field. Finally, we successfully developed protocols for the microcarrier-free expansion of naïve and lineage-biased PSCs as cellular aggregates.
iii) WP6 Outputs
This work package has resulted in the following outputs:
• 5 peer-reviewed research publications
• 1 review
• 1 published conference abstract
• 1 book chapter
• 2 additional manuscript is currently in preparation
• 0 patents
• 0 datasets
• 8 other products
• 0 trademarks
g) Work Package 7: Dissemination and Training
PluriMes aimed to promote and strengthen the general field of stem cell research and regenerative medicine in Europe. This has been achieved most effectively through coordinated activities with other European stem cell projects.
The extraordinarily high levels of interest and expectation make it crucial that accurate information on advances and challenges in stem cell research is widely disseminated. The PluriMes philosophy emphasised pro-active approaches to training and communication, for both academic groups and lay audiences.
i) WP7 Objectives
• Objective 1: Project Website
• Objective 2: Research Training, Dissemination and Communication
• Objective 3: Public Engagement and Outreach
ii) WP7 Results
Objective 1: Project Website
In 2014 the PluriMes Project launched a website which had both a public section as well as a secure members-only area (intranet). The public website has served as a platform for external communication. The intranet was designed to facilitate dissemination of information and communication between partners and also assisted with the general day-to-day management of the consortium. The website has had 8407 users, with 23,407 page views, over the duration of the project. In April 2018 the PluriMes website will be transferred to a hosting platform that can be maintained for 10 years after the project to ensure continued dissemination of project information.
Objective 2: Research Training, Dissemination and Communication
PluriMes placed a major emphasis on developing skills of stem cell researchers at all career stages. To accomplish this goal PluriMes put in place a training programme to provide rigorous high-level theoretical and practical training which would maximise synergy and interaction within the consortium whilst developing common standards of experimental practice and theoretical understanding.
By drawing on the complementary expertise of participating Institutions and like-minded consortia we established a cohort of European scientists who are trained to the highest standards in the state-of-the-art. This will serve to promote and strengthen the general field of stem cell research and regenerative medicine in Europe as well as provide a solid base for future developments in the field.
Our Training Programme comprised of three specific schemes:
1) A Mobility Programme
This programme enabled individuals to travel to members/non-members labs to test a specific technique, learn about a particular aspect of the project or conduct a collaborative experiment. This enhanced the collaborative nature of the project, raised the skills base and competitiveness of the junior team members and facilitated transfer of key technologies and comparative data analysis. During the Project nine exchanges took place including one SME-academic exchange and two external exchanges with another FP7 stem cell project; BioDesign.
2) Summer School on Stem Cells and Regenerative Medicine
The summer school is primarily aimed at advanced PhD students and early stage post-doctoral scientists. This training course has been run for the past 8 years supported by FP6 and FP7 projects and has provided advanced training to over 400 young researchers. The course is led by stem cell experts who share their experience in many facets of research and is a mixture of lectures, workshops, poster and discussion sessions. During the Project PluriMes collaborated with five other stem cell projects (ThymiStem, Neurostemcellrepair, HumEn, Intens and EuroStemCell) to provide core financial support for 4 Summer Schools (2014, 2015, 2016 and 2017). In total PluriMes sent 29 lab members to be trained during the lifetime of the project.
3) Workshops and Training Courses
PluriMes organised advanced training courses/workshops which were primarily for PluriMes members but with some places made available for external scientists. Specific workshop/training topics were developed throughout the project dependent on project members’ needs but PluriMes also joined forces with other like-minded consortia to run joint workshops on topics of mutual interest. During the Project PluriMes ran 8 workshops and 2 specialist training courses:
4) Dissemination Activities
PluriMes recognised that the high levels of interest and expectation about stem cells made it crucial that accurate information on advances and challenges in stem cell research be widely disseminated. The Plurimes philosophy emphasised pro-active approaches for communication to both academic groups and lay audiences. The project was committed to an active programme of dissemination activities which had maximum scientific reach and visibility and ensured effective and wide-spread dissemination of the project results. Accordingly PluriMes secured constructive relationships with other stem cell projects (Thymistem, HumEn, NeuroStemCellRepair, Intens, REVIVE, PSCP and EuroStemCell).
Our Dissemination strategy comprised of a number of avenues which were all actively employed:
I) External Dissemination
• Project Website
• Public Engagement
o Collaboration with EuroStemCell
o Outreach Champions for each Partner
o Outreach activities by partners
o Outreach materials to inform and engage the lay public including educational and role play materials for school children and college students
o Media Communication; News features for broadcast media such as euronews
o Contribution to, and endorsement of, books on stem cell biology for non-specialist audiences,
o Accurate information on clinical trials using (or claiming to use) pluripotent stem cells or mesenchymal stem cells.”
• Publication of peer-reviewed scientific publications in leading international journals
o Publication in Open Access journals or author deposition in repositories such as PubMed
o Data deposition in public database resources
• Presentations at European Symposia and International Conferences
• Production and release of new culture media and other products for stem cell research
• Dialogue/information/briefings for policy-makers, regulators, bioindustry, relevant clinicians and administrators
• EMBO Conference: Advances in Stem Cells and Regenerative Medicine
II) Internal Project Dissemination
• The Project Training Programme
• Annual Consortium Meetings
• Project intranet
5) Coordination with Other Stem Cell Consortia
Throughout the project PluriMes has built links with other stem cell projects across Europe as well as two other stem cell consortia (REVIVE and PSCP). PluriMes, ThymiStem, NeurostemcellRepair, HumEn , Intens and EuroStemCell have worked together on a number of coordinated activities including workshops and conferences. The LabEx consortium REVIVE has built close ties with the HEALTH-funded consortia and has been actively involved in a number of events. The PSCP has run one joint event with PluriMes.
6) Advances in Stem Cells and Regenerative Medicine Conference
The ‘Advances in Stem Cell Research’ conference series was originally pioneered by the FP6 Project EuroStemCell in 2007. The FP7 EuroSyStem Project then continued that series from 2009 to 2012. In 2017 the PluriMes Project took the leading role in organising and arranging the scientific programme for the next in the ‘Advances in Stem Cell Research’ conference series. For the latest in the series two major conference series (Advances in Stem Cell Research and Stem Cells in Cancer and Regenerative Medicine) merged to create the 2017 conference titled “Advances in Stem Cells and Regenerative Medicine”. This conference was held from 23rd to 26th May 2017 at the EMBL in Heidelberg, Germany. The event was supported by FP7 projects PluriMes and ThymiStem as well as the REVIVE Project and EMBO. Over 300 people participated in the conference.
The rationale for merging the two conference series was that stem cell research did not have a dedicated European conference series. The only equivalent is the annual Keystone Symposium on Stem Cells or the International Society for Stem Cell Research (ISSCR) Meeting, which although they are not always held in the US, are overwhelmingly US-dominated. If Europe is to remain competitive, European researchers need an opportunity to build and expand networks. This conference series aims to provide that opportunity by bringing together the European stem cell community and presenting it in a rigorous and dynamic format to the International stem cell community.
7) European Summer School on Stem Cell Biology
PluriMes, in collaboration with ThymiStem, Neurostemcellrepair, HumEn and Intens, has co-hosted the summer school each year since 2014. ThymiStem provided the administrative support for the event, with core funding being provided by the five stem cell research consortia.
These courses, which have been aimed primarily at pre- and early-stage post-doctoral scientists, have provided intensive theoretical training in key issues in fundamental stem cell biology. Emphasis was placed on human stem cells, and on future application of these disciplines. To ensure the students gained an appreciation of the broader context for the field, sessions on translational stem cell research; ethics; commercialisation; clinical application and science communication were included in the programmes. Each years’ faculty was drawn from the five stem cell consortia, and the wider European stem cell community, including basic scientists, clinicians, ethicists, regulators and biotechnologists. Plenary sessions and keynote discussions featured leading international speakers.
Each year, a maximum of 54 places have been offered on these 7-day schools. Participants from the five consortia funding the school were capped at 50%, and the remaining places advertised by open call. Each PluriMes partner was given the opportunity to send two lab members to the summer school across the lifetime of the PluriMes project. Over the four years of the PluriMes Project 29 PluriMes lab members, from 11 laboratories, have benefitted from the school. Over 220 junior researchers have been trained over the course of the four schools co-funded by PluriMes.
Objective 3: Public Engagement and Outreach
PluriMes laboratories were asked to nominate Outreach Champions who would lead each Institution’s contributions to the PluriMes public engagement activities. In 2014 lab members and PIs received training rom EuroStemCell on public engagement and many subsequently undertook public engagement activities on behalf of PluriMes and their host organization. During PluriMes 56 public engagement events/activities have taken place which have been led by PluriMes members. These events have taken place throughout Europe and in a variety of languages.
The PluriMes project developed three activities and some materials which were suitable for use with all age ranges, could easily be used in any language, are low cost and very easy to run. These activities were made available to anyone from PluriMes labs which removed a traditional barrier to doing public engagement. The PluriMes public activities are as follows.
• Activity One: Play-doh is used to show the public how pluripotent stem cells can make any type of the specialised cell.
• Activity Two: Coloured M&Ms are used to explain how specialised cells can be return to a naïve state using a method called reprogramming.
• Activity Three: Dylan Stavish, a lab member from the USFD, created a dance mat game which aims to introduce the public to Fluorescent Activated Cell Sorting (FACS). In Dylan’s game ‘cells’ fall from the top of the screen to ‘beakers’ at the bottom. When the cells reach the beaker the player needs to step onto the corresponding pad on a dance mat to ‘sort’ that cell. The game is played to music and the cells fall to the beat. The game will calculate how many cells the player sorts and how many cells the player misses and calculate a sorting efficiency at the end.
• Other Materials: The PluriMes Project Office worked with various lab members to create 2 banners for use at public events. One explains about the PluriMes project and the other about stem cells in general. These could be sent to any project partner for use at their own local events.
iii) WP7 Outputs
This work package has resulted in the following outputs:
• 0 peer-reviewed research publications
• 0 reviews
• 0 patents
• 4 other products
o PluriMes website: www.pluriMes.eu
o Dance mat game on cell sorting – Public Engagement Activity
o Play-doh stem cell – Public Engagement Activity
o M&M cell washing - Public Engagement Activity
• 0 trademarks
h) Work Package 8: Management
WP8 provided the structure for all management activities related to the consortium. The Project Office consisted of a Project Manager, Jenny Nelder, and a Project Administrative Assistant, Charlotte Butler.
The Project Office was responsible for the preparation and execution of all project meetings. The project has held a wide range of regular meetings including Work Package, Consortium, Technology oversight and General Committee meetings.
Under WP8 the Project Office also managed the project reporting though the submission of sub-deliverable, deliverable and milestone reports and periodic reports. The project progress was monitored through Situation Analysis Tables (SATs) that were regularly updated and circulated to all project partners.
The financial administration of the project was carried out by the Project Manager in collaboration with the University of Cambridge Research Operations Office and other beneficiary financial contacts. Funding was distributed to beneficiaries at the correct times, appropriate financial records were collected and retained and the relevant audits were undertaken.
Following signature of the consortium agreement and grant agreement there were two formal contract amendments made in consultation with the EC Officer. The Project also held three Technology Oversight Committee meetings to ensure that IPR arising from the project was appropriately commercialised.
In addition to the major project tasks the Project Office managed the general day-to-day project administrative tasks.
i) WP8 Objectives
• Objective 1: Project Implementation Meetings
• Objective 2: Project Reporting
• Objective 3: Financial Administration
• Objective 4: Administration of Legal Issues
• Objective 5: Management of the Project
ii) WP8 Results
Period 1 was very productive:
• The ‘Kick-off’ meeting was held in February 2014 in Paris, France, during which robust scientific working practices and management methodology for the Project were discussed and agreed by all partners.
• The PluriMes website was launched on 16th May 2014.
• The PluriMes Summer Workshop 2014 was held in July 2014 in Bled, Slovenia, during which PIs and lab members began networking and creating scientific collaborations. This also presented an opportunity to fully introduce the project to all lab members which they have all found very useful. PluriMes also collaborated with EuroStemCell to run public engagement training for all PIs and lab members.
• PluriMes’ First Annual Consortium Meeting took place in February 2015 in Kranjska Gora, Slovenia. The EC Officer for PluriMes attended for the first day and an informal review of the projects progress so far took place. The SAP were also present at the meeting and reviewed the progress made so far.
• The General Committee met 6 times
• The TOC met once
• 5 Deliverables were submitted to the EC
• 3 Milestones were submitted to the EC
• 25 Sub-deliverable reports were submitted to the Project Office.
• One Contract Amendment was executed.
• All ethical documentation and cell line registrations were processed as required.
In Period 2 – Months 19-36
• The Project has held four workshops, two of which have been followed by mini-consortium meetings:
o “Tissue engineering through stem cell-based self-organisation” – Cascais, Portugal. 22nd and 23rd September 2015 (included mini-consortium meeting)
o “CRISPR/Cas9” workshop at the 2nd Annual Consortium Meeting – Zell am See, Austria. 9th February 2015
o “Single cell RNA-seq” – Nice, France. 6th and 7th October 2016. (included mini-consortium meeting)
o “Stem cell culture: culture conditions and stability workshop” – Cambridge, UK. 28th and 29th November 2016.
• PluriMes Second Annual Consortium Meeting took place from 8th to 10th February 2016 in Zell am See, Austria. The SAP were present and reviewed the progress since the previous consortium meeting.
• The General Committee met 4 times
• The TOC met once.
• 15 Deliverables were submitted to the EC
• 10 Milestones were submitted to the EC
• 50 sub-deliverable reports have been submitted to the Project Office.
• One Contract Amendment was executed.
• All ethical documentation and cell line registrations were processed as required.
In Period 3 – Months 37-48
• The Project has held 2 workshops/training courses:
o “Genetic stability in human pluripotent stem cells” – Laax, Switzerland. 6th and 7th February 2017.
o SCT specialist workshop – Cambridge, UK. 10th and 11th October 2017.
• PluriMes Third Annual Consortium Meeting took place from 6th to 9th February 2016 in Laax, Switzerland. The SAP were present and reviewed the progress since the previous consortium meeting.
• The General Committee met twice
• The TOC met once.
• 24 Deliverables were submitted to the EC
• 2 Milestones were submitted to the EC
• 32 sub-deliverable reports have been submitted to the Project Office.
• A fourth Consortium Meeting/Wash-up meeting took place from 24th to 26th January 2018 at Cliveden House, London, UK. The SAP attended and reviewed the outcomes and potential impacts of the project.
• In July 2017 PluriMes underwent an internal EC ethics audit. All ethical documentation and paperwork required was submitted to the EC. No further action was requested by the EC.
iii) WP8 Outputs
This work package has resulted in the following outputs:
• 0 peer-reviewed research publications
• 0 reviews
• 0 patents
• 0 other products
• 0 trademarks
• An additional 0 manuscripts are currently under review or in preparation
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 and on future biomedical and clinical applications. The outcomes include new and advanced knowledge, improved technology platforms, standardised protocols compatible with GMP cell manufacturing, and a coherent body of highly skilled stem cell investigators.
The overall impact of PluriMes is in defining cellular parameters for therapeutic relevance and thereby setting the stage for future clinical implementation. This has been achieved by addressing key scientific and translational challenges:
• defining the expansion and differentiation potential of naïve ground state and lineage-biased sub-states of pluripotent stem cells
• establishing resources and platforms for directed differentiation and expansion of mesodermal stem/progenitor cells from renewable PSC resources;
• clarifying the lack of common identity between different classes of tissue-derived mesodermal progenitors (so-called MSCs);
• validating differentiation potency in animal models.
• defining culture conditions and high quality reagents suitable for future scale up and GMP cell production
Specific areas of impact are outlined below:
(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 range of musculoskeletal diseases.
(b) Clinical Trials
Two of the PluriMes Deliverables have direct significance for clinical trials:
• D5.5: In Vitro assessment of therapeutic potential of mesoangioblast in restoring dystrophin production. The overall outcome of this work will allow Partner 12 to proceed to a novel Phase II clinical trial, based upon intra-arterials systematic delivery of autologous, genetically corrected mesoangioblasts in very young patients with an optimized protocol.
• D6.4: Final media formulations and reagents manufactured in a cGMP facility for the scalable expansion of mesoangioblasts and selected iPSC-derived mesodermal progenitors. The outcome of this work is that the MHRA expressed an informal favourable opinion on the used of Myocult-SF in ongoing and future trials of cell therapy for Duchenne muscular dystrophy. Following this, Partner 12 will replace the previous “gold” standard medium, M5, with MyoCultTM- SF for future clinical trials.
(c) Knowledge to Harness the Potential of Human Pluripotent Stem cells
PluriMes addressed general hurdles associated with the fundamental character of pluripotent stem cells. New knowledge of the human ground state and of pluripotent sub-states provides a rational framework for complementary approaches to achieve optimal directed differentiation for potential disease modelling and clinical applications.
(d) New Technologies
The technologies developed and exemplified make available new ways to analyse and control stem cells, such as bioengineered stem cell culture methods to enable precision application of extrinsic factors to influence cell fate, or techniques that facilitate scale-up of mesodermal progenitors while minimising genetic variation. Such technologies offer new opportunities for commercialisation and application. Thus the naïve human pluripotent stem cell medium developed by Partner 1 has been licensed by Partner 11 and developed into a commercial product and also validated for cGMP production.
(e) Regulatory Standards
PluriMes considered regulatory issues in cell therapy, 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.
(f) Bioindustry
PluriMes includes two SME partners. Their involvement realised impacts for employment, competitiveness of the technological R&D base, and wealth creation in Europe. Notably, SCT UK located research activity related to PluriMes in a new facility in Cambridge.
(g) Skills and Training
PluriMes placed a major emphasis on developing skills of junior researchers and promoted this through laboratory exchanges, specialist workshops and active participation in consortium meetings.
(h) Employment.
In July 2014, prior to PluriMes project, Stemcell UK had 11 employees (no R&D staff). As of January 2018, Stemcell UK has 44 members (Sales, R&D, and support functions). The 5 employees, representing 4 FTEs funded by PluriMes Grant will remain in the Stemcell UK R&D team. Following PluriMes success, Stemcell UK is contributing to other EU Grants, having at January 2018 a total of 9 R&D employees
(h) Consolidation of the European Regenerative Medicine Community
A broad impact aimed at by PluriMes was to promote and strengthen the general field of stem cell research in Europe. This has been achieved by coordinating very effectively with related FP7-HEALTH projects and other stem cell groupings to run joint activities, such as the Hydra Summer School in Stem Cell Biology and Regenerative Medicine, and the Advances in Stem Cell Research Conference.
(i) Public Communication and Engagement
PluriMes worked with the FP7 coordination action EuroStemCell (www.eurostemcell.org) to ensure dissemination of accurate information in accessible formats to a diversity of academic, administrative and public audiences.
Dissemination Activities
In addition to open access publications and data deposition in public archives, PluriMes undertook a range of dissemination activities:
(a) Making project knowledge and information accessible to both specialist and public audiences via the worldwide web:
• PluriMes Project website: www.plurimes.eu
• EuroStemCell portal: www.eurostemcell.org
(b) Increasing scientific knowledge, interaction, training and communication within and beyond the consortium.
• 7 workshops
• 2 specialist training courses
• 4 consortium meetings
• 9 mobility exchanges between scientific laboratories
• 15 joint activities with other consortia
• 290 presentations at European Symposia & international conferences
• 17 instances of dialogue with policy-makers, bioindustry, regulators, relevant clinicians and administrators
• 3 contributions to books on stem cell biology for specialist/non-specialist audiences
• 7 media communications about the project
Communicating effectively to a range of stakeholders and public through outreach activities.
• 61 public engagement events throughout Europe
• creation of 3 new public engagement resources
• 51 PluriMes lab members trained in public engagement
Exploitation of results: production and release of new culture media and other products for stem cell research
In 2014 - 2015, SCT/SCTUK developed and launched several new culture media into the market including MesenCult™-ACF, a novel ACF culture medium and matrix system for deriving and expanding mesodermal progenitors from primary bone marrow without the use of serum; and STEMdiff™ Mesoderm Induction Medium for the differentiation of human pluripotent stem cells to early mesoderm and downstream mesodermal differentiation. These media were relevant to PluriMes and were tested and further optimized for specific applications in WP4 and WP6 projects.
MesenCult™-ACF and STEMdiff™ Mesoderm Induction Medium consisted of defined components and reduced animal derived materials. As a result, these medium formulations already met some of the initial requirements for higher compliance product development.
In 2015 – 2016, SCT launched RSeT defined medium for naïve-like human pluripotent stem cells to the life sciences market. RSeT™ reverts primed human pluripotent stem cells (hPSCs) and maintains cells in a naïve-like state. New media formulations to generate reset hPSCs developed in Work Package 1 were compared to RSeT™ media.
SCT launched the STEMdiff Mesenchymal Progenitor Kit, a defined culture kit consisting of animal component-free (ACF) induction medium, expansion medium, and attachment substrate. It is optimized for the derivation of cells with mesenchymal progenitor cell (MPC)-like properties from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. This kit provides a complete workflow of defined reagents for derivation and expansion of human ES- or iPS-derived MPCs. MPCs have potential utility in a range of applications. ES- and iPS-derived MPCs are a promising renewable cell source for academic and translational MSC research. This kit has been successfully tested in Work Packages 4, 5 and 6 to meet the deliverables in those work packages.
SCT launched MyoCult™ Expansion Kit (Human) for the culture and maintenance of human skeletal muscle progenitor cells (myoblasts). MyoCult™ Expansion Medium suppresses key myogenic differentiation genes while conserving myogenic progenitor markers. MyoCult™ was tested in Work Packages 4 and 5 and compared to media used in partner labs. SCT has also developed a serum-free version of MyoCult™ which was tested in Work Package 5 and showed high performance. MyoCult™ serum free medium may be a useful media for a future Phase I clinical trial planned by Partner 2a.
SCT launched MesenCult™-ACF Chondrogenic Differentiation Medium, an animal component-free (ACF) medium for the in vitro differentiation of human mesenchymal stem and progenitor cells (MSCs) into chondrogenic lineage cells, including chondrocytes. This medium is suitable for the differentiation of hPSCs-derived mesodermal progenitors to the chrondrogenic lineage. This medium has been tested in Work Packages 4, 5 and 6 using various cell sources.
SCT launched mTeSR™1 manufactured under cGMP, a feeder-free cell culture medium for human embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), mTeSR™1 is a highly specialized, serum-free and complete cell culture medium. With pre-screened raw materials that ensure batch-to-batch consistency and robust feeder-free protocols for ES and iPS cell culture, mTeSR™1 provides more consistent cultures with homogeneous, undifferentiated phenotypes. mTeSR™1 is manufactured under a cGMP quality management system compliant to 21 CFR 820, ensuring the highest quality and consistency for reproducible results.
SCT launched mTeSR™3D for expansion and scale-up of undifferentiated human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells as aggregates in 3D suspension culture with a novel fed-batch protocol. This culture system is compatible with a range of suspension culture vessels used for scale up and can be used in Work Package 6 to test expansion and differentiation of hPSCs.
In period 3, SCT launched a cell culture medium kit to convert human pluripotent stem cells (hPSC) that typically exists in post-implantation state to a naïve pluripotent state. NaïveCult™ Induction Kit (Catalog # 05580), developed based on Guo et al. (2017), is composed of defined cell culture media used sequentially for the transgene-independent or chemical reversion of primed hPSCs to a naïve-like, or reset, state. NaïveCult™ Induction Kit requires the use of a histone deacetylase inhibitor (HDACi), typically sodium butyrate or valproic acid, during the initial stage of naïve state induction. The development and commercialization of this kit is the result of research performed in Work Package 1.
In addition, SCT also launched another medium in period 3, for use in expanding naïve pluripotent stem cells. NaïveCult™ Expansion Medium (Catalog # 05590) has been developed for the optimal maintenance and expansion of transgene-independent, transgene-dependent, and embryo-derived naïve-like hPSCs in t2iL + Gö conditions. NaïveCult™ media contain pre-screened high-quality components and are compatible with hPSC including human embryonic stem (ES) and human induced pluripotent stem (iPS) cells. hPSCs maintained in NaïveCult™ Expansion Medium can be converted back to a primed state by culture in mTeSR™1 or TeSR™-E8™ and can then be differentiated downstream with STEMdiff™ products such as STEMdiff™ Definitive Endoderm Kit, STEMdiff™ SMADi Neural Induction Kit, STEMdiff™ Mesoderm Induction Medium, or STEMdiff™ Trilineage Differentiation Kit. The development and commercialization of this kit is also the result of research performed in Work Package 1.
For period 3, SCT also launched MesenCult™ Proliferation Kit (Human; Catalog #05411.). This kit is a standardized, serum-containing medium for the culture of human mesenchymal stromal derived cells or mesenchymal progenitor cells. MesenCult™ Proliferation Kit (Human) is optimized for the expansion of human mesenchymal progenitor cells in vitro as well as their enumeration using the colony-forming unit - fibroblast (CFU-F) assay. Components are pre-screened to minimize lot-to-lot variability. These media are relevant to PluriMes and were tested and further optimized for specific applications in WP4 and WP6 projects outlined in PluriMes.
A kit for the differentiation of human mesenchymal progenitor cells (MPCs) to osteocytes was also launched. The MesenCult™ Osteogenic Differentiation Kit (Human; Catalog # 05465) is specifically formulated for the in vitro differentiation of MPCs into cells of the osteogenic lineage. This kit is suitable for the differentiation of human bone marrow (BM)-derived MPCs previously culture-expanded in serum-containing medium (e.g. MesenCult™ Proliferation Kit [Human; Catalog # 05411] or MesenCult™-hPL [Human; Catalog # 05439]) or serum-free medium (e.g. xeno-free MesenCult™-XF Medium [Catalog # 05420] or animal component-free MesenCult™-ACF Medium [Catalog # 05440]). This medium has been tested in Work Packages 4, 5 and 6 using various cell sources.
Lastly in period 3, a kit for the culture and maintenance of human skeletal muscle progenitor cells (myoblasts) under serum-free conditions was manufactured by SCT. MyoCult™-SF Medium suppresses key myogenic differentiation genes while conserving myogenic progenitor markers in expanded myoblasts. MyoCult™-SF was tested in Work Package 5 and showed high performance. MyoCult™-SF Medium may be a useful media for a future Phase I clinical trial planned by Partner 2a. This medium was produced in period 3 and will available to the research market by mid-2018.
Ncardia (formerly Pluriomics, Partner 4) has established a manufacturing protocol for Pluricyte® Endothelial Cells at high purity and good functionality based on IP from the Leiden University Medical Centre. Within the scope of another EU project (European Union’s Horizon 2020 research and innovation programme; SME Instrument phase 2), Ncardia is translating the current adherent culture-based large-scale process for Pluricyte® EC production towards a better controlled and more robust method for further scale-up using fully controlled stirred tank bioreactors. Although Pluricyte® Endothelial Cells are not a commercial product at the moment, Ncardia has already used the cells in some customized service projects. Ncardia is currently reviewing the status of Pluricyte® Endothelial Cells to enter a product development program for customized safety and efficacy services. This will ultimately enable pharmaceutical companies to assess cardiovascular safety and efficacy of their potential drugs already early in development. Importantly, early screening and selection of drugs in predictive in cardiovascular in vitro model systems will lead to significant reduction of development costs and reduction of animal testing.
Partner 9 (EPFL) developed chemically defined hydrogel microcarriers for the large-scale expansion of mesodermal progenitors. These have applications in the areas of biotechnology, cell manufacturing, regenerative medicine. Partner 9 is currently exploring options with a possible commercial partner.
Partner 7 (USFD) developed a medium, named Primo, that supports the maintenance of mesoderm biased human pluripotent stem cells. Partner 7 is currently seeking patent protection for the formulation of Primo medium and exploring the possibility of licencing this to Partner 11.
List of Websites:
Website: www.plurimes.eu
Project Coordinator Details: Professor Austin Smith, Wellcome - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR
Tel: +44 (0) 1223 760233
Fax: +44 (0) 1223 760241
Email: ags39@cam.ac.uk
Project Office Contact Details: PluriMes Project Office, Wellcome - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR
Tel: +44 (0) 01223 746818
Fax: +44 (0) 1223 760241
Email: plurimes-office@stemcells.cam.ac.uk
The ambition of PluriMes is to advance understanding and control of human pluripotent stem cell differentiation in order to produce mesodermal progenitor cells for use in the study and treatment of diseases of muscle, bone and cartilage.
Cell replacement therapies offer a prospect for alleviating the enormous medical and economic burdens presented by injury and degenerative disease. Human pluripotent stem cells offer a renewable source of cells for cell therapy because they can, in principle, be converted into any cell type. In particular progenitor cells for mesodermal tissues such as muscle, bone and cartilage, may be produced through controlled differentiation in the laboratory. In contrast, current clinical trials in cell therapy are dominated by mesenchymal stromal cells (MSCs) harvested from post-natal tissues. MSCs are typically portrayed as universal stem cells and as a panacea for conditions ranging from heart failure and ischemic limb conditions to colitis and graft versus host disease. However, evidence of clinical efficacy is inconsistent and controversial (Bianco et al., Nature Medicine, 2013). Crucially, the MSC entity does not fulfil scientific criteria for an authentic stem cell but is arbitrarily defined by individual practitioners (Bianco et al., EMBO J, 2013). Critical properties such as cell purity, self-renewal capacity, differentiation stability, biological potency and clinical mode(s) of action are all indeterminate.
PluriMes aimed to provide a rigorous scientific foundation for prospective cell therapies by defining the cell state(s) and potency of MSCs compared to mesodermal progenitors generated from pluripotent stem cells. To reach this goal the project had to meet three key challenges:
(i) Provision of common platforms for human pluripotent stem cell self-renewal and directed differentiation based on isolation of naive and lineage-biased classes of stem cell.
(ii) Detailed characterisation of mesodermal progenitors generated during differentiation from pluripotent stem cells and comparison with MSC harvested from post-natal human tissues.
(iii) Growth of mesodermal progenitors with therapeutic potential in controlled environments suitable for pre-clinical manufacturing.
PluriMes combined the know-how of 10 academic and 3 bioindustry groups to reach each of these objectives, as summarised below:
(i) The project defined two new classes of human pluripotent stem cell; naïve cells representing the earliest embryonic founder cells with unlimited potential, and lineage-biased substates that are poised to develop along a particular developmental pathway. These fundamental advances in knowledge of pluripotency will guide efforts to further improve control of differentiation.
(ii) PluriMes teams developed several reagents and protocols for generating various mesodermal cell types, notably including functional skeletal muscle, by differentiation of pluripotent stem cells. In a parallel and comprehensive series of investigations, MSCs were obtained from different tissues but found to share no common molecular identity nor to exhibit regenerative properties in the body. These critical findings confirm other scientific data that MSCs are not a common cell type present in all tissues, do not contribute directly to tissue regeneration, and should not be termed stem cells.
(iii) Close collaborations between academic and industry partners resulted in new and improved media and devices for preserving and expanding stem cells. Selected formulations have been further refined to meet the high quality Good Manufacturing Practice regulations required to produce cells for therapeutic applications in humans.
PluriMes research results have been presented at numerous scientific conferences and are available in 41 publications in international peer-reviewed journals and books, with a further 16 manuscripts in preparation. The publications are open access and 11 high content datasets have been deposited in public databases. Two patents were filed and are in the process of being commercialised and one patent will be filed shortly. As a result of the project 11 new culture media and other products for stem cell research are being produced and released. Finally, in addition to the scientific activities, the PluriMes project engaged in an active programme of training and dissemination activities, promoting the field of stem cell research both within the consortium and externally to specialist and public audiences.
Project Context and Objectives:
Context
The PluriMes Project bought together leading European research teams and SMEs to create an integrated cross-disciplinary programme that would have a major impact on stem cell research in the dual fields of pluripotency and mesodermal progenitors for muscle, bone and cartilage.
Mesoderm derivatives comprise the largest proportion of the body mass and are central to a vast range of both system specific or organism-wide physiological functions. They are among the prime targets of aging-related involutional or degenerative changes. Diseases for which mesoderm derivatives represent the direct sites of morbidity include highly prevalent conditions, as well as rare, crippling or lethal diseases appearing in childhood, adolescence or adult life. Due to their role in growth, energy metabolism, hormonal integration of organ systems, reproductive function, control of hematopoiesis, circulation, and many other pivotal physiological functions, mesoderm-derived tissues are also central to a number of organism derangements such as obesity, menopause-related morbidity and vascular disorders.
Highly prevalent conditions such as osteoarthritis, osteoporosis, sarcopenia, frailty, and obesity represent some of the most significant determinants of morbidity that worsen quality of life in aging populations. These conditions arise from derangement in post-natal life of the function of connective tissue cells and their progenitors. They generate high social, medical and human costs. For example, the estimated direct healthcare cost attributable to sarcopenia in the United States in 2000 was $18.5 billion ($10.8 billion in men, $7.7 billion in women), which represented about 1.5% of total healthcare expenditures for that year. A 10% reduction in sarcopenia prevalence would result in savings of $1.1 billion (dollars adjusted to 2000 rate) per year in U.S. healthcare costs. In the United Kingdom direct cost of treatment for osteoarthritis exceeds £870 million per year, while cost of social and community services and loss of economic production exceeds £250 million. On the other hand, less prevalent or rare diseases such as muscular dystrophies and skeletal dysplasias typically emanate from genetic abnormalities that affect the morphogenesis, differentiation and growth of specialized cell types of mesodermal origin. Consequences for the affected individuals and their relatives are devastating.
Success of any therapy targeting postnatal mesoderm-derived tissues postulates a clear appreciation of the cellular tools at hand. This can only be based on a rigorous definition of their identity, biology, mode of culture, and functional properties. Major uncertainties have plagued the field in all these areas (reviewed in Bianco et al, Nature Med, 2013).
An important goal for PluriMes, therefore, was to define cellular parameters for therapeutic efficacy and thereby set the stage for future clinical implementation.
Key Challenges
At the outset, PluriMes needed to resolve state of the art challenges in pluripotent stem cell (PSC) biology in order to provide optimal platforms for production of efficacious differentiated progeny. Foremost among these issues was the fundamental nature of pluripotency in human. Studies in rodents had revealed that pluripotency is not a unitary identity but comprises at least two distinct stable states, naive ES cells and primed EpiSCs (Tesar et al, Nature, 2007; Brons et al, Nature, 2007; Nichols and Smith, Cell Stem Cell, 2009), together with heterogeneous, or metastable, sub-states (Chambers et al, Nature, 2007; Toyooka et al, Development, 2007; Hayashi et al, Cell Stem Cell, 2008; Hanna et al, Cell Stem Cell, 2009; Bao et al, Nature, 2009; Canham et al, PLoS Biology, 2010; Greber et al, Cell Stem Cell, 2011; Bernemann et al, Stem Cells, 2011). Importantly, however, a “ground state” of naïve pluripotency had been captured and stabilised by inhibition of the mitogen activated protein kinase (Erk) signalling pathway (Ying et al, Nature, 2008). Ground state PSC share common features across different strains of mice and rats. These characteristics include: high clonogenic efficiency from dissociated cells, near-homogenous expression of key pluripotency factors (Wray et al, Biochem Soc Trans, 2010); suppression through transcriptional pausing of many developmental genes (Marks et al, Cell, 2012); reduced levels of epigenetic histone modifications associated with gene silencing (Marks et al, Cell, 2012); global hypomethylation of DNA (Leitch et al, Nature Strut Mol Biol, 2014); and a distinct miRNA profile (Jouneau et al, RNA, 2012). These transcriptional and epigenetic features were postulated to underlie unbiased access to definitive germ layer differentiation (Smith and Nichols, Cold Spring Harbor Perspectives, 2012). Isolating human PSC that recapitulate all of these properties was therefore a key target for PluriMes. On the other hand, the heterogeneity of conventional human PSC was postulated to reflect differential lineage priming (Enver et al, Cell Stem Cell, 2009). An important goal for PluriMes therefore was to explore the existence of PSC sub-states, in particular with a lineage bias towards mesoderm. These two complementary approaches were anticipated to lead to definitive advances in understanding of the pluripotent constellation in human which could in turn shape downstream approaches to control differentiation.
Directed differentiation of PSC, particularly into skeletal mesoderm, was a further barrier in the field and a major focus for the collaborative research programme in PluriMes. This would entail the creation of a set of reporter lines, the development of new culture systems and functional assays, and detailed transcriptomic characterisation. In addition, there was a clear need to develop improved cell cryopreservation and expansion methods, compatible with clinical manufacturing (GMP) for cell therapy applications.
Most importantly, prior to PluriMes, and still today, post-natal mesodermal progenitors, variously described as mesenchymal stromal cells (MSC), mesenchymal stem cells (MSC), mesoangioblasts or pericytes, are proffered for a range of cell therapy applications. The different names alone, however, testify to the ambiguities and controversies in this field. In reality there is a dearth of reliable information on the origin and relatedness of these cell populations or their fate after transplantation (reviewed by Bianco et al, Nature Med, 2013). PluriMes therefore proposed a radical new approach: directed differentiation of human PSC would be exploited to establish developmental hierarchy, define cell identities, and determine biological activity and prospective therapeutic potency of specific mesodermal progenitors. PluriMes would then compare for the first time developmentally discrete mesodermal progenitors derived through PSC differentiation with cell preparations from post-natal human tissue that are presently considered for clinical use (so-called MSC). This exercise is designed in an unbiased way to provide new knowledge and advance understanding of mesodermal progenitor cell ontogeny, biology and function. PluriMes thereby aimed to provide defined cell classes and associated culture protocols to facilitate translational applications using both PSC-generated and post-natal tissue derived mesodermal progenitor cells.
The Consortium
Against this background, PluriMes sought to integrate knowledge in fields of stem cell biology, mammalian development and mesodermal 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 comprised world-leading academic expertise in each of these areas complemented with two bioindustry SMEs providing research and development for commercial exploitation. The overall multidisciplinary approach comprised a series of collaborative scientific workpackages in which PluriMes investigators addressed: (1) human ground state pluripotency; (2) Lineage biased sub-states (3) Genetic tools; (4) Lineage specification for paraxial and lateral mesoderm; (5) Comparative assessment of biological activity and therapeutic potential; (6) Bio-processing and scale-up. In addition, two work packages (7 and 8) were dedicated to management activities, and to dissemination and training respectively. See Project component schematic (Figure 1 of the attachment).
Overall the consortium aimed to maximise cross-fertilisation and synergy between the participating research teams. Thus the research plan was constructed to maximise collaboration rather than individual contributions. To stimulate and enable new directions and interactions, a contingency fund was set aside specifically to support innovative sub-projects proposed jointly by Principal Investigators.
Finally, to evaluate and advise on progress, PluriMes engaged a Scientific Advisory Panel of leading experts who reviewed the project at each of the annual consortium meetings and made recommendations on funding of new sub-projects.
Objectives
The over-arching goal of PluriMes was to exploit human pluripotent stem cell (PSC) differentiation to generate mesodermal progenitor cells with therapeutic potency for diseases of skeletal muscle, bone and cartilage, and to compare these with mesenchymal stromal cells (MSCs) isolated from various human tissues. To this end PluriMes set three primary 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 mesenchymal progenitor generated during developmental progression from pluripotent stem cells, and comparison with cells harvested from post-natal human tissues.
III. Scaleable expansion of mesenchymal progenitors in fully defined culture conditions suitable for pre-clinical manufacturing with retention of genetic integrity, identity, and engraftment potency
The PluriMes research plan was designed to achieve the objectives within the four years of the project, measured and verified by, amongst others:
• open access peer-review publications in leading international journals;
• data deposition in public archives;
• conference presentations;
• training courses and exchanges;
• patent applications;
• new protocols and commercial products for research;
• regulatory compliant protocols, materials and culture media for human stem and progenitor cells.
PluriMes also aimed to:
• Make Project knowledge/information accessible to both specialist and public audiences via a user-friendly website.
• Increase scientific knowledge, interaction, training and communication within and beyond the consortium.
• Engage effectively with a range of stakeholders and public through outreach activities
Project Results:
Overview
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 (www.plurimes.eu). 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. PluriMes also appointed an international Scientific Advisory Panel (SAP) of leading experts; Professor Margaret Buckingham (Chair), Professor Pam Robey (co-Chair), Professor Peter Zandstra, Professor Shahragim Tajbakhsh and Dr Glyn Stacey. The SAP attended the four annual consortium meetings and provided detailed reports with evaluations on progress and strategic guidance towards final outputs.
Objective (i) concerns the fundamental nature and regulation of pluripotent stem cells, which should be understood fully to achieve optimal control and translational efficacy. PluriMes has advanced the state of the art, firstly by generating and expanding human PSC in a naïve, or ground state, condition (Partner 1), and secondly by trapping a mesoderm-lineage biased sub-state of PSC (Partners 7 and 2b). Both of these new PSC culture systems are based on specific manipulations of the cell signalling environment. The resulting cell states have been validated by global transcriptome analyses and by in vitro differentiation. These findings constitute original advances in basic PSC biology and fulfil our aim to provide new routes to exploitation in biomedical research, whether for disease modelling or cell therapy applications. To this end, Partner 11 has developed commercial media products for naïve PSC and in addition a cGMP compatible media formulation.
Objective (ii) encompasses both directed differentiation of PSC into mesodermal progenitors, and characterisation of in vivo post-natal progenitors, including the so-called MSCs. For the former, a challenging but ultimately productive technical endeavour has been the generation of PSC carrying various lineage-specific fluorescent protein reporters (Partners 6, 8 and 10). In addition, the potential for autologous therapy with gene-corrected cells was established by using CRISPR/Cas 9 technology to correct the mutated α-sarcoglycan gene in iPSC derived from patients with Limb-Girdle Muscular Dystrophy 2D that were then differentiated into mesangioblasts (Partners 6 and 2a). Guided and validated by the reporters, Partner 8 refined protocols for the differentiation of lateral plate mesoderm derivatives (cardiac muscle and endothelial cells), while Partner 10 described for the first time robust generation of skeletal muscle from PSC, a major advance. Partner 9 developed a microfluidic device for fine environmental control over PSC differentiation and derivative mesodermal progenitors, while partner 2a developed skeletal muscle organoids for measuring functional differentiation properties. Finally, as a resource for testing the in vivo regenerative potency of human mesodermal progenitors, Partner 3 created the first genetically modified immunodeficient mouse model of skeletal disease.
In a critical study to examine the biological existence of the MSC entity, Partner 3 employed stringent in vivo assays to measure differentiation properties of cell populations derived from different sources. Assays for osteogenesis, chondrogenesis, angiogenesis, myogenesis, and establishment of hematopoietic niches revealed strikingly divergent differentiation capabilities of peri- and postnatal progenitors isolated from bone, muscle, umbilical cord blood, and other tissues, or produced by differentiation of PSC. Cellular diversity according to source was independently confirmed by transcriptome analysis. These published results demonstrate that only the bone marrow contains a multipotent skeletal stem cell fraction and disprove the notion of common stem cells in different tissues. As discussed in several review articles, the findings confirm that the concept of MSCs as mesenchymal stem cells lacks any solid scientific foundation.
Objective (iii) comprises the translational component of Plurimes. Partner 4 developed robust processes for upscaling and manufacturing of PSC-derived cardiomyocytes and endothelial cells based on directed differentiation methodology developed by Partner 8. Partner 9 designed and successfully tested a synthetic microcarrier system for bioreactor expansion of mesodermal progenitors. GMP-compatible cryopreservation media for mesodermal progenitors have been optimised in a collaborative effort between Partners 5, 11, 2a and 4. Partner 11, working with Partners 2a, 4 and 12, has demonstrated animal-component free media expansion of mesodermal progenitors. Importantly, the requisite high quality raw materials have been identified and documentation put in place for a GMP-compliant Technology Transfer Package to enable GMP production of this media. Finally, Partners 12 and 6 developed a strategy to engineer mesangioblasts to deliver exon-skipping RNA and thereby enhance their potency for cell therapy in muscular dystrophy.
In addition, over 48 months PluriMes has:
• Funded nine exchanges through the PluriMes mobility programme
• Co-organised four European Summer Schools on Stem Cells & Regenerative Medicine
• Run seven training workshops plus two specialist training courses
• Run four full consortium meetings and two mini-consortium meetings
• Worked cooperatively with 6 other stem cell consortia; HuMen, Thymistem, Neurostemcellrepair, Intens, REVIVE and UKRMP, to run two major conferences, and to provide places in PluriMes training workshops
• Collaborated with EuroStemCell on various public engagement activities and developed new outreach activities.
The principal results from PluriMes are presented in 44 Deliverables and 15 Milestones. Research discoveries are disseminated in 37 publications in peer-reviewed journals and 4 book chapters. An additional 16 manuscripts are currently submitted or in preparation. The publications are Open Access and high 11 datasets have been deposited in public databases. Intellectual property is being protected in 3 patent filings and 2 trademarks.
a) Work Package 1: Human ground state pluripotency
Pluripotent stem cell biology is based on the paradigm of mouse embryonic stem cells which consistently show the ability to produce all cell types of the body. These mouse cells can be propagated in a stable uniform condition of naivety, known as the ground state. Human pluripotent stem cells (PSC) by contrast are heterogeneous and show variable propensity for differentiation to particular lineages. In this workpackage we first determined conditions for capturing human PSC in a naïve condition both by conversion of conventional PSC and by reprogramming of somatic cells. We have characterised extensively the molecular properties of human naïve PSC and have examined their genetic stability. We then developed protocols to capacitate the naïve PSC for differentiation and determined their potential for multilineage development in vitro. Finally, in collaboration between academic and industry partners we have developed and refined media, substrates and protocols for generation and maintenance of human naïve PSC. These products are being commercialised for the research community and conditions have also been determined that are compatible with GMP cell manufacturing for future clinical applications.
i) WP1 Objectives
• Objective 1: To generate ground state (naïve) human PSC by conversion of extant ESC and iPSC and directly from somatic cells.
• Objective 2: Characterisation of ground state PSC
• Objective 3: Development of reagents and protocols suitable for pre-clinical application
ii) WP1 Results
For Objective 1 Generation of ground state PSC we demonstrated that conventional human PSC can be reset to a naïve state 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. Partner 1 went on to establish conditions for resetting to the naïve state using small molecules, avoiding the use of transgenes, and evaluated chromosomal stability with Partner 7. Finally, we showed that human somatic cells could be reprogrammed directly to naïve PSC and also identified a unique cell surface marker to discriminate naïve and primed PSC.
For Objective 2 Characterisation of ground state PSC we have shown that the reset PSC described above have metabolic features and global transcriptome and DNA hypomethylation features expected for ground state cells that are distinct from conventional PSC. Multilineage differentiation ability has been demonstrated in vitro and in vivo, for both transgenic and chemically reset naïve cells and compared with the potency of naïve PSC derived from human embryos. In addition, Partner 7 provided information on characterisation of available antibodies for PSC characterisation, and Partner 1 employed CRISPR/Cas9 to achieve targeted gene mutation in naïve PSC. Finally, Partner 11 received cell lines and protocols from Partner 1 and developed improved culture media for resetting and maintaining human naive PSC. This has resulted in new commercial media products.
Towards Objective 3 Development of reagents and protocols suitable for pre-clinical application Partner 9 and Partner 1 collaborated on a preliminary screen to identify substrates and Partner 1 extended this to develop conditions that support feeder-free propagation of reset PSC. Partner 11 identified higher grade, defined and quality controlled raw materials suitable for a GMP-compatible media formulation and produced a GMP-compliant Technology Transfer Package. Partner 1 and Partner 7 evaluated chromosomal stability and reset clinical grade primed PSC to a naïve condition.
In summary, this workpackage has established and validated a new type of human PSC that displays the features anticipated for a ground state stem cell closely related to naïve pre-implantation epiblast, and has laid groundwork for potential future applications in biomedical settings.
iii) WP1 Outputs
This work package has resulted in the following outputs:
• 2 peer-reviewed research publications
• 3 manuscripts are currently in preparation
• 0 reviews
• 2 patents
• 2 other products
• 3 data sets published in public database resources
• 0 trademarks
b) Work Package 2: Lineage biased sub-states
The body is made up of many tissues and cell types. These are generated during human development from stem cells that divide and produce cells of many different types and functions. This process can be recapitulated in the laboratory using cultures of pluripotent stem cells which retain the capacity to produce all cells of the body. These stem cells could be used to produce cells that could then be used clinically to replace tissues lost through disease. However, the challenge is to control the process of stem cell differentiation so that we can efficiently produce the cell types and tissues we require. Our work suggests that cultures of pluripotent stem cells are heterogeneous, so that some cells may be biased to produce one tissue type, while others may tend to differentiate into other tissues types. We think that this is not fixed but that the stem cells may transition between these different preferences with time. To make tissue generation more reproducible and efficient we have identified different stem cell states with different tissue preferences. We have characterised these different states at the molecular level and also the mechanisms that control their behaviour. From this we have now developed a fully defined culture medium which traps the stem cells in one of these states from which they can efficiently be directed to generated mesodermal cell types such as muscle bone and cartilage.
i) WP2 Objectives
• Objective 1: Characterisation of PSC substates
• Objective 2: Identification of mesoderm-biased substates of hPSC
• Objective 3: Trapping mesoderm biased hPSC subsets
ii) WP2 Results
The proposition underlying WP2 is that hPSC can exist in multiple interconvertible substates within the stem cell compartment but when occupying different substates they exhibit different properties with respect to self-renewal and differentiation. In particular, different substates may be associated with a bias in the potential fate of the cells upon differentiation. The work of WP2 was focused upon confirming this hypothesis, identifying substates with a mesoderm bias and elucidating molecular mechanisms that control the dynamics with which stem cells may transition between substates. Our ultimate goal was to identify conditions that may trap cells in a mesoderm biased substate, and assess whether doing so may offer practical advantages for expansion and derivation of mesodermal derivatives for safe clinical applications.
Initially, we carried out an extensive screen of several approaches to identify and isolate hPSC occupying distinct substates within the undifferentiated stem cell compartment, including assessment of surface antigen patterns, metabolic state and reporter genes. Using these tools we confirmed that hPSC can occupy different substates in which the cells exhibit different patterns of gene expression, and propensities for differentiation, while retaining the capacity to proliferate as an undifferentiated stem cells. Building on these results and using a combination of a cell surface antigen (SSEA3) characteristically expressed by undifferentiated hPSC, and a fluorescent reporter gene that indicates expression of MIXL1, a transcription factor turned on during the early phases of mesoderm specification, we found that a small proportion of cells in hPSC cultures expressed both SSEA3 and MIXL1. By isolating cells with these characteristics (i.e. SSEA3+/MIXL1+ cells) we were able to show that they would not only proliferate as undifferentiated cells but also that they would tend to towards mesodermal derivatives when allowed to differentiate. Further, from single cell transcriptomic analysis of SSEA3+/MIXL1+ cells we found that regulatory genes characteristic of both undifferentiated hPSC and of mesodermal pathways of differentiation could be found co-resident in the same cell, consistent with the notion of intermediate metastable states. Similar results were found using a reporter for another mesoderm related gene, BRACHYURY.
Importantly, we then discovered that whereas we could find these mesoderm biased stem cells when hPSC were grown under culture conditions that included inactivated feeder cells, they disappeared when hPSC were grown under fully defined conditions. This allowed us to identify specific signalling molecules that promote the differentiation of hPSC to mesoderm during which the SSEA3+/MIXL1+ appeared transiently. By screening other molecules that tended to inhibit differentiation, we were able to identify a combination of pro-differentiation and anti-differentiation factors that cross-antagonised each other and trapped the cells in the SSEA3+/MIXL1+ state. A transcriptomic analysis of these trapped cells showed that they correspond to a state on the trajectory of hPSC as they differentiate towards mesodermal derivatives. Using these data we were able to develop an optimised, defined medium formulation, called Primo, in which it were able to maintain mesoderm-biased hPSC over 10 passages while the cells retained karyotypic normality. We are currently seeking patent protection for the formulation of Primo medium.
iii) WP2 Outputs
This work package has resulted in the following outputs:
• 3 peer-reviewed research publications
• In addition, 1 manuscript is in press, 2 manuscripts have been submitted and 1 manuscript is in preparation
• 0 reviews
• 0 datasets
• 1 patent is in preparation
• 0 other products
• 0 trademarks
c) Work Package 3: Genetic Tools
Human pluripotent stem cells (hPSCs) have the capacity to produce other cell types, including cells belonging to the mesodermal lineage, such as cardiac and skeletal muscle. Defined protocols that allow a reproducible and efficient differentiation of hPSCs to specialized cell types in the culture dish are still under development. In WP3 we introduced into hPSCs fluorescent markers which make the cells glow green or red when they become a different cell type. Those so-called reporter cell lines carry fluorescent proteins that are under the control of tissue-specific genes and mark distinct cell types that arise during differentiation to mesoderm. Up to date we generated in PluriMes more than 10 reporter lines marking different cell types. In addition, we produced reporter lines carrying two independent colors under the control of specific genes, making possible to visualize the transition between differentiation steps. The reporter lines will facilitate the development of reliable differentiation protocols by other members of the PluriMes Consortium.
Moreover, in WP3 we developed techniques for changing the genetic makeup of the hPSCs. One of the aims is to correct genetic mutations causing diseases such as muscular dystrophies and in collaboration with other PluriMes partners we succeeded in repairing the mutation in patient specific induced PSCs with Limb-girdle-muscular dystrophy. Another reason for changing the genetic makeup is to model diseases such as Duchenne muscular dystrophy in the culture dish. The other PluriMes partners will use the modified cells to perform screens for therapeutic reagents.
i) WP3 Objectives
• Objective 1: Generation of PSC reporter lines
• Objective 2: Forward programming by inducible TFs
• Objective 3: Correction of mutations by gene targeting.
ii) WP3 Results
In Objective 1 we have generated a series of transposition compatible BAC vectors and targeting constructs carrying fluorescent proteins (FP; Venus, Cherry or TagBFP) under the control of mesodermal specific genes using recombineering technology. The reporters will facilitate the detection by live imaging and isolation by flow cytometry of relevant cell states during in vitro differentiation of hPSCs. This approach will be applied to analyse and optimise the mesodermal/mesenchymal trajectories in collaboration with Partners 7, 8 and 10. The reporters are specific for the pluripotent state (OCT4), presomitic mesoderm (MSGN1, BRACHYURY), paraxial mesoderm (MEOX1; PAX3, PAX7 for somatic skeletal muscle and PAX1, PAX9 for axial skeleton), lateral plate mesoderm (COUPTFII, PRRX1) and vascular derivatives (FOXC2). In summary, we generated 8 lines by BAC transposition, 10 single reporter lines by gene targeting and 4 dual reporter lines. Generation of reporter lines by gene targeting using CRISPR technology turned out to be more reliable for the expression of the fluorescent protein as compared to lines generated by BAC-transposition. Finally, the reagents that we have generated in PluriMes (targeting vectors, gRNA-expression vectors, hybridization probes) and protocols for gene-targeting and analysis of the clones can be easily applied to other human cell lines.
In Objective 2 we have generated a transposition compatible vector that allows the easy insertion of transcription factors by Gateway reaction under the control of a tetracycline inducible promoter. We cloned the cDNA of Mesogenin (MSGN1) together with the fluorescent protein mCherry. Induction of MSGN1 expression by Doxycycline administration in stable colonies resulted to changes in cell morphology already after 24 hours. The cells lost their epithelial morphology and acquired a mesenchymal phenotype, downregulated pluripotency markers (OCT4, NANOG) and upregulated epithelia to mesenchymal transition markers (SNAIL1, 2) and other mesodermal genes (MEOX1, PRRX1). The above results prove that it is possible to change cell fate by overexpression of specific transcription factors. For resetting hPSCs to the ground state of pluripotency we generated a vector for inducible expression of NANOG, KLF2 and mCherry. Stable NCRM1 hiPSCs formed dome shaped colonies and proliferated under culture conditions that support the ground state of mESC pluripotency in the presence of Doxycycline, whereas uninduced colonies could not be propagated under those conditions. Altogether, the functionality of the inducible vector was demonstrated in two different applications: a) forward programming and b) resetting to ground state.
In Objective 3 we have generated vectors for correcting mutations in the α-sarcoglycan gene (SGCA) in iPSCs derived from patients with Limb-Girdle Muscular Dystrophy 2D (LGM2D). In one of the two available LGMD2D iPSCs lines characterized by Partner 2a the mutation is a homozygous substitution (C>T) in exon 3, implying that correction in only one allele is sufficient to revert the disease phenotype. For correcting the mutation we used two types of targeting vectors: a) BAC based vectors, with one short and one very long homology arm and b) Cas9 compatible vectors with short homology arms. We successfully targeted the locus in patient iPSCs and corrected the mutation in one allele with a frequency varying between 5.5% and 31% and verified by sequencing. Targeting frequency was higher with CRISPR assisted targeting, however many colonies carried random integrations of the targeting vector in addition to the targeted event. Altogether, this study outlines a step towards autologous cell therapy in LGMD2D patients
iii) WP3 Outputs
This work package has resulted in the following outputs:
• 2 peer-reviewed research publications
• No additional manuscripts are currently under review or in preparation
• 0 datasets
• 0 reviews
• 0 patents
• 0 other products
• 0 trademarks
d) Work Package 4: Lineage specification for paraxial and lateral mesoderm
Human pluripotent stem cells can form any type of cell in the body but for some purposes it is very useful to be able to direct their differentiation to specialized cell types of interest. In Plurimes, the aim is to form cells of the blood vessels and various types of muscle. These are known to originate from different types of mesoderm in normal embryonic development. Our goal in this work package was to generate a set of cells lines that could “report” on the basis of expression of different colours where they are in their differentiation trajectory. To create these so-called transgenic human pluripotent cell lines we used gene editing to express green, red, yellow or blue fluorescent proteins in each different type of mesoderm. This allowed us to form endothelial and smooth muscle cells of the blood vessels very efficiently as well as heart and skeletal muscle. In addition, by separating populations of cells expressing several colours into cells expressing just one we were able to purify these cell types and identify proteins on the cell surface specific for each of the cells. This allowed us later to separate cells from pluripotent cells which did not express the coloured reporters. In this way we were able to refine and improve differentiation protocols so that they now contain no animal reagents (in principle applicable for further development in clinical applications) in a process that is applicable and efficient in multiple different pluripotent stem cell lines. This has been one of the bottlenecks in moving the field forward: how to differentiate all cell lines efficiently using similar protocols.
i) WP4 Objectives
• Objective 1: Defined conditions for differentiation of hPSC to lateral plate (LPM) and paraxial mesoderm (PM)
• Objective 2: Transcriptional profiling to identify surface markers, signalling pathways, transcriptional regulators and micro-environmental stimuli that promote mesoderm specification, patterning and expansion.
• Objective 3: Functional validation of regulatory pathways and surface proteins.
ii) WP4 Results
In this work package we aimed to define human PSC differentiation conditions for efficient derivation of early mesoderm and subsequently specific mesodermal cell types like heart cells, blood vessels and skeletal muscle relevant to tissue repair and regeneration. To make these different cell types, it is important that the early mesoderm that first forms as the hPSCs leave the pluripotent state receives the right signals to programme the later differentiation into the tissue types of interest. The work showed this is best done using defined differentiation conditions for the induction of lateral plate mesoderm (LPM) which gives rise to cells such as cardiomyocytes, endothelial cells, pericytes/vascular smooth muscle cells (vSMCs), and paraxial mesoderm (PM) which gives rise to skeletal and satellite muscle cells, cartilage and bone. Transgenic hPSC lines that become fluorescent through expression of lineage reporters generated in WP3 were used as an easy method to monitor differentiation and to determine exactly which time points were best for growth factor addition to result in the most efficient differentiation. The Partners thus developed culture conditions for the maintenance and expansion of clonal progenitors and the directed differentiation of presomitic mesoderm into skeletal muscle and satellite cells within 30 days. Functionally, these derivatives were examined using in vitro and in vivo assays in WP5.
The transgenic reporter lines were also exploited to obtain gene signatures for isolated cells at different differentiation stages of human mesoderm formation. Partners also identified sets of surface markers that can be used for the enrichment of mesoderm-derived endothelial cells and cardiomyocytes. The most significant findings were the development of methods to form different endothelial cell subtypes from hPSC. Each tissue and organ has its own type of endothelial cells and through the knowledge we developed on mesodermal patterning we were able to generate heart specific endothelial cells. When grown together with cardiomyocytes that had been selected from mixed differentiated cell populations using cell type specific surface markers we had identified in the study, we formed cardiac microtissues that were representative of human myocardium. In a similar way, close mimics of human skeletal muscle were developed from hPSC. The IP was licensed to one of the industrial partners (Pluriomics).
The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of (human) cardiomyocytes and vascular cells. We therefore established conditions for clonal expansion of cardiovascular progenitors (CPCs) (Birket et al. Nat Biotech 2015) In WP4, we used hPSCs, in which CPCs are genetically marked, to show that regulated MYC expression enables their robust expansion. These CPCs could be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes with distinctive physiological characteristics. The cells were clonogenic and could be expanded over 40 population doublings yet still be directed to differentiate to cardiomyocytes as well as vascular cells Together, this tractable model reveals the potential of hPSC-derived CPCs for precisely recreating elements of heart development in vitro.
Finally, we further confirmed functionality of ECs and cardiomyocytes (Giacomelli et al. Development 2017). The data following transplantation of these cells in zebrafish embryos was reported in Orlova et al, Nat Protocols, 2014). Additional transplantation data in chick embryos was also reported i(Guadix et al, Stem Cell Reports 2017). Various (combinations of) cell surface markers from transcriptomics analysis and other data now allow sorting of early mesoderm populations.
iii) WP4 Outputs
This work package has resulted in the following outputs:
• 7 peer-reviewed research publications (plus 1 pre-print)
• In addition 1 manuscript is in press and 1 manuscript is in preparation
• 0 reviews
• 0 patents
• 1 data set has been deposited in a public database resource. 2 other datasets will be deposited in a public database resource.
• 0 other products
• 0 trademarks
e) Work Package 5: Comparative assessment of biological activity and therapeutic potential.
The use of induced pluripotent stem cells (iPSCs) to regenerate damaged human tissues relies on a) complete knowledge of the characteristics and functions of natural stem/progenitor cells included in the human body b) perfect reproduction of those characteristics and functions in progenitor cells derived from iPSCs c) rigorous demonstration that progenitor cells obtained from iPSCs can effectively regenerate tissues in suitable animal models. We have demonstrated that stem/progenitor cells isolated from different human tissues such as, for example, skeletal muscle and bone, are able to regenerate only the tissue in which they reside, in contrast with the popular hypothesis that stem cells with identical functions (so called Mesenchymal Stem Cells) can be isolated from different sites of the human body. We have shown that, in many human tissues, stem/progenitor cells are located in the blood vessel wall and have identified some of the mechanisms regulating their behaviour and function at that site. We have generated and tested different cell types derived from iPSCs, such as endothelial and muscle cells, demonstrating their functionality in appropriate experimental conditions. Furthermore, we have started to explore the possibility of using iPSCs for regenerating specific components of the skeleton. All these results have been obtained through the development and application of multiple experimental models that represent powerful tools for future studies. Finally, we have tested different nutrient media for natural and iPSCs derived progenitor cells that do not contain any factor of animal origin and therefore do not carry any potential risk for human patients.
i) WP5 Objectives
• Objective 1: Differentiation potential of pre-, peri- and postnatal progenitors of mesoderm-derived tissues.
• Objective 2: Transcriptome of preogenitors of mesoderm-derived tissues
• Objective 3: Microvascular niche of mesoderm progenitors.
ii) WP5 Results
Objectives 1 and 2. We have developed tools and knowledge for the assessment of the effective regenerative potency of natural and hPSC-derived mesodermal progenitors.
We have established/refined multiple, rigorous cell differentiation assays based on heterotopic/orthotopic transplantation in the mouse. Our assays have demonstrated that progenitor cells isolated from different human post- and peri-natal mesodermal tissues represent distinct classes of tissue-specific progenitors rather than a uniform class of “mesenchymal stem cells”. The functional diversity observed in vivo based on the tissue origin of the cells, has been further validated by transcriptome analysis. These results are in contrast with the widely held concept of “mesenchymal stem cells” and may have a great impact on translational research focused on mesodermal tissue regeneration.
We have generated different hPSC-derived cell types, including hPSC-derived endothelial cells, hPSC-derived pericytes and hPSC-derived myogenic cells. The functional competence of all these cell populations has been demonstrated by transplantation in the developing Zebrafish and/or in the mouse and the expression of specific genes of interest has been confirmed by transcriptome analysis. We have also developed multiple populations of mesodermal progenitors derived from hPSCs through intermediate “mesenchymal” stages (hPSC-derived mesodermal/”mesenchymal” stem cells). In agreement with the results obtained with post-natal cells, we have observed that the expression of the “MSC” phenotype in hPSC-derived cells in vitro is not predictive of the ability to regenerate tissues in vivo, e.g. to form bone. We have also begun to develop new platforms for the generation of human muscle organoids, by using post-natal and hPSC-derived cells, and isogenic artificial muscle constructs containing myogenic cells, endothelial cells and pericytes derived from the same iPSCs. These constructs provide valuable experimental systems for regenerative medicine and drug development.
The gold standard in assessing the potential of mesodermal progenitors for clinical intervention is in vivo transplantation in animal models reproducing tissue disease/injury. Therefore, we have converted some of our in vivo experimental assays in the mouse into specific models of therapeutic intervention for skeletal and muscle disease. To this aim, we have developed the first model of human skeletal disease in immunocompromised mice (Fibrous dysplasia/SCID mice) thus providing a novel experimental tool in the skeletal field. Finally, since the clinical application of any progenitor cell type relies on the identification of suitable conditions for cells isolation/expansion, we have identified candidate chemically defined, animal-component-free medium formulations that can potentially be used for human natural and hPSC-derived mesodermal progenitors under GMP-compliant protocols.
Objective 3. Evidence has been provided that mesodermal progenitors in post-natal tissues are incorporated as pericytes in the blood vessel wall and that their differentiation/function at that site is regulated through molecular gradients. For this reason, we have developed cutting edge microfluidic systems that allow to reproduce and to investigate the molecular interaction of natural and hPSC-derived pericytes with the microvascular niche.
iii) WP5 Outputs
This work package has resulted in the following outputs:
• 8 peer-reviewed research publications
• 4 reviews
• 2 book chapters
• In addition there are 2 manuscripts in revision, 3 manuscripts in preparation and 4 published conference abstracts.
• 2 data sets have been deposited in a public database resource. 3 other datasets will be deposited in a public database resource.
• 0 patents
• 0 other products
• 0 trademarks
f) Work Package 6: Bio-processing and scale-up
Work Package 6 (WP6) is intended to lay the foundation for bio-processing and scale-up of the cell types relevant for the general aim of the Plurimes consortium. This comprises the production and validation of highly defined culture and cryopreservation conditions, as well as the development of strategies for the larger-scale production of clinically relevant cell types. New cell culture media formulations for the expansion and cryopreservation of several types of mesodermal progenitor cells were successfully developed, showing superior characteristics compared to the state-of-the-art. In parallel, bioengineers devised powerful biomaterials-based strategies for scalable expansion of the cells in bioreactors. To this end, synthetic hydrogels were formulated as micron-sized beads (‘microcarriers’) such as to expand the cells on their surface in suspension.
i) WP6 Objectives
• Objective 1: Development of GMP-compatible cryopreservation protocols
• Objective 2: Development of GMP-compatible expansion protocols
• Objective 3: Development of scale-up processes for large-scale production of cells in bioreactors.
ii) WP6 Results
In this work package, we aimed at developing Good Manufacturing Practice (GMP)-compatible protocols for the efficient and reliable cryopreservation, as well as the large-scale expansion of human mesodermal progenitors and pluripotent stem cells (hPSC) and their derivatives used in the PluriMes project.
Three cryopreservation medium variants containing GMP-compatible components were developed and tested for their effect on post-thaw cell viability, cell recovery and the re-establishment of successful cultures. One of the media, CryoStor® CS10, showed very good results for three sources of mesodermal progenitors (hPSC-derived MPCs, human mesoangioblasts, iPS- and ES cell-derived inducible myogenic progenitors) and human iPSC-derived vascular smooth muscle cells. All cell types stored for at least 6 months in CryoStor® CS10 maintained high cell viability, marker expression and morphology post thaw and culture.
Recent efforts by one of the PluriMes labs (A. Smith) have demonstrated that hPSCs can be modified, via small molecules and/or the exogenous expression of transcription factors, to adopt a state approximating the naïve state of murine pluripotent stem cells. A new commercial medium was developed to induce and maintain these naïve-like, or ‘reset’, hPSCs from primed hPSCs using a transgene-free or chemical-induction method. Human naïve reset PSCs generated and expanded using these media exhibited proper colony morphologies, expression of key naïve-associated genes and tri-lineage differentiation potential. In addition, an hPSC expansion medium containing high-quality components was developed and found to result in improved growth rates of multiple hPSC lines and compatibility with feeder-independent hPSC maintenance cultures.
Further, enhanced manufacturing protocols, relevant quality control and processes documentation, as well as key relationships with Contract Manufacturing Organizations (CMOs) manufacturing under cGMP compliance were successfully established. A “GMP-compliant Technology Transfer Package” detailing all required documentation and instructions to initiate the transfer of MesenCult™-ACF, a commercial research grade medium for the optimal derivation and expansion of Mesenchymal Progenitor Cells (MPCs), to a Contract Manufacturer Organization (CMO) for GMP-manufacturing.
Towards manufacturing cells in large-scale bioreactors, a chemically defined microcarrier system was developed and demonstrated successful expansion and maintenance of mesoangioblasts and iPSC-derived mesodermal progenitors. Compared to the gold standard commercially available microcarrier, the synthetic version showed improved cell detachment after bioreactor culture, overcoming a significant problem in the field. Finally, we successfully developed protocols for the microcarrier-free expansion of naïve and lineage-biased PSCs as cellular aggregates.
iii) WP6 Outputs
This work package has resulted in the following outputs:
• 5 peer-reviewed research publications
• 1 review
• 1 published conference abstract
• 1 book chapter
• 2 additional manuscript is currently in preparation
• 0 patents
• 0 datasets
• 8 other products
• 0 trademarks
g) Work Package 7: Dissemination and Training
PluriMes aimed to promote and strengthen the general field of stem cell research and regenerative medicine in Europe. This has been achieved most effectively through coordinated activities with other European stem cell projects.
The extraordinarily high levels of interest and expectation make it crucial that accurate information on advances and challenges in stem cell research is widely disseminated. The PluriMes philosophy emphasised pro-active approaches to training and communication, for both academic groups and lay audiences.
i) WP7 Objectives
• Objective 1: Project Website
• Objective 2: Research Training, Dissemination and Communication
• Objective 3: Public Engagement and Outreach
ii) WP7 Results
Objective 1: Project Website
In 2014 the PluriMes Project launched a website which had both a public section as well as a secure members-only area (intranet). The public website has served as a platform for external communication. The intranet was designed to facilitate dissemination of information and communication between partners and also assisted with the general day-to-day management of the consortium. The website has had 8407 users, with 23,407 page views, over the duration of the project. In April 2018 the PluriMes website will be transferred to a hosting platform that can be maintained for 10 years after the project to ensure continued dissemination of project information.
Objective 2: Research Training, Dissemination and Communication
PluriMes placed a major emphasis on developing skills of stem cell researchers at all career stages. To accomplish this goal PluriMes put in place a training programme to provide rigorous high-level theoretical and practical training which would maximise synergy and interaction within the consortium whilst developing common standards of experimental practice and theoretical understanding.
By drawing on the complementary expertise of participating Institutions and like-minded consortia we established a cohort of European scientists who are trained to the highest standards in the state-of-the-art. This will serve to promote and strengthen the general field of stem cell research and regenerative medicine in Europe as well as provide a solid base for future developments in the field.
Our Training Programme comprised of three specific schemes:
1) A Mobility Programme
This programme enabled individuals to travel to members/non-members labs to test a specific technique, learn about a particular aspect of the project or conduct a collaborative experiment. This enhanced the collaborative nature of the project, raised the skills base and competitiveness of the junior team members and facilitated transfer of key technologies and comparative data analysis. During the Project nine exchanges took place including one SME-academic exchange and two external exchanges with another FP7 stem cell project; BioDesign.
2) Summer School on Stem Cells and Regenerative Medicine
The summer school is primarily aimed at advanced PhD students and early stage post-doctoral scientists. This training course has been run for the past 8 years supported by FP6 and FP7 projects and has provided advanced training to over 400 young researchers. The course is led by stem cell experts who share their experience in many facets of research and is a mixture of lectures, workshops, poster and discussion sessions. During the Project PluriMes collaborated with five other stem cell projects (ThymiStem, Neurostemcellrepair, HumEn, Intens and EuroStemCell) to provide core financial support for 4 Summer Schools (2014, 2015, 2016 and 2017). In total PluriMes sent 29 lab members to be trained during the lifetime of the project.
3) Workshops and Training Courses
PluriMes organised advanced training courses/workshops which were primarily for PluriMes members but with some places made available for external scientists. Specific workshop/training topics were developed throughout the project dependent on project members’ needs but PluriMes also joined forces with other like-minded consortia to run joint workshops on topics of mutual interest. During the Project PluriMes ran 8 workshops and 2 specialist training courses:
4) Dissemination Activities
PluriMes recognised that the high levels of interest and expectation about stem cells made it crucial that accurate information on advances and challenges in stem cell research be widely disseminated. The Plurimes philosophy emphasised pro-active approaches for communication to both academic groups and lay audiences. The project was committed to an active programme of dissemination activities which had maximum scientific reach and visibility and ensured effective and wide-spread dissemination of the project results. Accordingly PluriMes secured constructive relationships with other stem cell projects (Thymistem, HumEn, NeuroStemCellRepair, Intens, REVIVE, PSCP and EuroStemCell).
Our Dissemination strategy comprised of a number of avenues which were all actively employed:
I) External Dissemination
• Project Website
• Public Engagement
o Collaboration with EuroStemCell
o Outreach Champions for each Partner
o Outreach activities by partners
o Outreach materials to inform and engage the lay public including educational and role play materials for school children and college students
o Media Communication; News features for broadcast media such as euronews
o Contribution to, and endorsement of, books on stem cell biology for non-specialist audiences,
o Accurate information on clinical trials using (or claiming to use) pluripotent stem cells or mesenchymal stem cells.”
• Publication of peer-reviewed scientific publications in leading international journals
o Publication in Open Access journals or author deposition in repositories such as PubMed
o Data deposition in public database resources
• Presentations at European Symposia and International Conferences
• Production and release of new culture media and other products for stem cell research
• Dialogue/information/briefings for policy-makers, regulators, bioindustry, relevant clinicians and administrators
• EMBO Conference: Advances in Stem Cells and Regenerative Medicine
II) Internal Project Dissemination
• The Project Training Programme
• Annual Consortium Meetings
• Project intranet
5) Coordination with Other Stem Cell Consortia
Throughout the project PluriMes has built links with other stem cell projects across Europe as well as two other stem cell consortia (REVIVE and PSCP). PluriMes, ThymiStem, NeurostemcellRepair, HumEn , Intens and EuroStemCell have worked together on a number of coordinated activities including workshops and conferences. The LabEx consortium REVIVE has built close ties with the HEALTH-funded consortia and has been actively involved in a number of events. The PSCP has run one joint event with PluriMes.
6) Advances in Stem Cells and Regenerative Medicine Conference
The ‘Advances in Stem Cell Research’ conference series was originally pioneered by the FP6 Project EuroStemCell in 2007. The FP7 EuroSyStem Project then continued that series from 2009 to 2012. In 2017 the PluriMes Project took the leading role in organising and arranging the scientific programme for the next in the ‘Advances in Stem Cell Research’ conference series. For the latest in the series two major conference series (Advances in Stem Cell Research and Stem Cells in Cancer and Regenerative Medicine) merged to create the 2017 conference titled “Advances in Stem Cells and Regenerative Medicine”. This conference was held from 23rd to 26th May 2017 at the EMBL in Heidelberg, Germany. The event was supported by FP7 projects PluriMes and ThymiStem as well as the REVIVE Project and EMBO. Over 300 people participated in the conference.
The rationale for merging the two conference series was that stem cell research did not have a dedicated European conference series. The only equivalent is the annual Keystone Symposium on Stem Cells or the International Society for Stem Cell Research (ISSCR) Meeting, which although they are not always held in the US, are overwhelmingly US-dominated. If Europe is to remain competitive, European researchers need an opportunity to build and expand networks. This conference series aims to provide that opportunity by bringing together the European stem cell community and presenting it in a rigorous and dynamic format to the International stem cell community.
7) European Summer School on Stem Cell Biology
PluriMes, in collaboration with ThymiStem, Neurostemcellrepair, HumEn and Intens, has co-hosted the summer school each year since 2014. ThymiStem provided the administrative support for the event, with core funding being provided by the five stem cell research consortia.
These courses, which have been aimed primarily at pre- and early-stage post-doctoral scientists, have provided intensive theoretical training in key issues in fundamental stem cell biology. Emphasis was placed on human stem cells, and on future application of these disciplines. To ensure the students gained an appreciation of the broader context for the field, sessions on translational stem cell research; ethics; commercialisation; clinical application and science communication were included in the programmes. Each years’ faculty was drawn from the five stem cell consortia, and the wider European stem cell community, including basic scientists, clinicians, ethicists, regulators and biotechnologists. Plenary sessions and keynote discussions featured leading international speakers.
Each year, a maximum of 54 places have been offered on these 7-day schools. Participants from the five consortia funding the school were capped at 50%, and the remaining places advertised by open call. Each PluriMes partner was given the opportunity to send two lab members to the summer school across the lifetime of the PluriMes project. Over the four years of the PluriMes Project 29 PluriMes lab members, from 11 laboratories, have benefitted from the school. Over 220 junior researchers have been trained over the course of the four schools co-funded by PluriMes.
Objective 3: Public Engagement and Outreach
PluriMes laboratories were asked to nominate Outreach Champions who would lead each Institution’s contributions to the PluriMes public engagement activities. In 2014 lab members and PIs received training rom EuroStemCell on public engagement and many subsequently undertook public engagement activities on behalf of PluriMes and their host organization. During PluriMes 56 public engagement events/activities have taken place which have been led by PluriMes members. These events have taken place throughout Europe and in a variety of languages.
The PluriMes project developed three activities and some materials which were suitable for use with all age ranges, could easily be used in any language, are low cost and very easy to run. These activities were made available to anyone from PluriMes labs which removed a traditional barrier to doing public engagement. The PluriMes public activities are as follows.
• Activity One: Play-doh is used to show the public how pluripotent stem cells can make any type of the specialised cell.
• Activity Two: Coloured M&Ms are used to explain how specialised cells can be return to a naïve state using a method called reprogramming.
• Activity Three: Dylan Stavish, a lab member from the USFD, created a dance mat game which aims to introduce the public to Fluorescent Activated Cell Sorting (FACS). In Dylan’s game ‘cells’ fall from the top of the screen to ‘beakers’ at the bottom. When the cells reach the beaker the player needs to step onto the corresponding pad on a dance mat to ‘sort’ that cell. The game is played to music and the cells fall to the beat. The game will calculate how many cells the player sorts and how many cells the player misses and calculate a sorting efficiency at the end.
• Other Materials: The PluriMes Project Office worked with various lab members to create 2 banners for use at public events. One explains about the PluriMes project and the other about stem cells in general. These could be sent to any project partner for use at their own local events.
iii) WP7 Outputs
This work package has resulted in the following outputs:
• 0 peer-reviewed research publications
• 0 reviews
• 0 patents
• 4 other products
o PluriMes website: www.pluriMes.eu
o Dance mat game on cell sorting – Public Engagement Activity
o Play-doh stem cell – Public Engagement Activity
o M&M cell washing - Public Engagement Activity
• 0 trademarks
h) Work Package 8: Management
WP8 provided the structure for all management activities related to the consortium. The Project Office consisted of a Project Manager, Jenny Nelder, and a Project Administrative Assistant, Charlotte Butler.
The Project Office was responsible for the preparation and execution of all project meetings. The project has held a wide range of regular meetings including Work Package, Consortium, Technology oversight and General Committee meetings.
Under WP8 the Project Office also managed the project reporting though the submission of sub-deliverable, deliverable and milestone reports and periodic reports. The project progress was monitored through Situation Analysis Tables (SATs) that were regularly updated and circulated to all project partners.
The financial administration of the project was carried out by the Project Manager in collaboration with the University of Cambridge Research Operations Office and other beneficiary financial contacts. Funding was distributed to beneficiaries at the correct times, appropriate financial records were collected and retained and the relevant audits were undertaken.
Following signature of the consortium agreement and grant agreement there were two formal contract amendments made in consultation with the EC Officer. The Project also held three Technology Oversight Committee meetings to ensure that IPR arising from the project was appropriately commercialised.
In addition to the major project tasks the Project Office managed the general day-to-day project administrative tasks.
i) WP8 Objectives
• Objective 1: Project Implementation Meetings
• Objective 2: Project Reporting
• Objective 3: Financial Administration
• Objective 4: Administration of Legal Issues
• Objective 5: Management of the Project
ii) WP8 Results
Period 1 was very productive:
• The ‘Kick-off’ meeting was held in February 2014 in Paris, France, during which robust scientific working practices and management methodology for the Project were discussed and agreed by all partners.
• The PluriMes website was launched on 16th May 2014.
• The PluriMes Summer Workshop 2014 was held in July 2014 in Bled, Slovenia, during which PIs and lab members began networking and creating scientific collaborations. This also presented an opportunity to fully introduce the project to all lab members which they have all found very useful. PluriMes also collaborated with EuroStemCell to run public engagement training for all PIs and lab members.
• PluriMes’ First Annual Consortium Meeting took place in February 2015 in Kranjska Gora, Slovenia. The EC Officer for PluriMes attended for the first day and an informal review of the projects progress so far took place. The SAP were also present at the meeting and reviewed the progress made so far.
• The General Committee met 6 times
• The TOC met once
• 5 Deliverables were submitted to the EC
• 3 Milestones were submitted to the EC
• 25 Sub-deliverable reports were submitted to the Project Office.
• One Contract Amendment was executed.
• All ethical documentation and cell line registrations were processed as required.
In Period 2 – Months 19-36
• The Project has held four workshops, two of which have been followed by mini-consortium meetings:
o “Tissue engineering through stem cell-based self-organisation” – Cascais, Portugal. 22nd and 23rd September 2015 (included mini-consortium meeting)
o “CRISPR/Cas9” workshop at the 2nd Annual Consortium Meeting – Zell am See, Austria. 9th February 2015
o “Single cell RNA-seq” – Nice, France. 6th and 7th October 2016. (included mini-consortium meeting)
o “Stem cell culture: culture conditions and stability workshop” – Cambridge, UK. 28th and 29th November 2016.
• PluriMes Second Annual Consortium Meeting took place from 8th to 10th February 2016 in Zell am See, Austria. The SAP were present and reviewed the progress since the previous consortium meeting.
• The General Committee met 4 times
• The TOC met once.
• 15 Deliverables were submitted to the EC
• 10 Milestones were submitted to the EC
• 50 sub-deliverable reports have been submitted to the Project Office.
• One Contract Amendment was executed.
• All ethical documentation and cell line registrations were processed as required.
In Period 3 – Months 37-48
• The Project has held 2 workshops/training courses:
o “Genetic stability in human pluripotent stem cells” – Laax, Switzerland. 6th and 7th February 2017.
o SCT specialist workshop – Cambridge, UK. 10th and 11th October 2017.
• PluriMes Third Annual Consortium Meeting took place from 6th to 9th February 2016 in Laax, Switzerland. The SAP were present and reviewed the progress since the previous consortium meeting.
• The General Committee met twice
• The TOC met once.
• 24 Deliverables were submitted to the EC
• 2 Milestones were submitted to the EC
• 32 sub-deliverable reports have been submitted to the Project Office.
• A fourth Consortium Meeting/Wash-up meeting took place from 24th to 26th January 2018 at Cliveden House, London, UK. The SAP attended and reviewed the outcomes and potential impacts of the project.
• In July 2017 PluriMes underwent an internal EC ethics audit. All ethical documentation and paperwork required was submitted to the EC. No further action was requested by the EC.
iii) WP8 Outputs
This work package has resulted in the following outputs:
• 0 peer-reviewed research publications
• 0 reviews
• 0 patents
• 0 other products
• 0 trademarks
• An additional 0 manuscripts are currently under review or in preparation
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 and on future biomedical and clinical applications. The outcomes include new and advanced knowledge, improved technology platforms, standardised protocols compatible with GMP cell manufacturing, and a coherent body of highly skilled stem cell investigators.
The overall impact of PluriMes is in defining cellular parameters for therapeutic relevance and thereby setting the stage for future clinical implementation. This has been achieved by addressing key scientific and translational challenges:
• defining the expansion and differentiation potential of naïve ground state and lineage-biased sub-states of pluripotent stem cells
• establishing resources and platforms for directed differentiation and expansion of mesodermal stem/progenitor cells from renewable PSC resources;
• clarifying the lack of common identity between different classes of tissue-derived mesodermal progenitors (so-called MSCs);
• validating differentiation potency in animal models.
• defining culture conditions and high quality reagents suitable for future scale up and GMP cell production
Specific areas of impact are outlined below:
(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 range of musculoskeletal diseases.
(b) Clinical Trials
Two of the PluriMes Deliverables have direct significance for clinical trials:
• D5.5: In Vitro assessment of therapeutic potential of mesoangioblast in restoring dystrophin production. The overall outcome of this work will allow Partner 12 to proceed to a novel Phase II clinical trial, based upon intra-arterials systematic delivery of autologous, genetically corrected mesoangioblasts in very young patients with an optimized protocol.
• D6.4: Final media formulations and reagents manufactured in a cGMP facility for the scalable expansion of mesoangioblasts and selected iPSC-derived mesodermal progenitors. The outcome of this work is that the MHRA expressed an informal favourable opinion on the used of Myocult-SF in ongoing and future trials of cell therapy for Duchenne muscular dystrophy. Following this, Partner 12 will replace the previous “gold” standard medium, M5, with MyoCultTM- SF for future clinical trials.
(c) Knowledge to Harness the Potential of Human Pluripotent Stem cells
PluriMes addressed general hurdles associated with the fundamental character of pluripotent stem cells. New knowledge of the human ground state and of pluripotent sub-states provides a rational framework for complementary approaches to achieve optimal directed differentiation for potential disease modelling and clinical applications.
(d) New Technologies
The technologies developed and exemplified make available new ways to analyse and control stem cells, such as bioengineered stem cell culture methods to enable precision application of extrinsic factors to influence cell fate, or techniques that facilitate scale-up of mesodermal progenitors while minimising genetic variation. Such technologies offer new opportunities for commercialisation and application. Thus the naïve human pluripotent stem cell medium developed by Partner 1 has been licensed by Partner 11 and developed into a commercial product and also validated for cGMP production.
(e) Regulatory Standards
PluriMes considered regulatory issues in cell therapy, 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.
(f) Bioindustry
PluriMes includes two SME partners. Their involvement realised impacts for employment, competitiveness of the technological R&D base, and wealth creation in Europe. Notably, SCT UK located research activity related to PluriMes in a new facility in Cambridge.
(g) Skills and Training
PluriMes placed a major emphasis on developing skills of junior researchers and promoted this through laboratory exchanges, specialist workshops and active participation in consortium meetings.
(h) Employment.
In July 2014, prior to PluriMes project, Stemcell UK had 11 employees (no R&D staff). As of January 2018, Stemcell UK has 44 members (Sales, R&D, and support functions). The 5 employees, representing 4 FTEs funded by PluriMes Grant will remain in the Stemcell UK R&D team. Following PluriMes success, Stemcell UK is contributing to other EU Grants, having at January 2018 a total of 9 R&D employees
(h) Consolidation of the European Regenerative Medicine Community
A broad impact aimed at by PluriMes was to promote and strengthen the general field of stem cell research in Europe. This has been achieved by coordinating very effectively with related FP7-HEALTH projects and other stem cell groupings to run joint activities, such as the Hydra Summer School in Stem Cell Biology and Regenerative Medicine, and the Advances in Stem Cell Research Conference.
(i) Public Communication and Engagement
PluriMes worked with the FP7 coordination action EuroStemCell (www.eurostemcell.org) to ensure dissemination of accurate information in accessible formats to a diversity of academic, administrative and public audiences.
Dissemination Activities
In addition to open access publications and data deposition in public archives, PluriMes undertook a range of dissemination activities:
(a) Making project knowledge and information accessible to both specialist and public audiences via the worldwide web:
• PluriMes Project website: www.plurimes.eu
• EuroStemCell portal: www.eurostemcell.org
(b) Increasing scientific knowledge, interaction, training and communication within and beyond the consortium.
• 7 workshops
• 2 specialist training courses
• 4 consortium meetings
• 9 mobility exchanges between scientific laboratories
• 15 joint activities with other consortia
• 290 presentations at European Symposia & international conferences
• 17 instances of dialogue with policy-makers, bioindustry, regulators, relevant clinicians and administrators
• 3 contributions to books on stem cell biology for specialist/non-specialist audiences
• 7 media communications about the project
Communicating effectively to a range of stakeholders and public through outreach activities.
• 61 public engagement events throughout Europe
• creation of 3 new public engagement resources
• 51 PluriMes lab members trained in public engagement
Exploitation of results: production and release of new culture media and other products for stem cell research
In 2014 - 2015, SCT/SCTUK developed and launched several new culture media into the market including MesenCult™-ACF, a novel ACF culture medium and matrix system for deriving and expanding mesodermal progenitors from primary bone marrow without the use of serum; and STEMdiff™ Mesoderm Induction Medium for the differentiation of human pluripotent stem cells to early mesoderm and downstream mesodermal differentiation. These media were relevant to PluriMes and were tested and further optimized for specific applications in WP4 and WP6 projects.
MesenCult™-ACF and STEMdiff™ Mesoderm Induction Medium consisted of defined components and reduced animal derived materials. As a result, these medium formulations already met some of the initial requirements for higher compliance product development.
In 2015 – 2016, SCT launched RSeT defined medium for naïve-like human pluripotent stem cells to the life sciences market. RSeT™ reverts primed human pluripotent stem cells (hPSCs) and maintains cells in a naïve-like state. New media formulations to generate reset hPSCs developed in Work Package 1 were compared to RSeT™ media.
SCT launched the STEMdiff Mesenchymal Progenitor Kit, a defined culture kit consisting of animal component-free (ACF) induction medium, expansion medium, and attachment substrate. It is optimized for the derivation of cells with mesenchymal progenitor cell (MPC)-like properties from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. This kit provides a complete workflow of defined reagents for derivation and expansion of human ES- or iPS-derived MPCs. MPCs have potential utility in a range of applications. ES- and iPS-derived MPCs are a promising renewable cell source for academic and translational MSC research. This kit has been successfully tested in Work Packages 4, 5 and 6 to meet the deliverables in those work packages.
SCT launched MyoCult™ Expansion Kit (Human) for the culture and maintenance of human skeletal muscle progenitor cells (myoblasts). MyoCult™ Expansion Medium suppresses key myogenic differentiation genes while conserving myogenic progenitor markers. MyoCult™ was tested in Work Packages 4 and 5 and compared to media used in partner labs. SCT has also developed a serum-free version of MyoCult™ which was tested in Work Package 5 and showed high performance. MyoCult™ serum free medium may be a useful media for a future Phase I clinical trial planned by Partner 2a.
SCT launched MesenCult™-ACF Chondrogenic Differentiation Medium, an animal component-free (ACF) medium for the in vitro differentiation of human mesenchymal stem and progenitor cells (MSCs) into chondrogenic lineage cells, including chondrocytes. This medium is suitable for the differentiation of hPSCs-derived mesodermal progenitors to the chrondrogenic lineage. This medium has been tested in Work Packages 4, 5 and 6 using various cell sources.
SCT launched mTeSR™1 manufactured under cGMP, a feeder-free cell culture medium for human embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), mTeSR™1 is a highly specialized, serum-free and complete cell culture medium. With pre-screened raw materials that ensure batch-to-batch consistency and robust feeder-free protocols for ES and iPS cell culture, mTeSR™1 provides more consistent cultures with homogeneous, undifferentiated phenotypes. mTeSR™1 is manufactured under a cGMP quality management system compliant to 21 CFR 820, ensuring the highest quality and consistency for reproducible results.
SCT launched mTeSR™3D for expansion and scale-up of undifferentiated human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells as aggregates in 3D suspension culture with a novel fed-batch protocol. This culture system is compatible with a range of suspension culture vessels used for scale up and can be used in Work Package 6 to test expansion and differentiation of hPSCs.
In period 3, SCT launched a cell culture medium kit to convert human pluripotent stem cells (hPSC) that typically exists in post-implantation state to a naïve pluripotent state. NaïveCult™ Induction Kit (Catalog # 05580), developed based on Guo et al. (2017), is composed of defined cell culture media used sequentially for the transgene-independent or chemical reversion of primed hPSCs to a naïve-like, or reset, state. NaïveCult™ Induction Kit requires the use of a histone deacetylase inhibitor (HDACi), typically sodium butyrate or valproic acid, during the initial stage of naïve state induction. The development and commercialization of this kit is the result of research performed in Work Package 1.
In addition, SCT also launched another medium in period 3, for use in expanding naïve pluripotent stem cells. NaïveCult™ Expansion Medium (Catalog # 05590) has been developed for the optimal maintenance and expansion of transgene-independent, transgene-dependent, and embryo-derived naïve-like hPSCs in t2iL + Gö conditions. NaïveCult™ media contain pre-screened high-quality components and are compatible with hPSC including human embryonic stem (ES) and human induced pluripotent stem (iPS) cells. hPSCs maintained in NaïveCult™ Expansion Medium can be converted back to a primed state by culture in mTeSR™1 or TeSR™-E8™ and can then be differentiated downstream with STEMdiff™ products such as STEMdiff™ Definitive Endoderm Kit, STEMdiff™ SMADi Neural Induction Kit, STEMdiff™ Mesoderm Induction Medium, or STEMdiff™ Trilineage Differentiation Kit. The development and commercialization of this kit is also the result of research performed in Work Package 1.
For period 3, SCT also launched MesenCult™ Proliferation Kit (Human; Catalog #05411.). This kit is a standardized, serum-containing medium for the culture of human mesenchymal stromal derived cells or mesenchymal progenitor cells. MesenCult™ Proliferation Kit (Human) is optimized for the expansion of human mesenchymal progenitor cells in vitro as well as their enumeration using the colony-forming unit - fibroblast (CFU-F) assay. Components are pre-screened to minimize lot-to-lot variability. These media are relevant to PluriMes and were tested and further optimized for specific applications in WP4 and WP6 projects outlined in PluriMes.
A kit for the differentiation of human mesenchymal progenitor cells (MPCs) to osteocytes was also launched. The MesenCult™ Osteogenic Differentiation Kit (Human; Catalog # 05465) is specifically formulated for the in vitro differentiation of MPCs into cells of the osteogenic lineage. This kit is suitable for the differentiation of human bone marrow (BM)-derived MPCs previously culture-expanded in serum-containing medium (e.g. MesenCult™ Proliferation Kit [Human; Catalog # 05411] or MesenCult™-hPL [Human; Catalog # 05439]) or serum-free medium (e.g. xeno-free MesenCult™-XF Medium [Catalog # 05420] or animal component-free MesenCult™-ACF Medium [Catalog # 05440]). This medium has been tested in Work Packages 4, 5 and 6 using various cell sources.
Lastly in period 3, a kit for the culture and maintenance of human skeletal muscle progenitor cells (myoblasts) under serum-free conditions was manufactured by SCT. MyoCult™-SF Medium suppresses key myogenic differentiation genes while conserving myogenic progenitor markers in expanded myoblasts. MyoCult™-SF was tested in Work Package 5 and showed high performance. MyoCult™-SF Medium may be a useful media for a future Phase I clinical trial planned by Partner 2a. This medium was produced in period 3 and will available to the research market by mid-2018.
Ncardia (formerly Pluriomics, Partner 4) has established a manufacturing protocol for Pluricyte® Endothelial Cells at high purity and good functionality based on IP from the Leiden University Medical Centre. Within the scope of another EU project (European Union’s Horizon 2020 research and innovation programme; SME Instrument phase 2), Ncardia is translating the current adherent culture-based large-scale process for Pluricyte® EC production towards a better controlled and more robust method for further scale-up using fully controlled stirred tank bioreactors. Although Pluricyte® Endothelial Cells are not a commercial product at the moment, Ncardia has already used the cells in some customized service projects. Ncardia is currently reviewing the status of Pluricyte® Endothelial Cells to enter a product development program for customized safety and efficacy services. This will ultimately enable pharmaceutical companies to assess cardiovascular safety and efficacy of their potential drugs already early in development. Importantly, early screening and selection of drugs in predictive in cardiovascular in vitro model systems will lead to significant reduction of development costs and reduction of animal testing.
Partner 9 (EPFL) developed chemically defined hydrogel microcarriers for the large-scale expansion of mesodermal progenitors. These have applications in the areas of biotechnology, cell manufacturing, regenerative medicine. Partner 9 is currently exploring options with a possible commercial partner.
Partner 7 (USFD) developed a medium, named Primo, that supports the maintenance of mesoderm biased human pluripotent stem cells. Partner 7 is currently seeking patent protection for the formulation of Primo medium and exploring the possibility of licencing this to Partner 11.
List of Websites:
Website: www.plurimes.eu
Project Coordinator Details: Professor Austin Smith, Wellcome - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR
Tel: +44 (0) 1223 760233
Fax: +44 (0) 1223 760241
Email: ags39@cam.ac.uk
Project Office Contact Details: PluriMes Project Office, Wellcome - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR
Tel: +44 (0) 01223 746818
Fax: +44 (0) 1223 760241
Email: plurimes-office@stemcells.cam.ac.uk
