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European Consortium for Stem Cell Therapy for Neurodegenerative Diseases

Final Report Summary - NEUROSTEMCELL (European Consortium for Stem Cell Therapy for Neurodegenerative Diseases)


Executive Summary:

NEuroStemCell is formed to create a world-leading consortium that can take stem cell based therapies for Parkinson´s disease (PD) and Huntington´s disease (HD) to the clinic. The consortium brings 13 together elite European and American research teams and 2 SMEs from 6 EU member ountries representing the broad range of expertise necessary to reach this goal, including stem cell specialists, developmental neurobiologists, scientists and clinicians with expertise in animal models of PD and HD and in vivo imaging, with the goal to develop safe and validated cells and clinical grade reagents to be used in clinical trials and eventually also in drug discovery. The regulatory, ethical and societal issues associated with the use of stem cells for therapy will be carefully considered as science progresses from bench to bedside.

PD and HD are ideal candidate diseases for restorative stem cell-based therapies. In both diseases the pathology is slowly progressive and characterized by the preferential loss of one type of neuron, i.e. the mesencephalic dopamine (mesDA) neurons in PD and the GABAergic medium sized spiny neurons in HD. The cell replacement strategy aims at substituting the lost mesDA and GABA neurons, respectively, by implantation of new functional cells. Although other cell types are ultimately affected, experimental evidence obtained in rodent and primate models of PD and HD, as well as the experience gained from clinical trials using grafts of fetal mesDA and striatal GABAergic progenitors, indicate that effective restorative therapies may be possible to achieve by neural transplantation in these two diseases. Further development of this approach, however, will critically depend on the development of alternative sources of therapeutically effective cells derived from stem cells.

NeuroStemCell is formed to create a world-leading consortium that can take stem cell based therapies for Parkinson´s disease (PD) and Huntington´s disease (HD) to the clinic. The consortium brings 13 together elite European and American research teams and 2 SMEs from 6 EU member ountries representing the broad range of expertise necessary to reach this goal, including stem cell specialists, developmental neurobiologists, scientists and clinicians with expertise in animal models of PD and HD and in vivo imaging, with the goal to develop safe and validated cells and clinical grade reagents to be used in clinical trials and eventually also in drug discovery. The regulatory, ethical and societal issues associated with the use of stem cells for therapy will be carefully considered as science progresses from bench to bedside.

PD and HD are ideal candidate diseases for restorative stem cell-based therapies. In both diseases the pathology is slowly progressive and characterized by the preferential loss of one type of neuron, i.e. the mesencephalic dopamine (mesDA) neurons in PD and the GABAergic medium sized spiny neurons in HD. The cell replacement strategy aims at substituting the lost mesDA and GABA neurons, respectively, by implantation of new functional cells. Although other cell types are ultimately affected, experimental evidence obtained in rodent and primate models of PD and HD, as well as the experience gained from clinical trials using grafts of fetal mesDA and striatal GABAergic progenitors, indicate that effective restorative therapies may be possible to achieve by neural transplantation in these two diseases. Further development of this approach, however, will critically depend on the development of alternative sources of therapeutically effective cells derived from stem cells.

NEuroStemCell is focused on the identification and systematic comparison of progenitor cell lines with the most favourable characteristics for mesDA and striatal GABAergic neuronal differentiation, generated either directly from human embryonic stem (ES) cells1, from Neural Stem (NS) cells2 derived from ES cells or fetal brain, from induced Pluripotent Stem (iPS) cells3 or from in vitro short-term expanded neural progenitors from ventral midbrain grown as neurospheres (VMN, Ventral Midbrain Neurospheres)4, and perform rigorous and systematic testing of the most prominent candidate cells in appropriate animals models. The consortium will engage in parallel into a number of educational activities and promote the development of resources for patient groups, regulators and lay public.

Project Context and Objectives:

In line with the purpose of the call the NeuroStemcell consortium made use of alternative sources of in vitro expandable embryonic and neural stem cell lines for the development of cell-based therapies for two major neurodegenerative diseases, PD and HD. The core research program was focused on the comparison of the survival, differentiative and migratory capacity of stem cell-derived neural progenitors, and the optimization of their survival and function after transplantation in appropriate animal models. The work was focused on cells of human origin, i.e. the development of cells that can be used clinically, but in the initial phase of the program experiments on rodent stem cells (mouse and rat) was performed in parallel as a convenient tool to assist in defining the optimal conditions for the generation of transplantable mesDA and striatal GABAergic progenitors. Imaging and a full repertoire of behavioural studies assisted in deciphering the pattern of donor-host interactions so as to improve the understanding of the biological processes necessary to standardize the survival, differentiation and safe functional integration of the donor cells in vivo.

The translational part of the program was focused on the manufacture and safety assessment of clinical grade reagents and cells for use in clinical trials by the implementation of clinical, regulatory, societal and ethical issues at all stages of the pre-clinical work, and the development of protocols for phase-I clinical trials in PD and HD.

NeuroStemcell represents, in part, a continuation of work carried out within the framework of the FPVI Integrated Project EuroStemCell in which neural stem cell lines have been developed and optimized for transplantation. NeuroStemcell continued to build on the resources provided by this network and also engage constructively with other FPVII stem cell projects in fundamental stem cell biology, regenerative medicine and cell therapy.

NeuroStemcell established an international Scientific Advisory Panel (SAP) to provide high level advice and monitoring of the project. Four internationally leading scientists accepted to join this board: Prof William Langston, The Parkinson’s Institute, Sunnyvale (leading PD neurologist and clinical scientist), Prof Nancy Wexler (leading HD researcher and chairman of the Hereditary Disease Foundation in New York), Prof Ole Isacson (stem cell researcher at Harvard University in Boston), and Prof Ron McKay (international leader in the stem cell field at the NIH). The SAP reviewed the progress of the project annually and provide advice and guidance on the work.

The consortium was designed to maximize synergy between the participating teams and foster a productive interface between experimentalists and clinicians. The research plan was constructed primarily around collaborative projects and is closely linked to the requirements associated with the clinical use of stem cells. Furthermore, in order to stimulate and enable new directions and interactions a reserve fund was set aside in the budget specifically to support innovative sub-projects proposed by the PIs, or for the recruitment of new PIs, if needed. This strategy has proven extremely valuable and successful in the EuroStemCell consortium, encouraging the conversion of new leads into productive collaborations.

The work plan was structured into six closely linked workpackages (WPs):

In Work-package 1 the goal was to improve the understanding of the biological processes involved in the controlled differentiation of embryonic and fetal stem cells and of the most recent iPS cells and, based on this knowledge, to devise optimized protocols for the generation of mesDA and striatal GABAergic neurons in clinically relevant numbers for transplantation.

In Work-package 2 we studied the in vivo performance of stem cell-derived mesDA and striatal GABAergic cell preparations with the goal to identify promising candidate cell lines that have the ability to differentiate into authentic mesDA and striatal GABAergic neurons in high numbers after transplantation, in the absence of any tumor formation.

In Work-package 3 we performed a comprehensive pre-clinical testing of the functional efficacy of the stem cell-derived mesDA and striatal cell lines in relevant animal models of PD and HD, with the goal to identify and validate the most promising cell preparations to be used for further development towards clinical use.

In Work-package 4 we explored the usefulness of MR and PET imaging techniques for monitoring, non-invasively, the survival, growth, function and potential adverse effects of the grafted cells, with the goal to devise a combination of MR and PET imaging that can be included as assessment tools in the clinical transplantation protocols.

In Work-package 5 the criteria, technology, and protocols for manufacture of stem cells and their progeny to clinical grade standard were addressed with the ultimate goal to generate a cell bank for fully validated, safe and efficacious cells, ready for clinical use and drug discovery.

Work-package 6, finally, addressed the complex clinical, societal, regulatory and ethical issues that are associated with the development of stem cells for clinical use. The scientific and regulatory requirements for cells to be useful for transplantation in PD and HD patients were defined, linked to a road-map aimed at the implementation and design of clinical phase-I clinical trials in patients with PD and HD.

In summary, NeuroStemcell while pursuing the following ten major goals, ensured the corresponding expected outcomes:

- To develop protocols for production of transplantable mesDA and striatal GABAergic progenitors from human ES and NS cell lines, building on recent developments in stem cell biology (Peer-reviewed scientific publications, Patents, Commercial licenses);
- To carry out pre-clinical testing of stem cell-derived mesDA and striatal GABAergic progenitors in rodent models of PD and HD with the goal to generate efficient and safe candidate cells for clinical use (Peer-reviewed scientific publications);
- To identify procedures and safety systems that will allow complete elimination of proliferative and/or tumour-forming cells from the graft cell preparations (Peer-reviewed scientific publications, Patents, Commercial licenses)
- To apply in vivo imaging tools for non-invasive monitoring of the survival and growth of the grafted cells, as well as tools to reveal adverse immune/inflammatory reactions to the graft (Peer-reviewed scientific publications, Protocols for use in clinical trials, Validated new imaging ligands)
- To develop criteria, procedures and protocols for reproducible, safe and large-scale manufacture of stem cells and their progeny to clinical grade standard (Peer-reviewed scientific publications, Patents, Commercial licenses)
- To establish a banked stock of validated, safety-qualified and traceable cells for clinical use (Peer-reviewed scientific publications, Cell bank for use in clinical trials, Commercial licenses)
- To develop clinical protocols to be used in phase-I trials in patients with PD and HD (Peer-reviewed scientific publications, Protocols for clinical phase-I trials)
- To establish ethical and safety criteria to guide implementation of stem cell therapies in the clinic (Peer-reviewed scientific publications, Principles for assessment of safety in clinical stem cell trials in neurodegenerative diseases, Ethical guidelines for use of stem cells in PD and HD therapy)
- To develop an interface with bio-industry and devise a product-specific regulatory strategy that is compatible with a future marketing authorization (Guidelines, Commercial licenses,- Defined regulatory road-map)
- To provide accessible information to patient organizations and lay public about the advancement in the field and future critical steps to be fulfilled (Public web site, Workshops, Media releases)

enable new directions and interactions a reserve fund was set aside in the budget specifically to support innovative sub-projects proposed by the PIs, or for the recruitment of new PIs, if needed. This strategy has proven extremely valuable and successful in the EuroStemCell consortium, encouraging the conversion of new leads into productive collaborations.

The work plan was structured into six closely linked workpackages (WPs): WP1 focused on the generation and in vitro characterization of mesDA and striatal GABAergic progenitors from human ES, NS and 2i-iPS cells and from transiently expanded human VMN. In WP2, 3 and 4 the Partners performed extensive testing and comparison of the cells in the most appropriate in vivo disease models, including detailed phenotypic characterisation and assessment of the neuroanatomical, immunological, behavioral and functional properties of candidate therapeutic cell lines. The risk of overgrowth or tumor formation was addressed by procedures for selection of non-proliferating lineage-restricted progenitors, in combination with in vivo imaging and exploration of “safety switches” that allowed us to eliminate proliferative cells from the graft cell before and after transplantation. WP5, led by the SME Partners, focussed on the manufacture and safety of clinical grade cells and reagents, and work out criteria for defining the suitability of the cells for clinical application. WP6 guided the work performed in WP1-5 so that issues critical for clinical application were adequately and specifically addressed, integrated aspects of regulatory requirements as well as societal and ethical issues, and developed protocols for phase-I safety trials to be conducted in PD and HD patients.

Project Results:

In the first 12 months NeuroStemcell has fully and consistently established and activated all of its scientific committees, management structures, direction boards, with reference to both Consortium internal resources (Partners) and external advisors/contractors (Scientific Advisory Panel-SAP, subcontractor, website development).

The project has started with the kick off meeting in Copenhagen (Dec. 14-15 2009), where all the managing committees met (Management Group-MG, Board of Director-BoD, Steering Committee-SC) to set out a final internal system of science control based on Progress Check Points (PCP) leading to a final deliverable (DEL), to identify involvement of each PI, to plan outreach & training activities for the first year and to identify the tasks for each member of the MG and finalize and approve the composition and role of the technical committees (Good Manufacture Practice-GMP and Technological Evaluation Panel-TEP). To facilitate good communication and interaction between partners, an internet conferencing plan has been implemented for all work packages.

The Project Office in Milan has been fully established: the team is composed by Gianni Munizza, Project Manager (PM), Simona Castiglioni assisting the PM on general enquiries and administrative tasks and Valentina Brambilla, as part time assistant to outreach & training activities, communication issues and website update and control. The Outreach & Training Office in Lund has been established under the guidance of the Networking Director Malin Parmar and involving Eva Nordin for all logistics. The website www.neurostemcell.org has been put in existence and full activity, project logo and graphic communication materials have been designed and produced as tools of the 4 year project. The web site has a public domain that provides general information about the project and its goals, in addition to promoting NeuroStemcell dissemination activities. The members-only area is for deposition and exchange of research results between the partners.

The consortium funds were distributed by the Coordinating unit and activity commenced immediately at all sites as planned in the TA. During the first 12 months, NeuroStemcell has:

- appointed a distinguished SAP of leading scientists and clinicians (Ronald McKay, Nancy Wexler, Ole Isacson, William Langstone)
- held at month 5, the first annual consortium meeting (3 days, 62 researchers), during which all PIs and several junior scientists presented their initial data and reinforced the planned collaborations.
- Activated the procedures for and achieved recruitment of a subcontractor for regulatory issues
- Established all procedures for the planned Outreach & Training programme
- Participated in the first joint Hydra Summer School on Stem Cell Biology (sept 2009) together with two other consortia (EuroSyStem and Optistem)
- Organized the first NeuroStemcell workshop in Hydra (sept 2009; 2 days, 37 participants)
- Begun the activities of two (of the four) WINGS groups on “In vivo” and “Patternability”, these being small teams of postdocs and PhD students from the participating laboratories focussed on specific research objectives.

During months 13-24 (period II) NeuroStemcell Committees have operated according to their role as described in Annex I to ECGA and according to practise and internal regulations established during Year I of the project. The project office has continued its tasks in coordinating all day to day aspects of the project, especially verifying that all deadlines both on scientific and management tasks are respected. Under the supervision of the Coordinator and the Networking directors has organised meetings, webconferences, and helped to promote interactions between the groups with existing and refined programmes. The overall budget of the Consortium was carefully monitored for each partner, and the first payment was organised according to the level of expenditure of the Partners. Three amendements were positively concluded with the entry of a new Beneficiary and the partial reorganisation of work package 5 with the inclusion of a new subcontractor. The website was provided with new tools and documents both for the consortium members and the general public. Interactions with other Consortia were also enhanced through the organisation of common outreach activities.

In the first 12 months NeuroStemcell fulfilled commitments to 14 Deliverables and 7 Major Milestones (and to 38 internal Progress Check Points). The Consortium has already published or had accepted for publication 12 peer reviewed publications (which acknowledge European Commission support). One manuscript is currently submitted and 9 are in preparation. The research programme of NeuroStemcell is aimed at the comparison of the survival, differentiative and migratory capacity of stem cell-derived neural progenitors and the optimization of their survival and function after transplantation in PD and HD animal models. Major advancements in this direction has occurred in WP1, as partners 2a and 7a have produced and validated Lmx1a- and Nurr1-eGFP knock in ESC lines for the generation of mouse dopaminergic neurons and then successfully translated this approach to human cells. Differentiating human ES cells transduced with a Lmx1a expressing lentivirus were also shown to develop into TH positive neurons co-expressing transcription factors typically found in the ventral mesencephalon (this work has been published in a leading journal; Friling et al., PNAS, 2009). Partner 4a has uncovered the role of oxysterol in augmenting the generation of DA neurons from human ES cells (this was reported in another leading journal, Sacchetti et al., Cell Stem Cell, 2009) and also demonstrated that Wnt/beta-catenin signalling blockade promotes neuronal induction and dopaminergic differentiation in mouse ES cells (Cajanek et al., Stem Cells, 2009). Partner 1 reported the derivation of NS cells from iPS which were mouse fibroblasts derived and described their neurogenic capacity (Onorati et al., Mol Cell Neurosci., 2009). Under the support of two other EU consortia, partner 3 has recently generated human ES cell derived neural stem cells which exhibit extensive self-renewal (these cells have been named long-term (lt-)hESNSC) and potential for synaptic integration. Within NeuroStemcell, partner 3 has joined forces with partner 7a to first generate reliable and safe human iPS cell lines with reduced numbers of reprogramming factors and excisable constructs and then converted them into clinically relevant primitive long-term self-renewing neuroepithelial cells (lt-NES, Figure 1) capable of giving rise to functionally active neuronal subtypes. This work is ready for submission to a leading journal (Falk, A., Koch P et al.).

Figure 1 Lt-NES cell lines derived in partners 3 and 7a laboratories from a variety of human iPS cell lines stain positive for neural stem cells markers Nestin, Sox1, Sox2, and Pax6 as well as for neural rosette markers Dach1 and PLZF. NES cells grow in a rosette-like organisation with apical expression of ZO-1.

In WP3, partner 5 has progressed considerably in the longitudinal analyses of the behavioral phenotypes in four different HD transgenic mouse lines. This work is critical in order for the partners to perform cell transplantation trials in a fully characterized HD mouse line. In WP4, partners 6 and 8a have already established an experimental paradigm to study adverse side-effects (rejection/overgrowth) associated with human hNSC intrastriatal grafting into a rat model of Huntington’s disease. High field MRI and MRS data from early time points of these longitudinal follow-up of partner 8a’s neural stem cells grafts demonstrate the feasibility and pertinence of such paradigm to address these questions. In the context of WP5 a cell bank has been established at partner 11 in which stem cell lines from several partner laboratories have already been deposited. Work in WP6 has led to a document which is the first description of what will be required from stem cell-derived cells for moving them to clinical application in PD and HD patients. Some of this information is included in a manuscript by the WP leader which is published in a leading journal (Lindvall and Kokaia, Journal of Clinical Investigation, 2010).

Networking and training. NeuroStemcell has organized one workshop and co-organized a Summer School. The WINGS activities are already fully operational and a series of exchange schemes have been set up to facilitate interaction and sharing of informations.

During months 13-24 (period II) NeuroStemcell fulfilled commitments to 19 Deliverables and 12 Major Milestones (and to 67 internal Progress Check Points). The Consortium has published or hadaccepted for publication 17 peer reviewed publications which acknowledge European Commission support. 8 manuscripts are currently submitted and 7 are in preparation. Among these, 6 are joint publications among different NeuroStemcell PI/laboratories.

Major advancements in WP1 have been made in the generation of series of relevant cell-specific and stage-specific human reporter lines, in the combinatorial transcription factors expression to promote lineage specification and in the optimization of protocols which employ cell extrinsic factors and transcription factors expression for driving terminal differentiation.

In particular:

(i) hESC BAC transgenic Hes5::GFP, Nurr1::GFP, Pitx3::GFP reporter lines relevant to midbrain DA neuron differentiation have been generated by partner 14 (Ganat Y. et al., submitted) and one (Nurr1::GFP) validated and already distributed to partner 2a. Further constructs (FoxA2::GFP, Corin::GFP, Lmx1A::GFP, WNT1::GFP) and lines (Dll1::eGFP) have been generated and/or have entered subsequent in vivo studies in partners 2a and 14 laboratories. Three other reporter lines that are specific for ventral forebrain precursors (Nkx2.1::GFP Dlx2::tVA, Islet::GFP) have been generated/incorporated by partners 1, 8a, 14 into the work flow and one (GSH2::GFP) is in its advanced construction by partner 8a and 14.
(ii) The work by partner 2a on using microRNA regulated lentiviral vectors for tracking differentiating neural progenitors obtained from hES has been completed and published in a leading journal (Sachdeva R. et al., PNAS 2010). Further developments of this work by partner 2a have already led to a new, validated GFP-mir292T vector that includes target sequences for the neuron specific mir-124, allowing selection for transplantable human neural progenitors.
(iii) Partners 2a, 3, 4b have reported conclusively on the impact of Lmx1a- and, partner 9, Dmrt5- forced expression in promoting mesDA neuronal differentiation (Kirkeby A. et al., in preparation; Gennet N. et al., submitted revised) and demonstrated the efficacy of the combinatorial expression of Lmx1A, Otx2, FoxA2, En1 in driving mesDA reprogramming of human fetal neural progenitorss (Panman L. et al., submitted revised).
(iv) Partners 3 and 7 have worked to apply the lt-hESNES protocol generated in 2009 by partner 3, to other human ES and human iPS cell lines (Falk A. et al., under review).
(v) Partners 4a, 7a and 2b further established a method for propagating human ventral midbrain (hVM) neural stem/progenitor cells present in the VM tissue and subsequently differentiating them by addition of Wnt5a, with a resulting 4-fold more DA neurons than the starting VM preparation (Ribeiro D. et al., submitted).
(vi) Partner 4 has reported a novel differentiation protocol based on sequential Wnt3a and Wnt5a treatment while partner 14 has reported a novel DA neuron differentiation protocol based on the combined early exposure to SHH, FGF and canonical Wnt signals that drive DA neuron specification via a FOXA2+ floor plaste intermediate stage. This protocol is now being applied to Nurr1::GFP reporter lines. For the generation of striatal donor cells one major advancement is represented by a new protocol developed by partner 14 which permit the derivation of FOXG1 positive anterior precursors which can be maintained in culture for more than 200 days. In addition, a modification of a Dual SMAD-inhibitor induction system has been successfully applied by partners 1, 8a to generate striatal progenitors.
(vii) Partner 7a has developed a new reprogramming strategy for the generation of transgene-free iPS cells.

Collectively, new tools and optimized protocols for hES have been developed in WP1 that have allowed a substantial advancement in term of number and quality of mesDA and GABAergic progenitors and differentiated neurons obtained from hES.

Advancements in WP2 have been made in defining consensus criteria for selection of cells for transplantation in models of PD and HD. Some of the main concepts were tested experimentally by isolating and FACS purifying subpopulations of neurogenic cells and neurons obtained from BAC transgenic human ES reporter lines established in WP1. Work in this direction performed by partner 14 has led to the identification of miR371-3 level as a critical predictive marker enabling the pre-selection of highly neurogenic human pluripotent cells (Kim H. et al., submitted revised). A further improvement in the purification of transplantable CNS cell types has been achieved by addition of NP1 molecule identified by partner 14 (Lafaille F. et al., in preparation) and further improvements are expected as a follow up of the comparative transcriptomic profile on NPC versus lt-hESNES completed by partners 3, 8a, 14.

Partners 2a, 4b and 14 have reported on the successfull transplantation of (mouse) reporter cell lines in PD animals (Ganat Y. et al., submitted revised). In contrast, transcription factor-overexpressing or morphogen treated human cells have yet to demonstrate efficient survival and differentiation. However, partner 14 together with partner 2a have new transplantation trials ongoing with Nurr::eGFP cells exposed to the protocols identified in WP1. In fact, data in mouse have shown that the Nurr1 stage of ES cell derived midbrain DA differentiation is most suitable for the survival of TH+ cells (Ganat Y. et al., submitted revised).

Partner 3 has reported that transient Notch inhibition promotes neuronal differentiation of lt-hESNES cells after transplantation (Borghese L., et al., 2010) and partners 3 and 14 have identified strategies to enhance migration and innervation of stem cell derived donor cells.

Taken together the results in this WP provide important steps towards the development of a cell replacement strategy in PD.

In WP3 partner 3 has shown that neurons from human pluripotent stem cells are endowed with a remarkable potential to establish orthotopic long-range projections in the adult mammalian brain (Steinbeck J. et al., submitted). Partners 5 has tested a number of HD transgenic mice in grafting experiments. Partners 5, 3 and 7 have reported substantial differentiation of lt-hESNES cells after transplantation into HD models, however not stable differentiation into DARPP32 striatal specific phenotypes. New protocols and lines from WP1 are now transferred to WP3. For PD, partner 14 has conducted a number of studies with unsorted HES5::GFP, Nurr1::GFP and Pitx3::GFP cells and has defined the conditions and protocols that eliminate the rosette stage cells that lead to graft overgrowth.

In WP4 a new radioligand has been identified and validated for its ability to detect microglial activation in the HD rat model.

In the manufacturing and safety arena (WP5), trehalose has been validated by partner 11 as an alternative to DMSO to cryopreserve human neural stem cells (Dessi S.S et al. patent in preparation at BioRep s.r.l.). In addition, partner 11 has fullfilled commitment to act as a cell bank for the Consortium (and beyond) through a consolidated procedure (partners 1, 3 and 7 have already deposited cell lines at partner 11’s biorepository) (Diaferia G.R. et al., in press; Diaferia G.R. et al., submitted). Standard operating procedures (SOPs) have been defined and released). Furthermore, partner 1 and 11 have completed a chromosomal analyses on mouse neural stem cells establishing the conditions for a similar work on human cells (Diaferia G.R. et al., 2011).

Work within WP6 has progressed according to plans, with the production and delivery of a consistent report on the “Regulatory requirements and strategies for cell therapies for neurodegenerative diseases” produced by ERA Consulting under the coordination of partner 2b. A second deliverable is represented by a working paper produced by partner 12 on “Protocols for risk assessment and safety testing of stem cell therapy in PD and HD”. A working paper on “The cases of PD and HD: ethical and epistemic considerations” is also in advanced phase of preparation by partner 2 and will be submitted after the scheduled Ethical Workshop (middle 2011) (Hug K. et al., in preparation). A fourth report has been prepared by all partners involved in WP6 on the “Advancements of and problems with generated cell lines from clinical perspective”.

TRAINING AND OUTREACH (YEAR II)

In addition to its contracted activities (workshops, summer school, annual meeting) NeuroStemcell has successfully created new opportunities for interaction among young scientists and new creative ways of communicating science to the general public. The WINGS programme was developed at the end of year I and fully implemented during year II: the groups have collaborated under the supervision of the Networking and Training director to further explore specific areas defined by the WINGS Committee. As public engagement is concerned, NeuroStemcell and the Consortia EuroSyStem, EuroStemCell, OptiStem, EsTools have communicated science in unexplored ways: a theatre play (Staminalia: a dream and a trial) to represent the complex palette of emotions and human feelings of a scientist and a documentary (Behind the Science) to recognize that modern scientists do not work alone and to show how the complex EC funded networks work.

During months 25-36 (period III) NeuroStemcell Milano project office has continued its tasks in coordinating and monitoring activities, deadlines and reports, while verifying that new scientific needs and interactions among the partners were duly considered and, where appropriate, put into action. After April 2011, precise elements of the research program were discussed and reshaped based on the mid-term report,. Changes include addition of new deliverables and the opening of two new internal calls aimed at strengthening and broadening the scope of neurostemcell. The project manager monitored all phases of the submissions and of the evaluation, announced the awardees and verified that the new collaborations started. In addition, as a consequence of the reshaping, a new inter-consortia activity has started between NeuroStemcell and the Transeuro Consortium.

Under the supervision of the Coordinator in Milano and of the Networking Director in Lund, the two offices have also promoted scheduled meetings and web conferences. Furthermore, the overall budget of the consortium was constantly monitored for each partner by the project manager. The web site was refined and constantly updated and interaction with other consortia in term of outreach and public communication well coordinated and effective. In the last few months of year III, recruitment of a science communicator allowed the organization of an extra level of communication enabeling communication of major break throughs as well as involvement in current issues debates.

During months 25-36 NeuroStemcell fulfilled commitments to 22 Deliverables and 7 Major Milestones (in addition to 44 Internal Progress Check Points, PCPs). The consortium has published or has accepted for publication 30 peer reviewed publications which acknowledge NeuroStemcell support, among these, 2 are joint publications among different NeuroStemcell PIs. Another 11 papers are currently submitted, under revisions or in preparation..

In Year III NeuroStemcell activities have shifted robustly towards clinical translation with major roadblocks passed. In Major achievement includes a new protocol for the generation of substantia nigra DA neurons from human pluripotent stem cells that ameliorated PD symptoms in animal models without forming tumors, novel strategies for prescreening of progenitors based on the level of a specific miR in order to select lines with better differentiative performance in vitro and in vivo, the ability of a network of transcription factors to implement dopaminergic differentiation in ES cells. Additionally, a new differentiation protocol is under refinement for the generation of striatal medium spiny projection neurons from human pluripotent stem cells. Additional major advancements are represented by the development of a chemical-based strategy that enhances migration of donor derived neurons and by parallel sorting strategies that allow to exclude the risk of overgrowth. Finally, new sensitive tasks of striatal function have been developed for both rat and mouse models of PD and HD allowing to test any donor cells for motor and cognitive improvements. Collectively, these achievements, in addition to the recently developed tolerization model, represent key assets in NeuroStemcell final year’s effort to move stem cell therapy towards the clinics.

In particular, significant progress in WP1 include:

(i) The development of transgene-free human iPS cells (Partner 7a) which are able to differentiate into TH+, FoxA2+ and Pitx3 + neurons
(ii) The generation and propagation of forebrain neural stem and precursor cells from the above and other human iPS and ES cells (Falk A. et al. PLos One, 2011) (Lafaille, in preparation)
(iii) the generation of authentic midbrain dopamine neurons directly from human pluripotent stem cells using a floor-plate-based strategy that does not require further genetic modification and does not involve an NSC intermediate (Partner 14). More work is undergoing in Partner 2a lab with dual SMAD inhibitors, SHH and wnt agonists under fully defined conditions. (Kriks et al., Nature 2011) (Kirkeby et al. under submission).
(iv) the subsequent demonstration, impacting WP2, of excellent survival of grafted DA neurons in mouse, rat and monkey models of PD, which was associated with significant behavioral improvement, including complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia (Partner 14) (Kriks et al., Nature 2011 and Kirkeby et al. under submission).
(v) The refinement of a protocol for the differentiation of human pluripotent stem cells into striatal medium spiny neurons carrying electrophysiological properties (Partner 1, 5, 7b) (DelliCarri, in preparation)
(i) The demonstration of the role of transcription factor Lmx1a (Partners 2a, 3, 4b, Bjorklund, Brustle, Perlmann) and Dmrt5a and FGF signalling (partner 9) in the specification of ventromedial midbrain neural progenitors in vitro (Gennet et al., PNAS 2011; Jaeger et al., Development 2011; Deng Q. et al., Development. 2011).
(vi) The use of optogenetics to show that grafts of Wnt5a-overexpressing neural stem cells from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice become functionally integrated in the DA-denervated striatum (Partner 2b) (Tonnesen et al., PLoS ONE 2011).
(vii) The direct conversion of human fibroblasts into dopaminergic neurons (Partner 2a) (Pfisterer et al., PNAS 2011).
(viii) Global transcriptome analyses in combination with the expression of various transcription factor determinants for mesDA, serotonergic neurons and motoneurons (Partner 4b) (Panman et al., Cell Stem Cell 2011) while Partner 14 conducted comparative transcriptome analyses using different mesDA neuron differentiation protocols and demonstrated that early fate specification predicts proper dopaminergic differentiation and best in vivo engraftment.

Advancements in WP2 include:

(i) The identification of miR-371-3 level as diagnostic of the differentiation capacity of pluripotent stem cell lines (Partner 14) (Kim H et al. Cell Stem Cell. 2011), including in iPS from PD patients with genetic diseases (PINK1, LRRK2 and Parkin mutations).
(ii) The successful employment of MACS sorting of PSA-NCAM+ post-mitotic neurons to separate postmitotic mDA neurons from contaminating rapidly dividing cells in ESC cultures with demonstration of no overgrowth after transplantation (Partner 4, 2a)
(iii) The demonstration that GCV treatment efficiently excludes proliferative HSV-TK expressing human ES derived cells after grafting in HD models (Partners 5, 8)
(iv) The development of a tolerization protocol that allowed human cells to be tested in vivo in rodent models of neurodegeneration over an extended period (up to 25 weeks so far tested) (Partner 5).
(v) The identification of grafts intrinsic mechanisms inhibiting donor cell migration and integration with demonstration that a small molecule inhibitor of receptor tyrosine kinase successfully overcome the autoattraction of the donor cells (Partner 3) (Ladewig J et al. in preparation) (Ladewig, J., Koch, P., Brüstle, O., 2009, patent PCT/EP2009/002149; PCT/EP2010/001841)
(vi) A number of solutions to the unsolved issues of the authenticity, integration and functional efficacy in rigorous animal tests of human pluripotent stem cells derived DA neurons, as described in points (iii) and (iv) of WP1. (Partners 2a, 14) (Kriks et al., Nature 2011).

Furthermore, a collaboration has been established with the Transeuro consortium to develop an improved protocol for the short-term expansion of DA progenitors and their efficient differentiation into midbrain DA neurons in order to reduce the number of foetal tissue required to promote functional recovery in the 6-OHDA model of PD and subsequent translation to the clinic (NeuroStemcell Partners 4a, 2b, 7 and Transeuro Partners 2b and 7).

Progress in WP3 include:

(i) The development of at least three protocols for the successful derivation of TH expressing cell lines that survive engraftment in vivo, with no evidence of overgrowth while alleviating the drug-induced rotational bias observed after the dopamine-depleting lesion of the nigrostriatal pathway (Partners 2a, 9, 14).
(ii) The development of behavioural tasks sensitive to striatal dysfunction (Partner 5) (Lelos M.J. et al, Behav Brain Res. 2011) (Trueman R.C. et al. Brain Res Bull, 2011)
(iii) Two large-scale experiments aimed at assessing in vivo the functional efficacy of ES-derived cells differentiated towards a striatal phenotype (Partners 5, 1)
(iv) The in vivo assessment of two potentially viable cell lines (Partners 5, 1).
(v) The demonstration that one of these lines has been established to successfully tolerise the rat (i.e. allowing for long-term assessment of cells in vivo without need for immune suppression treatment) (Partner 5)
(vi) The application of labelling techniques to visualise integration of the graft (Partner 3)

Advancements in WP4 include:

(i) The successul in vivo characterization by atraumatic imaging tools of the 6OH-DA induced lesion in rat using: proton NMR spectroscopy to identify possible changes in the metabolite profile induced by dopaminergic deafferentiation at the striatal level; neuroinflammatory PET markers (18F-DPA714) to characterize potential detection of astrocytic/microglial activations at the striatal and midbrain level; the D2-receptor antagonist 18F-fallypride, as a means to monitor presynaptic dopaminergic deficiency.
(ii) The demonstration of the usefulness of various PET ligands to monitor phenotypic differentiation and the occurrence of potential adverse effects (neuroinflammatory reaction, proliferation, astrocytic reaction) following intrastriatal grafting of various human stem cell preparations in immunocompetent and athymic nude rats. These PET tracers include: 11C-DPA713/18F-DPA714/11C-SSR180575 as specific markers of the microglial/astrocytic reaction observed either within the grafted previously excitotoxically-lesioned striatum, or within the graft undergoing rejection; 18F-FDG as a specific marker of glucose metabolism revealing that graft implanted at various stages of in vitro differenciation and leading to hyperproliferation are not hypermetabolic but hypometabolic instead; 18F-fallypride as a specific marker of terminal differenciation of striatal progenitors into mature striatal neurons expressing the post-synaptic dopaminergic D2-receptors.

Progress in WP5 include:

(i) The development of a protocol for the microcarrier-based expansion of lt-NES® in spinner flask bioreactors (Partner 3, with the subcontractor).
(ii) The development of a non toxic cryoprotectant succesfully tested on several neural stem cell lines. (Patent MI2011A000678 I.S.E srl/CNR on “Composizione e Metodo per la Crioconservazione di Cellule” by Dessi SS and Biunno I.)
(iii) The establishment of cell bank quality control methods for bacteria, mycoplasma, senescens, genetic stability and phenotypic stability, as well as methods for detection of virus such as HIV-1, HCV, HBV, CMV, EBV, HSV2, HHV6, HHV8 and Human erythrovirus (parvovirus B19) (Diaferia et al., J Cell Physiol 2012)(Diaferia et al., Biopreservation & Biobanking, 2011)

Finally, in WP6:

(i) A working group for designing Phase 1 clinical trials with transplantation of candidate cell lines in PD and HD patients has been created and tasks have been assigned. The need for a dopaminergic cell potency assay has been established (Partners 2b, 4a, 5, 7)
(ii) A report has been published summarizing achievements and unsolved issues in stem cell transplantation for PD (Lindvall O. Nature Biotechnology, 2012)
(iii) The clinical translation of short-term expansion of human midbrain DA neuron progenitors has been promoted via a joint activity with Transeuro (see WP2). (Patent MI2011A000678 I.S.E srl/CNR on “Composizione e Metodo per la Crioconservazione di Cellule” by Dessi SS and Biunno I.)
(iv) The ethical and societal aspects of stem cell therapy in PD and HD patients have been discussed in a workshop held in Lund in May 2011 (Hug K and Hermerén G. submitted, 2012) (Hug K and Hermerén G. Current Molecular Medicine, 2012)

Importantly, in addition to the inter-consortia activity described in WP2, to further streamline NeuroStemcell objectives, seven new internal projects have been approved through 2 internal calls:

1. Striatal fate instruction of human neural stem cells via forced expression of striatal lineage determinants (Partner 1 and 8, Cattaneo and Perrier) (impacting WP1, WP3)
2. Using iN technology to generate human dopamine neurons (Partner 2a, Parmar) (impacting WP1, WP3)
3. A combinatorial and sequential approach to instruct midbrain da neurons (Partner 3 and 4a, Brustle and Arenas) (impacting WP1)
4. Improved survival, maturation and integration of hESC-derived graft for PD and HD by cotransplantation with hESC-derived astrocytes (Partner 8a and 14, Perrier and Studer) (impacting WP1, WP3)
5. Identification and isolation of midbrain dopamine neural cells using cell surface markers (Partner 9, Li) (impacting WP1)
6. A new approach to functional validation of human iN and ES cell-derived dopamine (DA) neurons in the rat PD model: Head-to-head comparison with primary human midbrain DA neurons (Partner 2a, Parmar and Björklund) (impacting WP2)
7. Epigenomics features iPSCs and somatic progenitor lines (Partner 11, Biunno) (impacting WP5)

These focussed projects will collectively produce 13 new Deliverables in year 4 contributing to 4 out of 6 WPs (five in WP1, two in WP2, three in WP3 and three in WP5)

WP1

Among the key progress in WP1 during the last funding period, Partner Biorklund/Parmar reported on the direct conversion of human fibroblasts into dopaminergic neurons. These directly converted neurons were found to exhibit immunochemical and functional properties of mesDA neurons in vitro. Partner Li validated a series of antibodies for their suitability to identify ESC-derived mDA progenitors or neurons. These studies identified an antibody raised against Folate Receptor alpha (FolR1) as promising candidate for the identification of DA progenitors or neurons.

Succeeding in an internal call during the forth funding period, Partner 3 launched a study on the impact of microRNA modulation on the in vitro generation of mesDA neurons. The data gathered in this study revealed that certain miRNAs are differentially regulated during the specification of floor plate precursor cells and in dopaminergic neurons derived thereof. Specific miR were tested for their potential to impact on the generation of dopaminergic neurons in a cell culture paradigm specifically devised for the induction of this neuronal fate. It was found that miR-181a promoted DA specification, while miR-124 exhibited an inhibitory effect. The data suggest that some miRNAs could be used to further enhance the generation of midbrain DA neurons from pluripotent stem cells.

Following the demonstration that authentic midbrain dopamine neurons can be obtained from human pluripotent stem cells using a floor-plate-based strategy (Kriks, Nature 2011) work performed by Partner Parmar using dual SMAD inhibitors, SHH and wnt agonists under fully defined conditions has led to a new protocol and to DA neurons that were able to survive and significantly ameliorate amphetamine-induced rotation behaviour.

Partners Cattaneo, Dunnett and Barker have worked jointly towards the generation of human striatal GABAergic neurons by applying an ontogeny-recapitulating protocol based on the application of the appropriate morphogens regimen in order to evoke a striatal fate in differentiating human pluripotent and neural stem cells. They have published jointly the first demonstration that an authentic DARPP32/CTIP2/GABA positive MSN character can be obtained from human pluripotent stem cells. In a parallel effort, Partner Li has developed an additional strategy for generating transplantable DARPP-32 positive striatal projection neurons from human pluripotent stem cells (submitted).

In particular, significant progress in WP1 include:

(i) continous demonstration that overexpression of Ascl1, Brn2, and Myt1l along with Lmx1a and FoxA2 leads to efficient conversion of human fibroblasts into DA neurons

(Parmar, in preparation)

(ii) The identification and validation of FolR1 as a specific mouse DA cell surface marker to be exploited for the isolation of midbrain dopamine neural lineage derived from human pluripotent stem cells (Li et al., manuscript in preparation)
(iii) The demonstration of the role of liver X receptor ligands in midbrain neurogenesis (Theofilopoulos et al., Nat Chem Biol 2013)
(iv) The generation by Partner Cattaneo of the cellular and molecular tools to allow the forced expression of key striatal specific transcription factors in hES and ltNES cells (Faedo et al., unpublished).
(v) The demonstration by Partner Brustle that miR-181a, can be used to further enhance the generation of midbrain DA neurons from pluripotent stem cells (Stappert 2013).
(vi) A protocol for the generation of scalable amounts of nearly pure populations of hES-derived astrocytes which have been used by several labs to try to implement their neuronal diff protocol (Perrier, Studer et al., unpublished).
(vii) The completion of a dual SMAD inhibitors, SHH and wnt agonists based protocol for the generation of DA neurons from human pluripotent stem celsl (Kirkeby et al., Front Cell Neuroscience 2012)
(viii) The completion and publication of new consortium protocols for the generation of medium sized spiny neurons from human pluripotent stem cells (Delli Carri et al., Development 2013; Nicoleau et al., Stem Cells 2013; Arber et al., unpublished).
(ix) A review of the literature on direct programming and iPS cell technologies (Ladewig et al., 2013; Peitz et al., 2013)
(x) Derivation and characterization of neuroepithelial-like stem cells from human foetal hindbrain and demonstration of their similarity to ES/iPS cell derived ltNES cells (Tailor et al., J Neurosci, 2013)

WP2

The objective of this WP was to characterize and improve the ability of the cells developed in WP1, to survive, grow, and differentiate in vivo into fully mature mesDA and striatal GABAergic neurons, in the absence of tumour formation or adverse immunological/inflammatory host response.

Results from WP1 indicated that it is possible to generate authentic midbrain DA neurons without the need of genetic modification and that the use of morphogenic factors and small molecules regulating Wnt/b-catenin signalling is essential in order to achieve correctly specified human midbrain DA neurons. Using these protocols (Kriks et al., 2011, Nature; Kirkeby 2012, Cell Reports), we are now in the position to generate large numbers of mesDA neurons with a phenotype that is closely similar to their fetal counterparts. These DA neurons survive transplantation into animal models of PD, mature into the desired neuronal type, innervate appropriate structures within the host brain and induce significant recovery of motor behaviour. The extent of recovery seen with the hESC-derived cells matches the recovery that can be obtained with primary mesDA neuroblasts obtained from human fetal VM. Importantly, no overgrowths or adverse events have been seen so far, indicating that these cells may be safe to use for further clinical translation.

Furthermore, the new results obtained during the IV funding period show that the developmentally relevant midbrain factors such as Shh, FGF and Wnt can also be used to increase the number and maturation of human midbrain dopamine neurons derived from neural stem/progenitor cells present in human ventral midbrain fetal tissue. Notably, the new procedure increases the number of midbrain dopamine neurons obtained from one fetal tissue by 6-fold. We think that by reducing the need for multiple donor fetuses for grafting individual PD patients, we will be able to increase the feasibility and accessibility of this technology and facilitate the development of cell replacement therapy for Parkinson’s disease.

In conclusion, in our view, the hESC-derived mesDA neurons studied reported by the Consortium represent a significant step toward a safe, reliable and efficacious source of transplantable DA neurons, and hold promise for future use in cell replacement therapy.

Significant progress in WP2 include:

(i) The advancement in consensus criteria identifying and selecting DA progenitor cells suitable for transplantations as to enable elimination of tumour-forming cells and the time points suitable for best survival, maturation and integration (all).
(ii) The succesfull use of morphogenic factors (including Wnt5a) and small molecules to regulate Wnt/b-catenin signalling in order to achieve correctly specified human midbrain DA neurons that survive transplantation, do not form tumours and induce long term behavioural recovery in animal models of PD (Kikerby et al. Cell Rep, 2012; Andersson et al., 2013).
(iii) The first report of the generation of induced neurons via direct conversion in vivo (Torper 2013)
(iv) The first reports on the in vivo survival and differentiation of human striatal progenitors generated with the protocols developed in WP1 (Delli Carri et al., Development 2013; Nicoleau et al., 2013; Arber unpublished)
(v) The identification of the stage of development of midbrain dopamine progenitors that will survive and mature after grafting (Kricks et al.,Nature 2011; Kikerby et al., 2012)
(vi) The demonstration that human fetal ventral midbrain progenitors can be expanded in the presence of morphogens (Ribeiro et al., 2012)
(vii) The development of sorting methods that allows elimination of tumour-forming cells from the graft cell preparations (Arenas, Perlmann, Bjorklund, Studer, unpublished)
(viii) The identification of graft intrinsic mechanisms inhibiting donor cell migration and integration (Brustle, unpublished)
(ix) The initiation of a collaboration with the Transeuro consortium to develop improved protocols for short-term expansion of dopamine progenitors and their efficient differentiation in order to reduce the number of foetal tissue required for transplantation to promote functional recovery (Lindvall, Arenas, Barker, unpublished).

WP3

The tasks set for WP3 have evolved around the functional assessment of novel cell sources in vivo. Three cell lines differentiated towards a DAergic phenotype and three lines for the GABAergic medium spiny neurons phenotype were utilised in WP3 for assessment of their functional efficacy in vivo.

Significant progress in WP3 include:

(i) DAergic phenotype: The first cell line was produced by Partner Studer and a comprehensive series of experiments confirmed the ability of the cells to survive, to mature into a DAergic phenotype and to integrate into the host tissue in mouse, rat and primate brains. Moreover, in both the mouse and the rat models of PD, the cells were capable of alleviating simple motor dysfunctions, including amphetamine-induced rotational bias (Kriks et al, 2011). The second cell line was produced by Partner Parmar, and shown to survive, reduce the rate of proliferation and mature into a DAergic-like cell. Thus, this cell line has been shown to successfully alleviate motor dysfunctions in a rat model of PD (Kirkeby et al., 2012). Neither groups saw indication of overgrowth. A third cell line, which was developed by Partner Li, consists of mouse epiblast derived cells which have been differentiated to a DAergic phenotype. After grafting into a mouse model of PD, the cell graft revealed TH +ve cells and reduced rotational bias, although this difference disappeared with continued testing (Li, in preparation).
(ii) Partner Parmar also showed that transplanted human fibroblasts converted to iN cells, and with low efficiency into TH + iN cells (Torper et al. PNAS, 2013).
(iii) Improved efficiency of neuronal (TH) conversion in vitro by combining the Ladewig et al, Nature Methods, 2012 protocol with a delay in transgene activation, reaching conversion efficiencies of 100%. In subsequent transplantation studies, were shown to survive and maintain their neuronal phenotype in vivo (Pereira, In preparation, 2013).
(iv) In the case of HD, one line, developed by Partner Cattaneo, was found to graft efficiently into a rodent model of HD, maintain a neural phenotype and integrate into host tissue. Only very few DARPP-32 +ve cells were observed in the grafts by 12 weeks post-transplantation, but despite this, there was evidence of reduced rotational bias on the apomorphine-induced rotation task (Delli Carr et al.,, Development 2013). The second cell line, developed by Partner Li, was found to survive and integrate into the host tissue in a rat model of HD. Encouragingly, there was no evidence of proliferation of the ES-derived cells, and a significant proportion of the cells had matured to a MSN-like phenotype (as indicated by expression of DARPP-32 as a marker, Arber et a.l unpublished). A third protocol developed by Partner Perrier led to human ventral-telencephalic precursors committed to differentiation into striatal projection neurons in vivo after homotypic transplantation in quinolinate-lesioned rats (Nicoleau, 2013).

WP4

One of the major achievement of workpackage 4 over this last period was to demonstrate the usefulness of PET and MR imaging to assess in vivo the degree of maturation/differentiation and the functionality of human embryonic stem cells in a rodent models of Parkinson’s diseases.

Through a collaborative effort between the Parmar, Perrier and Hantraye groups, a transfer of know-how and implementation of the Lund's differentiation protocol (Kirkeby et al. 2012) in the I-Stem laboratory in Evry has been possible. Beside demonstrating the effective feasibility of transferring the capacity of generating dopaminergic neurons from hPSC, this enabled the French partners to produce the necessary hPSC-derived dopaminergic cells to be grafted in 6OHDA lesioned nude rats in MIRCen and to image these grafted rats using a combination of three different PET radioligands and T2-weighted MR imaging.

Significant progress in WP5 include demonstration that:

(i) grafted cells survive and expand normally in the grafted striatum (T2-weighted MR imaging)
(ii) some grafted cells differentiate into dopaminergic neurons expressing normal dopamine reuptake system (as judged by 18F-LBT999 PET)
(iii) cells release de novo a significant level of dopamine (18F-Fallypride PET) and
(iv) are not subjected to major adverse effect like rejection phenomena or neuroinflammatory reaction (as judged by 18F-DPA714 PET)

These PET imaging data are in preparation for publication (Hantraye, Parmar and Perrier).
Importantly, all these techniques and methods have been selected because they all are translational by nature and can readily be applied to patients. PET tracers equivalent to the 18F-LBT999, 18Ffallypride and 18F-DPA714 are available in most European countries for preclinical and clinical use. This work is expected to serve as a scientific basis for the preparation of a stem cell intracerebral tranplantation clinical trial.

WP5

WP5 was focused on the manufacturing processes and safety aspects of cell production for PD and HD cell therapies, including establishment of criteria for defining the suitability of the cell lines for clinical application, stability over long term passage, media development, and scale-up of manufacturing under GMP conditions.

The achievements in WP5 have provided criteria for stem cell lines suitable for clinical applications, which will be important for future identification, characterisation and usage of new stem cell lines. The development of a non-toxic cryoprotectant, cell bank GMP manufacturing conditions and up-scaling protocols under GMP condition will be of importance for future optimisation towards a permanent source of human ESC- and/or NS- and/or iPS-derived cell lines and committed progenitors for transplantation trials in PD.

Significant progress in WP5 included the

(i) Establishment of cell bank quality control methods for bacteria, mycoplasma, senescence, genomic and phenotypic instability and viral detection
(ii) The banking and culturing pluripotent stem cells
(iii) The identification and implementation of critical quality control programs for the generation of clinical grade pluripotent stem cells
(iv) A protocol for the up-scaling and automation of lt-NES cells that will also be the basis for further optimisation and testing of other stem cell sources (Badenas et al., in preparation).

WP6

The work within WP6 has defined the steps necessary to move a clinical candidate cell, derived from stem cells, from basic studies to clinical application in patients with PD and HD. By investigating how each step of the clinical translation should be performed in a responsible way from the scientific, clinical, ethical and regulatory perspective, the work in WP6 has served as a model for how to promote the clinical translation of stem cells not only in PD and HD but also in other brain disorders. This work has already given rise to articles in influential top journals such as Nature Biotechnology, Cell Stem Cell and The Journal of Clinical Investigation. Furthermore, WP6 activities have been followed up by the International Society for Stem Cell Research (ISSCR), which created a working group, comprising PIs from Neurostemcell and WP6, and produced, to a large extent based on work performed in the Neurostemcell project, a white paper on the “Clinical translation of stem cells in neurodegenerative disorders.” Finally, WP6 has directly influenced the work in the California Institute of Regenerative Medicine (CIRM). Following the workshop on "Designing Phase I clinical trial with transplantation of candidate cell line in Parkinson's disease" arranged by WP6 in London in September 2012, an almost identical workshop with participation of PIs from WP6 in Neurostemcell was arranged by CIRM in San Francisco in March 2013.

Significant progress in WP6 include the definition of:

(i) the scientific, clinical, and regulatory requirements on the specific candidate cells to be suitable forimplantation in PD patients (Hug et al., 2012 Journal of ethical clinics, 2013).
(ii) the design of the first clinical trial with stem cell-derived dopaminergic cells in the perspective of previous clinical studies with grafting of human fetal mesencephalic tissue and of the ongoing TRANSEURO trial (Lindvall,Nat Biotechnology 2012; Barker 2012).

TRAINING AND OUTREACH IN PERIOD IV

NeuroStemcell has successfully achieved its contracted activities (annual meeting 2012, wrap up meeting 2013, London workshops, summer school organized jointly with EuroSyStem and Optistem in September 2012). Besides, the Consortium has completed a number of additiona activities which are fully reported in Section 4 of this report.

Potential Impact:

Stem cell technology holds the promise to turn cell transplantation from a highly experimental procedure to a clinically useful therapy for major neurodegenerative diseases. NeuroStemcell promoted and speeded up this development. Provided that not all CNS diseases are equally suitable targets for cell replacement therapy, NeuroStemcell identified the best chances for success in those applications where the leading cause of disability may be linked to a defined, localized degeneration of neurons, such as the loss of midbrain DA neurons in PD and the early loss of striatal neurons in HD. The encouraging results obtained in open-label clinical trials with transplants of fetal midbrain in PD and ganglionic eminence tissue in HD gave further reasons to believe that a stem cell based cell replacement approach could provide significant clinical benefit and develop into a clinically useful therapy for large numbers of patients with hitherto intractable neurodegenerative diseases.

Chronic debilitating diseases, such as PD and HD, constitute a major and increasing health problem and a heavy burden on the health care system in Europe. PD is the second most common neurodegenerative disease (after Alzheimer’s disease) affecting 1–2 percent of all individuals over the age of 50, and HD is the most common mono-genetic disease of the nervous system with a prevalence of 3–5 individuals/100 000 in Europe. HD is a progressive devastating disease that develops slowly over 10–20 years leading, directly or indirectly, to death. The pharmacological treatment available today is only palliative and of little help to the patients. In case of PD, current pharmacological and neurosurgical therapies are effective in most patients, but they are essentially symptomatic treatments and do not modify the long-term evolution of the disease. In both these cases, therefore, there is a great need of new forms of therapy that are able to modify the underlying disease processes and/or restore function in affected individuals. Cell therapy, alone or in combination with disease modifying interventions, represents a novel neurorestorative approach that, if successful, may revolutionize the way we treat neurodegenerative diseases and injuries to the CNS.

Contribution to the Topic

NeuroStemcell brought together 13 leading European research teams and 2 SMEs to form an internationally competitive consortium with the broad range of basic and clinical expertise necessary to make significant progress in this field. The program took advantage of the progress that has been made during the last few years in the generation of homogenous and stable stem cell lines from embryonic, fetal and adult sources, and built on the major developments made in the elucidation of the role of extrinsic and cell-intrinsic factors in the differentiation and specification of immature stem cells towards specific neuronal fates. The participants in NeuroStemcell have contributed significantly to these recent developments and will now join together in order to speed up further development towards the clinic. The Work-packages final deliverables represented a significant advance in the field, and the overall structure of the program is designed to have maximal impact on the translation of stem cell biology to clinical therapies for neurodegenerative disorders.

Major Impacts

The program had a considerable impact, first, in developing protocols for efficient production of fully validated human stem cell-derived mesDA and striatal GABAergic cell lines, an secondly, in the establishment of a bank of fully validated cells derived under GMP conditions, that can be used not only in clinical transplantation trials but also by the research community as large and important tools in pharmacological research and drug discovery.

The consortium made a concerted effort to work out scientific, regulatory and ethical standards for application of stem cells in nervous system diseases. Careful assessment of all elements involved in the design of clinical stem cell-based transplantation trials in PD ad HD paved the way for how such trials should be planned and conducted in the future, and close interactions with the major players, key experts and stakeholders in the stem cell field in Europe ensured that this part of the program had full impact. In addition, by involvement of two key biotech SMEs NeuroStemcell created a productive academic/industry interface that helped to establish a stronger biotech industry base on the European scene. Such a base in the longer perspective is necessary for Europe to become competitive and successful in the stem cell field.

With respect to the international perspective, the global research community has entered a phase of intense effort to accelerate the rate of discovery in embryonic and tissue stem cell biology with the ultimate goal of developing new pharmaceutical and cell-based therapies. European scientists are in a very good position to take a lead in the development and translation of advances in stem cell research towards clinical application. However, for European science and bio-industry to maintain a competitive edge it is essential to maximize added value and synergy by building successful multidisciplinary partnerships at a trans-national level. A willingness to create a sustainable, attractive and internationally competitive research sector will ultimately yield considerable dividends for European bio-industry and healthcare.

Chronic debilitating diseases, such as PD and HD, constitute a major and increasing health problem and a heavy burden on the health care system in Europe. PD is the second most common neurodegenerative disease (after Alzheimer’s disease) affecting 1–2 percent of all individuals over the age of 50, and HD is the most common mono-genetic disease of the nervous system with a prevalence of 3–5 individuals/100 000 in Europe. HD is a progressive devastating disease that develops slowly over 10–20 years leading, directly or indirectly, to death. The pharmacological treatment available today is only palliative and of little help to the patients. In case of PD, current pharmacological and neurosurgical therapies are effective in most patients, but they are essentially symptomatic treatments and do not modify the long-term evolution of the disease. In both these cases, therefore, there is a great need of new forms of therapy that are able to modify the underlying disease processes and/or restore function in affected individuals. Cell therapy, alone or in combination with disease modifying interventions, represents a novel neurorestorative approach that, if successful, may revolutionize the way we treat neurodegenerative diseases and injuries to the CNS.

Contribution to the Topic

NEeuroStemCell brings together 13 leading European research teams and 2 SMEs to form an internationally competitive consortium with the broad range of basic and clinical expertise necessary to make significant progress in this field. The program will take advantage of the progress that has been made during the last few years in the generation of homogenous and stable stem cell lines from embryonic, fetal and adult sources, and build on recent major developments made in the elucidation of the role of extrinsic and cell-intrinsic factors in the differentiation and specification of immature stem cells towards specific neuronal fates. The participants in NeuroStemcell have contributed significantly to these recent developments and will now join together in order to speed up further development towards the clinic. Each of the six Work-packages has a final deliverable that will represent a significant advance in the field, and the overall structure of the program is designed to have maximal impact on the translation of stem cell biology to clinical therapies for neurogenerative disorders.

In Work-package 1 the goal is to improve our understanding of the biological processes involved in the controlled differentiation of embryonic and fetal stem cells and of the most recent iPS cells and, based on this knowledge, to devise optimized protocols for the generation of mesDA and striatal GABAergic neurons in clinically relevant numbers for transplantation.

In Work-package 2 we will study the in vivo performance of stem cell-derived mesDA and striatal GABAergic cell preparations with the goal to identify promising candidate cell lines that have the ability to differentiate into authentic mesDA and striatal GABAergic neurons in high numbers after transplantation, in the absence of any tumor formation.

In Work-package 3 we will perform a comprehensive pre-clinical testing of the functional efficacy of the stem cell-derived mesDA and striatal cell lines in relevant animal models of PD and HD, with the goal to identify and validate the most promising cell preparations to be used for further development towards clinical use.

In Work-package 4 we will explore the usefulness of MR and PET imaging techniques for monitoring, non-invasively, the survival, growth, function and potential adverse effects of the grafted cells, with the goal to devise a combination of MR and PET imaging that can be included as assessment tools in the clinical transplantation protocols.

In Work-package 5 the criteria, technology, and protocols for manufacture of stem cells and their progeny to clinical grade standard will be addressed with the ultimate goal to generate a cell bank for fully validated, safe and efficacious cells, ready for clinical use and drug discovery.

Work-package 6, finally, will address the complex clinical, societal, regulatory and ethical issues that are associated with the development of stem cells for clinical use. The scientific and regulatory requirements for cells to be useful for transplantation in PD and HD patients will be defined, linked to a road-map aimed at the implementation and design of clinical phase-I clinical trials in patients with PD and HD.

Major Impacts

The program will have considerable impact, first, in developing protocols for efficient production of fully validated human stem cell-derived mesDA and striatal GABAergic cell lines, an secondly, in the establishment of a bank of fully validated cells derived under GMP conditions, that can be used not only in clinical transplantation trials but also by the research community as large and important tools in pharmacological research and drug discovery.

The consortium will make a concerted effort to work out scientific, regulatory and ethical standards for application of stem cells in nervous system diseases. Careful assessment of all elements involved in the design of clinical stem cell-based transplantation trials in PD ad HD is likely to set the standard for how such trials should be planned and conducted in the future, and close interactions with the major players, key experts and stakeholders in the stem cell field in Europe will ensure that this part of the program will have full impact. In addition, by involvement of two key biotech SMEs NeuroStemcell will create a productive academic/industry interface that will help to establish a stronger biotech industry base on the European scene. Such a base will in the longer perspective be necessary for Europe to become competitive and successful in the stem cell field.

The International Perspective

The global research community is entering a phase of intense effort to accelerate the rate of discovery in embryonic and tissue stem cell biology with the ultimate goal of developing new pharmaceutical and cell-based therapies. In the United States, California alone has committed $3,000,000,000 to stem cell research over the next 10 years, and similar initiatives are in development is several other US states. Canada has established a nationwide Stem Cell Network with generous funding over 14 years to link stem cell researchers and clinicians with systems biologists, biotechnologists and bioengineers. Similarly, in Singapore, Japan and China, major funding is being invested in basic and translational stem cell research.

European scientists are in a very good position to take a lead in the development and translation of advances in stem cell research towards clinical application. However, for European science and bio-industry to maintain a competitive edge it is essential to maximize added value and synergy by building successful multidisciplinary partnerships at a trans-national level. A willingness to create a sustainable, attractive and internationally competitive research sector will ultimately yield considerable dividends for European bio-industry and healthcare.

List of Websites:

Website: www.neurostemcell.org

PROJECT CO-ORDINATOR CONTACT DETAILS:

Professor Elena Cattaneo

Università degli Studi di Milano
Dipartimento di Bioscienze
Via Viotti, 3/5, 20133 Milano
Tel. +39.2.50325830 Fax. +39.2.50325843
E-mail: elena.cattaneo@unimi.it

PROJECT OFFICE CONTACT DETAILS:

NeuroStemcell Project Office

Università degli Studi di Milano
Dipartimento di Bioscienze
Via Viotti, 3/5, 20133 Milano
Tel. +39.2.50325841/2 Fax. +39.2.50325843
E-mail: neurostemcell.pm@unimi.it