Community Research and Development Information Service - CORDIS



Project ID: 602278
Funded under: FP7-HEALTH
Country: Italy

Periodic Report Summary 2 - NEUROSTEMCELLREPAIR (European stem cell consortium for neural cell replacement, reprogramming and functional brain repair)

Project Context and Objectives:
Neurostemcellrepair is a world-leading Consortium that aims at taking stem cells through the final pre-clinical steps necessary before their clinical use in trials for Parkinson’s Disease (PD), as well as achieve substantial advancements in their adoption as cell replacement strategies for Huntington’s Disease (HD). These two disorders are at different stages in their clinical translation for stem cell treatment, yet they have common paths and requirements that need to be fulfilled. We anticipate that addressing one condition will benefit the other and will also be useful for establishing a platform by which we can treat a wider spectrum of neurodegenerative disorders. PD is taken as the prototypical disease because it has been successfully treated with dopamine cell therapies and, in addition, recent developments have provided cells and protocols ready for optimization for GMP compatibility and translation to the clinic. At the same time, we aim to further advance the field by implementing emerging stem cell programming tools and methods for enhanced tissue integration of grafted cells in order to develop new approaches, validated at pre-clinical stages, for the treatment of PD and HD.

Experience gained from clinical trials using grafts of fetal mesencephalic dopaminergic (DA) and striatal GABAergic progenitors have shown that effective repair can be achieved by neural transplantation. Notably, transplanted DA neurons, derived from the ventral mesencephalon (VM), can functionally reinnervate the denervated striatum, restore dopamine release and, at least in some PD patients, induce substantial long-term clinical improvement (Politis, Sci Transl Med, 2010; Barker, Lancet Neurology, 2013). On the basis of these observations a new clinical trial has been initiated (Transeuro,, aimed at developing efficacious and safe conditions for fetal VM-based therapies for PD. However, to move to large-scale applications, readily available, renewable, and bankable cells are needed.

Recent discoveries, stemming from members of this Consortium, have identified morphogens and transcription factors (TF) critical for successful phenotype specification and differentiation of therapeutically relevant DA neurons from human pluripotent stem cells. These findings have led to cell differentiation protocols that currently represent the gold standard for cell therapy with ventral mesencephalic (mes) DA neurons (Kriks, Nature, 2011; Kirkeby, Cell Reports, 2012) and, very recently, to novel ontogenetic factors that promote the dopaminergic phenotype (Theofilopoulos, Nat Chem Biol, 2012; Andersson, PNAS, 2013). Inspired by this work, a protocol to generate authentic striatal GABAergic medium spiny neurons (MSNs) has also been recently published by other members of this Consortium (Delli Carri, Development, 2013). Furthermore, partners in this Consortium have identified a scalable source of neural progenitors able to mature into functional neurons (Koch, PNAS, 2009). With the advent of induced pluripotent stem (iPS) cell technologies and direct reprogramming of somatic cells, further sources of transplantable cells have become available. One partner in this Consortium was one of the first groups to report on the reprogramming of fibroblasts into DA neurons (Pfisterer, PNAS, 2011), while work by another partner has identified small molecules that increase efficiency of neuronal conversion (Ladewig, Nat Methods, 2012). Hence, this Consortium brings together several groups that are leaders in the world and are capable of moving this field forward, towards a safe clinical translation of stem cell-based interventions, especially for PD.

In particular, the project aims at fulfilling the final requirements for progressing stem cells towards clinical translation for dopamine cell replacement in PD, while addressing crucial issues on the way to future applications also in HD. Thus, our main goal will be to optimise and standardise the current cell-generating protocols and incorporate tools, methods and technologies to ensure efficient, accurate and safe integration and functional efficacy of human stem cell-derived neurons, to promote brain repair and functional recovery in PD and HD.


Neurostemcellrepair has the following six objectives and measurable outcomes

1. Increase the yield and purity of transplantable mesDA and striatal GABAergic MSN progenitors from human pluripotent and neural stem cell lines.
2. Improve anatomical incorporation and functional integration of donor cells in the recipient tissue, by promoting their survival, differentiation, distribution, directed axonal growth and connectivity.
3. Explore alternative donor cell sources by testing the reprogramming potential of human fibroblasts for generating authentic and subtype specific neurons.
4. Test and compare the enhanced efficacy of stem cell-derived progenitors in pre-clinical studies in animal models of PD and HD with the goal to select the most efficient and safe candidate cells for clinical use.
5. Validate new imaging technologies to demonstrate stable performance and integration of the transplanted cells.
6. Fulfil all requirements to enable the large-scale GMP-compatible manufacturing and banking of stem cells and their progeny according to clinical grade standards, including cytogenetic and epigenetic characterization.

Project Results:

In order to improve the quality of MesDA cell preparations, we have implemented the culture conditions and examined the properties of the cells. In particular, we have focused our work on the following three aspects:
We worked on the implementation of novel developmental factors to existing stem cell differentiation protocols. We (Partners 2-ULUND, 3-UKB and 4-KI) have explored the impact of selected factors, among which transcription factors, microRNAs and morphogens. We showed that the transcription factor PBX1 and the microRNA MIR181 promote mDA differentiation when overexpressed in human neural stem cells (lt-NES cells). We also found that application of the morphogen Wnt5a together with other patterning factors induced mDA differentiation of lt-NES cells as assessed by morphological, gene expression and electrophysiological analyses. We also explored the effect of OTX2 overexpression in hESC-derived mDA cultures with the aim to generate A10 mDA neurons.
Another task aimed at identifying small molecules capable of improving direct neuronal conversion of fibroblasts. In particular, 6 compounds were found to increase the neuronal conversion in a dose-dependent manner and, when combined, gave a high neuronal purity (50%) and an outstanding conversion efficiency (almost 500%) (Partner 2-ULUND).
We (Partner 3-UKB) then focussed on the identification and characterization of more amenable cell sources for direct cell reprogramming by investigating the use of neonatal human cord blood (CB) and adult peripheral blood (PB) cells. We reported the generation of stable human induced neural progenitor cells capable of differentiating into glial and electrophysiologically active neuronal subtypes. In particular, iNSCs can be specified and differentiated into a midbrain dopamine-like neuronal population.
We (Partner 1-UMIL) have optimized the existing version of hES-to-MSNs protocol that generates human ventral telencephalic progenitors starting from hES cells. With this approach, stem cells eventually differentiate into mature, electrophysiologically active neurons, (Darpp32+/Ctip2+ 10-15%). We therefore aimed at establishing a hES cell-based inducible gain-of-function (iGOF) system whereby transcription factors expressed in the developing striatum can be harnessed to improve MSNs differentiation. This model allowed us to collect insights about the role of TFs during striatal development and how to exploit them for the protocol optimisation.
A study by Partner 4 (KI) performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse, finding 25 molecularly defined human cell types among which new undescribed subsets. The study also developed a method to assess fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. Therefore, this study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies for PD.
In sum, our studies have identified novel factors and tools that, when applied to existing differentiation protocols, allow to improve mDA differentiation.

Cell transplantation trials performed with dopaminergic progenitors obtained from hES cells through the established protocols gave variable functional outcome. Hence, we (Partner 2-ULUND) took an unbiased approach to identify predictive markers expressed in dopamine neuron progenitors correlating with graft outcome in an animal model of PD. A novel set of markers associated with the precise and desirable caudal midbrain phenotype was identified and will be implemented. These markers have therefore aided the optimization of the DA differentiation protocol in order to avoid batch-to-batch variability in in vivo outcome of the DA progenitors. This is an important step forward in making these cells ready for clinical trial. In addition, the newly identified markers will be implemented as future quality control assays in order to assess the in vivo potential of each clinical DA cell batch prior to transplantation. This will in the future provide an easy accessible way of monitoring the cell quality and predicting the in vivo performance of each cell batch.
As a further step toward clinical application, we (Partner 2-ULUND) developed a highly efficient GMP-compatible hESC differentiation protocol for production of pure cultures of caudal VM progenitors which give rise to DA rich and functional graft in vivo. This protocol enables manual production of a large number of DA progenitors. The protocol has been transferred to Partner 11 (ROSLIN), where the first 3 test runs of differentiation were produced, and to Partner 12 (MILTENYI), in order to test the protocol on H9 for enabling closed-system production (Prodigy) for future clinical upscaling.

Another main achievement (Partner 2-ULUND) was the establishment of a procedure for the tracing of synaptic connections from grafted DA neurons based on the use of mutated rabies virus. It was shown that host neural circuitry form synaptic contacts with grafted hESC-derived neurons and are stably maintained. It was also proven that grafted hESC-derived DAergic neurons provided synaptic input to distal and proximal host structures. These data confirmed that functional connectivity and circuit integration is established between grafted hESC-derived DAergic neurons and host neural circuits.

We demonstrated the successful employment of optogenetic and chemogenetic technologies to hESCs for evaluation of graft function in long-term in vivo transplantation studies. We could specifically elicit and detect DA release from grafted hESC-derived DA neurons post-transplantation after >20 weeks. We identified that DREADD technology was the most reliable method for specifically stimulating hESC-derived DA neurons post-transplantation to release DA, as detected using in vivo amperometry. In our hands chemogenetic technology gave the most reliable and robust evidence of triggered DA release in vivo 24 weeks post-transplantation as assessed using amperometry in a rat model of PD.

We developed a new protocol for highly efficient generation of functional iNs from human fibroblasts, of both post-natal and adult origin. The conversion efficiencies described in this new protocol are greater than those previously published for hiNs, and most notably can be obtained in the absence of a selection step. We demonstrate that hiNs can be converted first in vitro prior to transplantation and survive and give rise to an equal number of hiNs as with those transplanted as primed fibroblasts and converted in vivo.

When using human fetal VM as a gold-standard, we demonstrated that hESC-derived DA neurons are almost indistinguishable from bona fide human fetal DA neurons in term of their efficacy and efficiency of maturation in vivo in PD animal models. These data form a significant driver for the pursuit of the first in-man clinical trials using hESC-derived DA neurons in patients with PD and provide a platform of experiments through which other novel cell sources.

We (Partner 6-UCAMB, 8-UEDIN) also examined the expression profile of extracellular matrix (ECM) molecules in the post-mortem brain of PD patients. A significant number of ECM components are prominent in mediating both the innate and adaptive immune response, thus providing new potential insight into disease mechanisms and the ability to devise strategies to manipulate ECM signalling for transplanting cells in the PD brain.

Initial observations suggest that hES-derived MSN-progenitors transplanted in QA-lesioned athymic, adult rats were able to differentiate into striatal neurons in vivo showing optimal survival up to 2 months after transplantation. However only a small portion of donor cells correctly mature to MSN phenotype. Histological analysis of human-specific fibres from the graft showed that hES-derived MSN send out projections and connect with the endogenous tissue.
Furthermore, we (Partner 1-UMIL and 11-ROSLIN) have been also working on the identification of other responsive human embryonic cell lines that are GMP-compliant for future clinical applications. The analysis of two of the GMP hES lines (RC9 and RC17) has confirmed that they are able to reach full maturation in a proportion of cell similar to that seen in validated lines (H9).

Different teams (Partner 6-UCAMB and 8-UEDIN) have joined forces and worked on analyzing ECM expression in brain sections of both HD and PD patients. The most notable finding is the lack of overlap between HD and PD in terms of ECM protein expression profile. ECM molecules identified will offer potential targets for modulation of the innate and adaptive immune response as a possible therapeutic strategy.

A key challenge in cell replacement strategies is the limited axonal outgrowth and integration of donor cells. To overcome these challenges we (Partner 8-UEDIN) addressed the question as to how effective integrin manipulation will be in enhancing axonal outgrowth from human embryonic stem cell derived grafts and from preliminary results we can conclude that integrin expression is correlated with the regenerative potential of axons.

We (Partner 3-UKB) increased the PSA-NCAM levels in the transplant region by PST-overexpression using lt-NES cells as vehicle and we have further shown that overexpression of PST leads to enhanced neurite outgrowth in lt-NES cells. Finally, we improved axonal growth of transplanted MSN neurons by local application of ECM degrading enzymes (Partner 8-UEDIN).

Finally, we (Partner 6-UCAMB and 10-ISENET) sought to identify new inflammation-related genetics variants contributing to the disease process and clinical expression in HD as well as validate these markers at the histopathologic level in order to refine our understanding of the neurodegeneration processes and thus of the host environment for future neural grafting. We genotyped a large cohort of HD patients for inflammation-related genetic modifiers. In addition we have undertaken a TMA high-throughput study to look at the changes in protein expression of the inflammatory markers identified with the genetic analysis as well as standard inflammatory markers in the striatum of HD patients. This will be useful not only in defining possible new therapeutic targets but will also have direct implications in any cell based reparative approach involving grafting cells to the HD striatum.

In order to standardise the behavioural tasks across laboratories, we (Partner 5-UCARD and 7-UNITO) achieved to define the optimal behavioural battery for assessment of motor function in rodent models of Parkinson’s and Huntington’s disease by reviewing the multiple behavioural tests available. A comprehensive investigation into the functional efficacy of the hESC-derived DA neurons has been completed, while the examination of the ability of the ES- or iPS-derived MSNs will start after issues associated with survival are addressed sufficiently to ensure appropriate graft survival, maturation and integration.

We reported on the survival and differentiation of a novel lt-NES derived DA cell therapy product in vivo, as well as a systematic histological and behavioural investigation of hVM tissue grafts and ES-DA cells: stem cell-derived dopamine neurons engraft efficiently, survive and integrate into the brain, contributing to the growing body of evidence that demonstrates the safety and efficacy of ES-derived dopamine cells and suggests that we are now very near to clinical application of these cells.

It is also essential that we assess the different ES-derived MSN neurons in rodent models of disease, in order to ensure their safety, survival and functional efficacy. We reported a comprehensive assessment of the functional efficacy of rat and human whole ganglionic eminence (WGE) grafts on a range of motor tasks. It remains critical to establish a clear baseline characterisation of the "gold-standard" fetal medium spiny neurons, to determine the level of functional improvement that can be expected from a novel ES-derived MSN cell therapy product.

To promote functional recovery in Huntington disease, we asked whether training procedures can further promote functional recovery. To address this task, we (Partner 7-UNITO) set up two different protocols of training, whose our results indicate that both protocols provide positive effects on motor symptoms related to voluntary movement and spontaneous movement.

We (Partner 3-UKB and 13-LIFE&BRAIN) were also able to develop imaging technologies for assessing functional integration of grafted neurons. In particular, we adopted successfully the light-sheet fluorescence microscope (LSFM, MPI Heidelberg) for the analysis of neural grafts and their connectomes. The acquisition platform is suitable to permit whole-mount visualization of cleared whole mouse and rat brains at single cell resolution. We expect this technology to facilitate the standardized analysis of grafted neurons in the host brain, providing essential information on the integrative potential and functionality of the grafts.
We also devised an approach suitable for quantitative assessment of human transplant innervation in the context of an entire mouse brain. The results obtained indicate that, for example, lt-NES cell grafts in the dentate gyrus and the striatum receive afferent innervation highly similar to that of endogenous hippocampal and striatal neurons. Finally we report the development a set of tools that substantially improve the speed and accuracy of neural circuit reconstructions in the context of LSFM-generated whole brain preparations.

In Period II, In order to select the hESC GMP line with best response to hES-to-mesDA protocol and in order to verify transferability of the procedure to clinical grade GMP cell lines, we (Partner 2-ULUND) executed two independent and complete differentiation experiments on the GMP cell lines RC9 and RC17. We (Partner 11-ROSLIN) created a qualified bank of RC17 hESC and distributed vials of this bank along with SOPs for their culture to partners to be used as starting material. We (Partner 11-ROSLIN) also created a Research Grade Distribution Bank (RGB) for the selected RC17 hESC cell line. This bank has been created, quality testing performed and has now been distributed to Consortium members (MTA approval in place). GMP gap/risk analysis of the process of differentiating hESC into mesDA progenitors was initiated. As the first step towards qualifying the process, the reagent risk assessment (RA) process has been initiated and the majority of these have been drafted, along with developing a target product profile.
Consortium members have also evaluated whether a cryopreservation strategy for clinical delivery is feasible and confirmed that this might be the best approach as final differentiated cell product can be shipped as frozen product to transplantation clinics, where no further culturing or manipulation of the cells will be needed prior to transplantation. Cryopreservation strategies have been designed also in order to accommodate the manufacturing scale. Assessment of the viability of the samples will show the robustness of the cryoprotocol and cryopreservation strategy.

In order to meet the regulatory requirements for advanced-therapy medicinal products (ATMPs), the Consortium is working to set standardized reagents and highly reproducible production procedures, where automation may contribute to the successful development of therapies. We could show that pluripotent stem cells (PSCs) can be expanded with equal or better yield and quality as comparable bench protocols and that the differentiation of PSCs to mesDA neurons is feasible on the Prodigy system at Partner 12. By adopting this system, we have established and technically evaluated a magnetic sorting process for mesencephalic dopaminergic neuron progenitor cells (mesDA) derived from pluripotent stem cells (PSCs). Toward the manufacture of DA neurons according to a GMP-compatible protocol, as part of transferring manufactured dopaminergic (DA) neuron to clinic, we (Partner 2-ULUND) have tech-transferred the Neurostemcellrepair (NSR) protocol to Partner 11 (ROSLIN).

Potential Impact:
Stem cell technologies hold the promise of taking cell transplantation all the way from a highly experimental procedure to a clinically applicable therapy for major neurodegenerative diseases. However, neuronal replacement in the adult brain poses a number of challenges with respect to donor cell generation, delivery and functional integration, and not all CNS diseases may be equally suitable targets for such an approach. The best candidate diseases for neuronal transplantation are thought to be those where the leading cause of disability is linked to a defined, localized degeneration of neurons, such as the loss of midbrain DA neurons in PD and the early degeneration of striatal neurons in HD. Stem cell technologies have evolved at a very rapid pace during the last years and it is nowadays possible to produce human cells capable of variable degree of functional recovery in animal models of PD and HD. The main goal of this project is, therefore, to take human stem cells through the final steps towards their clinical application in cell replacement therapy for PD, and to further enhance functional cell replacement for the treatment of both, PD and HD. In that regard, our work will also benefit from the Transeuro project (, which aims at developing an efficacious and safe cell replacement therapy methodology for PD using MesDA neurons from human ventral midbrain fetal tissue. These developments will facilitate the application of human stem cell derived-DA neurons developed by our Consortium, enabling their rapid and effective translation from the laboratory to clinical trials.

The project will contribute to the topics detailed under HEALTH.2013.1.4.1 and will cover a significant number of crucial issues of basic, clinical and industrial research, as indicated below:
• The expansion of intermediate progenitors capable of generating fully differentiated and phenotypically stable mesDA neurons or MSNs. This goal constitutes a considerable step forward as more homogenous cell preparations will be obtained, while the time necessary to differentiate cells to the desired stages for transplantation is reduced.
• To develop strategies to control optimal donor cell adaptation in the host milieu. To date, engraftment and differentiation of transplanted cells rely on the spontaneous unfolding of developmental programs imposed onto donor cells in vitro, but adaptation to the host milieu is not guaranteed. Controlled expression of key molecules involved in differentiation will be used to control in vivo the maturation of the donor cells and enhance their connectivity. Ultimately, the project will provide critical advancements in the engraftment and functional integration of human stem cell-derived mesDA neurons and MSNs in animal models of PD and HD.
• To develop strategies to improve the reprogramming of human fibroblasts into mesDA neurons and MSNs and their engraftment in models of PD or HD. While pluripotent and neuropotent stem cells represent an exquisite and unlimited donor source, recent developments in direct cell fate conversion may provide an even faster and potentially in vivo applicable route for the generation of replacement cells. While focusing on foreseeable clinical applications of stem cells, our Consortium will not ignore these recent developments and will integrate them in a comparative manner alongside classic stem cell-based differentiation paradigms.
• To provide novel and urgently needed procedures and tools for detailed analysis of engrafted donor cells in the host tissue. Our project has a strong component of technological development, which will also relate to monitoring of grafted stem cells with innovative microscopic imaging technology. In particular, the assessment of different donor populations to engage in functional synaptic interactions with the host brain will enable an efficient and unprecedented visualization of the complete functional connectivity of grafted neurons. A key goal will be the visualization of functional host-graft connectivity, a crucial issue for graft efficacy, which has not been sufficiently documented. These technologies might also open new opportunities for commercialisation and distribution to a wider scientific community.might also open new opportunities for commercialisation and distribution to a wider scientific community.
• The development of second-generation and clinical-grade protocols for the differentiation of mesDA neurons from human pluripotent stem cells in GMP conditions is a major objective of the project. Our project will focus on further improving currently existing protocols for the differentiation of mesDA neurons and MSNs not only by incorporating new factors and improving the efficiency and quality of the cells we produce, but most importantly by engaging to adopt and standardise best in practice protocols, we will secure appropriate transfer and implementation of our protocols in GMP in order to deliver GMP-translated manufactured DA neurons by the end of project.
By taking advantage of our most recent discoveries and expertise in the field of regenerative medicine, Neurostemcellrepair aims to close the gap between development and clinical implementation of cell replacement therapies for PD and HD. The encouraging results obtained in open-label clinical trials with transplants of fetal midbrain in PD and ganglionic eminence tissue in HD clearly indicate that cell replacement can provide significant clinical benefit and be developed into a clinically useful therapy for large numbers of patients with hitherto intractable neurodegenerative diseases.

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