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Home » Cahier Charcot » Press report Symposium 2006
European Charcot Foundation Symposium, 16-18 Nov 2006, Taormina , Italy .
"Mending the brain. Stem cells and repair in Multiple Sclerosis”
   
Backgrounder for the press 
 
SUMMARY
Multiple sclerosis (MS) is an inflammatory and degenerative disease of the central nervous system leading in time to severe disability. Treatment today is initially focussed on reducing the inflammatory process in the brain that causes the breakdown of the protective myeline sheaths around neurons. Spontaneous repair of brain damage also occurs and is the reason that many patients recover partially or completely from their first MS attacks. On the long run however, spontaneous recovery and pharmacological treatment cannot prevent the disease from progressing. Alternative methods are therefore under research. A promising novel treatment option for MS might be stem cell therapy. Many investigations in animal models of MS demonstrated successful repair of brain damage and the return of neuronal functions. The best results have been obtained using embryonic stem cells, since these are most plastic and have the best properties to adapt to host tissue environments. Since embryonic stem cells are not widely available and their use is subjected to ethical and legal debate, other types of stem cells have been considered. In adult humans, stem cells can be found in several tissues, eg. bone marrow, blood, brain, fat tissue, pancreas and skin. Transplantation of autologous (from the patient self) bone marrow stem cells has been performed in the clinic for 20-30 years for the treatment of haematological malignancies and is a proven safe procedure. These cells have been administered in animal models of MS and here they know how to migrate into the brain and find the exact location of the brain damage. These cells induce restoration of the myelin sheath and hence neuronal function. Since the use of bone marrow stem cells from the patient self is a well-known safe and very promising procedure, this European Charcot Foundation symposium discussed the start of a clinical trial to assess 1. safety/tolerance and 2. effectiveness in 60 patients across 5-6 international centres. A number of preconditions were recommended by the international forum of MS experts:
·     To only use autologous stem cells derived from bone marrow;
·     Cells should be harvested, cultured, purified and stored in a central specialised
 lab under strict GMP-regulations;
·     Cells should not undergo genetic modification;
·     Clinical procedures of stem cell trials should be standardised and centrally coordinated;
·     Reporting of all side effects and efficacy results should be managed in a central database.
 
Some scientists at the conference, pledged for more fundamental research before stem cell treatment in man should start at all. It is beyond doubt that there is lots of research work to be done on the nature of brain damage and natural repair mechanisms of nerve tissue, on the interaction between immune system and stem cells and on various other aspects in this complex neurobiological arena.
However, there is growing, although sometimes inconclusive or casuistic evidence of clinical relevant brain-repair and protective properties of transplanted stem cells. Given the urgency of finding a cure for this widespread, disabling disease, most scientists argued that it is justifiable to arrange a rapid onset of well-managed trials, concluded Prof. Hommes, chairman of the independent European Charcot Foundation. "The proof of concept in animal experiments is available. Now it is time to proceed to the clinic.”
 
Background information – summary of lectures
 
 

MULTIPLE SCLEROSIS FACTS:
·     MS is a chronic (auto)immune-mediated CNS-confined demyelinating disease affecting 2.500.000 people worldwide; disease onset, 20-40 years of age.
·     MS is clinically characterized by a relapsing-remitting course usually followed by a progressive phase.
·     MS is pathologically characterized by both inflammation (demyelination) and neurodegeneration (axonal loss and neuronal damage).
·     Although ‘spontaneous’ repair occurs (40-50% of demyelinated lesions are fully or partially remyelinated), MS invariably progresses (ambulatory problems in 70-80% patients at 25 years from onset).
Lancet Neurol, 2002
 
 
XII European Charcot Foundation Lecture
 
BLAKEMORE, Cambridge   U.K. : Regeneration and repair in multiple sclerosis. Evidence from experimental pathology.
 
In multiple sclerosis (MS) the myeline sheath that allows neuronal conduction is destroyed. Early in the disease new myelin sheaths can be formed by cells generated from endogenous precursor cells. However, as the disease progresses this remyelinating process fails. Means are therefore needed to stimulate remyelination. There are two possible approaches: firstly, by stimulation of endogenous remyelination, and secondly by transplanting exogenous remyelinating cells. In animal models, attempts to stimulate endogenous remyelination have so far proved unsuccessful. Remyelination can, however, readily be achieved by transplanting exogenous remyelinating cells. Prof. Blakemore suggested that the time is right to extend this research to the clinic.
 
 
SESSION   I. Basic mechanisms of regeneration
 
BRÜCK, Göttingen, Germany : Spontaneous remyelination in multiple sclerosis. How and Why?
Repair of damage to the isolating myelin sheath around neurons is a spontaneously occurring natural process. Especially in the early stages of MS there is extensive repair, but also in the chronic phase about 40% of all lesions show signs of remyelination. Patients may differ considerably in the magnitude of this repair. Some patients show an almost complete spontaneous recovery from brain damage whereas others remain impaired, and these differences occur at early as well as chronic stages of the disease. The extent of remyelination depends on the availability of oligodendrocytes and susceptible axons. Prof. Brück continued his lecture to show that especially the cortex has a remarkable rapid and efficient capacity to mend demyelinated neurons in the rat and human brain. Lesions in the cortex are overall less destructive than white matter lesions.
 
MARTINO, Milan, Italy : How does the brain repair itself?
Three processes are crucially involved in brain repair. First of all, inflammation plays a key role in the degenerative, but also in the regenerative phase of MS. Further, plasticity of the brain is required when functions of lost neurons have to be taken over by other neuronal pathways. Finally, the body itself produces new neurons, nerve connections (synapses) and supporting tissue (glia) in a process called neurogliogenesis. Stimulation of neurogliogenesis is the subject of much research efforts today. To a certain extent the newly manufactured neurons and glia cells migrate from their ‘home spots’ to the sites of damage. However, maturation and hence migration can be negatively influenced by inflammatory processes. The stimulation by endogenous stem cells to repair brain damage will in that case not be very effective.
   
LUBETZKI, Paris, France: Why does an axon not remyelinate in multiple sclerosis?
Sometimes the brain shows little remyelination and the question is why? There is no single explanation for this since many different factors contribute. One crucial factor is the availability of precursor cells around the lesion. Guidance molecules that either attract the precursor cells, or prevent their migration towards the demyelinated area could influence repair capacity.   Another main factor is the propensity of neurons to respond to the precursors. The interaction between the precursor cells and neurons is a delicate process in which time and place is pivotal.
 
SELMAJ, Lodz, Poland : Overcoming failure to repair demyelination in EAE: the Notch inhibition.
Failure of tissue repair contributes to the progression of MS. In an inflammatory milieu the oligodendrocyte precursor cells (OPCs) that play a role in recovery are being inhibited. This recovery failure is regulated by the so-called Notch receptor. Stimulation of this Notch system blocks the differentiation of OPCs which is a necessary step for repair, and stops myelination of the rat optic nerve during embryonic development. Blocking the Notch system by a gamma-secretase inhibitor on the other hand, enhances recovery and myelin repair, reduces axonal loss and glial scarring and also reduces the severity of symptoms in the EAE animal model.
 
FRANKLIN, Cambridge, U.K. : Stimulating remyelination in chronic demyelination.
CNS remyelination is mediated by adult stem cells/precursor cells (OPCs). In response to demyelination there is a rapid activation of microglial cells which produce factors that activate the astrocytes, which in turn activate the OPCs. Morphological changes occur in the OPCs, they start expressing different genes and differentiate into myeline producing oligodendrocytes.
Inflammation and a rapid removal of myelin ‘waste’ fragments (debris) are key to bringing about successful remyelination. The presence of macrophages -which is a sign of tissue inflammation- is helpful to the repair process, as depletion of macrophages leads to a significant reduction in remyelination. Injection of an inflammatory agent (zymosan) enhances remyelination when OPCs are transplanted in an animal model of chronic (less inflamed) demyelinated plaque (non-myelinated nerve fibre layer of the retina).
Removal of myelin debris by macrophages is important for remyelination and facilitates the differentiation of OPCs. Adding this debris impairs the remyelination process.
 

COMI, Milan, Italy : Is it clinically relevant to repair focal multiple sclerosis lesions?

Originally, plaques were regarded as the major cause of MS disability. Recently, it has become apparent from pharmacological and MRI studies that -besides plaque formation- there is a widespread degenerative process ongoing in normally appearing white and grey matter. Prof. Comi points out that this type of damage which continues to accumulate inside the CNS, may have a closer relationship to irreversible disability than the focal lesions. The cause of this degenerative process is largely unknown, the possibility of a remote effect of lesions has been disputed.

The evolution of MS starts with an early acute phase in which the attacks are usually followed by an almost complete recovery of neurological dysfunctions, because of the efficiency of remyelination and the neuronal redundancy in the CNS. Half of the attacks however, have been shown to lead some sequelae, irrespective of earlier or later stages of the disease. MRI studies of optic nerves that suffered an acute attack demonstrated that in patients with poor recovery there was irreversible tissue damage. Moreover some abnormality occurred also in patients with an apparently normal visual function. The study of the evolution of new lesions visible on MRI showed that about 10% had a very good repair and disappeared in subsequent MRI scans, 60% remained visible, but had a good recovery, whereas 30% became a so-called black hole, indicating a poor efficiency of repair mechanisms.  

Apart from these individual patient differences, the type and location of the lesion also determines whether the lesion damage will remain permanent. For instance, in a recent study, 56% in paraventricular lesions turn into a black hole, while eg. in the infratentorial this is 29%. Larger lesions (>6mm) and longer enhancement within the lesion increase the risk of conversion into a black hole. Pharmacological treatment with glatiramer acetate (GA) or natalizumab led to less conversion of lesions into black holes. The progression of MS damage appears to be both patient- and lesion specific. It is apparent that MS attacks have to be controlled in order to minimize permanent loss of function. This is in accordance with the observation that the frequency of attacks in the early phase of the disease and the severity of the attacks are predictors of long-term prognosis.

Inflammation plays a complex role in MS lesions because it has both positive and negative effects. For instance early treatment of the attack with high dose steroids reduces the duration of inflammation, whereas it may affect the neuroprotective effects of the final phase of inflammation. In conclusion, cellular therapy to support recovery from attack should preferentially be performed in the early phase of the disease.
 

Early clinical prognostic factors for disability in MS

·     Male sex

·     Age at onset > 40 years

·     Polysymptomatic

·     >5 relapses in first 5 years

·     short interval between 1st and 2nd attack

·     incomplete recovery from 1st attack

                                                                Comi 2006

 

PALACE, Oxford , U.K. : Regeneration and neuroprotection.

Axonal damage occurs in acute lesions early in MS. In the treatment of brain damage neuroprotection is necessary besides an anti-inflammatory therapy. We observed that patients can recover spontaneously although this process may take up to a year or more.

The brain self repair mechanisms of the brain include:

·     The capacity of inflammation to produce neurotrophic factors and cytokines that stimulate cell growth. For example, in an animal spinal cord transection the implantation of activated macrophages promoted repair;

·     Cellular repair, which includes local remyelination by OPCs, differentiation and migration of ‘repair cells’ (adult neural stem cells) although whether this leads to replacement of neurones and glia is unclear.

·     Reorganisation of cortical neuronal pathways. Compensatory pathway in response to damage may develop by unmasking of latent pathways or development of new pathways. Compensatory pathways can be found in the same region (as the lesion) or in the same region of the other hemisphere, in functionally associated regions, or in adjacent parts of the cortex. It has been demonstrated that MS patient use different brain areas for specific tasks as compared to controls. This can be rapidly reversed by pharmacologically stimulating the cholinergic system.

 

These repair mechanisms often fail in MS and the reason is multifactorial. Possible factors are the ongoing damage, the exhaustion of stem cell supply, aging of the brain, the damage of migratory pathways for adult stem cells, scar formation that inhibits sprouting and a reduction of reparative inflammation with anti-inflammatory treatment.

 

 
  
SESSION II. Repairing the brain
 
BARON VAN EVERCOOREN, Paris, France: Activation of adult neural stem cells in the demyelinated brain: from rodent to man.
This lecture addressed the role of endogenous neural stem cells in remyelination. How can these cells be activated? This was first investigated in animal models of diseases. The adult neural stem cells located in the subventricular zone (SVZ) of rats can be activated eg. in response to trauma, neurodegeneration or autoimmune demyelination (EAE). There was a large increase of cells in the SVZ and in the rostral migratory stream, which was accompanied by an increase in proliferation and mobilisation throughout the brain. Then, depending on the pathological environment, they can differentiate into astrocytes, neurones or oligodendrocytes and participate in the repair process. Would this work in humans as well? The human SVZ is not entirely comparable to the rat making the translation of results from animal models to the human situation more difficult. The human SVZ is a more simple organized region as compared to the rodent. In MS, this region is activated by the disease process leading to an increase in very early progenitors. Further investigation of adult neural stem cells and their progenitors in the brain of non-human primates that are more closely related to man should help to gain insights in their process of activation in response to demyelination and their role in myelin repair.
 
BARKER, Cambridge , U.K. : Sources of stem cells for brain repair.
Barker gave an overview of the standing of cell therapies for brain repair in Parkinson’s disease (PD), another neurodegenerative disease. The typical motoric dysfunction in PD is a result of the loss of dopaminergic cells in the nigra. Simply restoring these cells by transplantation is however, troublesome. Autograft transplantation (with cells of the patient itself) does not create the problem of tissue rejection and avoids ethical questions, but to date the results have been disappointing. Endogenous neural precursor stem cells from adult brain areas could be made to develop into dopamine-producing cells, but it hasn’t been feasible to harvest enough such cells from the adult brain. Allograft transplantation (from other people) of human fetal brain cells has led to positive results in some PD patients, but generates many ethical problems. In addition, there are immunological problems of rejection as well and practical ones in that a lot of fetuses are required for each grafted patient (4-5 needed per patient). Xenograft transplantation involves cells of animal origin and this raises difficulties of tissue rejection, ethics and politics, and very importantly the possibility of spreading animal viruses into the human population (zoonoses).
 
BLAKEMORE, Cambridge, U.K. : Endogenous versus exogenous OPC for remyelination.
Prof. Blakemore continues his lecture of yesterday by summarizing the advantages transplanted OPCs have over endogenous oligodendrocyte progenitor cells for remyelination in situations where this is failing. Using rat models he showed that the rate at which endogenous OPCs repopulate in damaged tissue declines with age and is significantly slower than that achieved by transplanted neonatal cells. Because of this, transplanted neonatal and embryonic OPCs remyelinate areas of demyelination more quickly than endogenous cells. Both endogenous and exogenous OPCs can only repopulate normal tissue when there are no host OPCs and both loose their remyelinating potential in the absence of inflammation. He concluded that exogenous neonatal OPCs have a far greater remyelinating potential than endogenous cells, however they are subjected to most –but not all- the restraints that effect endogenous cells.
 
BRÜSTLE, Bonn, Germany : Engineering embryonic stem cells for neural repair.
Human embryonic stem (hES) cells are very promising as transplantation material since they are pluripotent and can give rise to various cell types, they can be multiplied endlessly, and have the possibility to be genetically modified. Neural cells derived from hES cells integrate successfully with surrounding host tissue and are fully functional.
Culturing these neural cells in vitro has led to complete differentiation and stable colonies of pre-OPC cells. Once transplanted, these cells receive synaptic input and hook up with their neighbours. Also in an inflammatory environment the hES cells engulf axons and form myeline sheaths around them. They fully integrate into the host tissue.
These hES cells can also be used to deliver genes to a deficient host CNS. As such, it was possible to transfect genes into ES cells and transplant them successfully into rodents.
The culturing and differentiation of hES cells is relatively easy. The conditions to grow hES cells have successfully been sorted out, they can be frozen and thawed, propagated through many passages and still they remain stable. They differentiate predictably into 3 cell types: neurons, astrocytes and oligodendrocytes. The hES cell-derived neural stem cells exhibited however, a limited differentiation with regard to transmitter system (only GABAergic), which could be modulated over time (3-4 weeks) by regional factors. The transplanted hES cells transmit neuronal signals and receive neuronal input although full maturation may take up to 6 months.
Using genetic manipulation techniques, cells can be generated that can be used for gene therapy.
 
RICE, Bristol, U.K. : Autologous bone marrow stem cells: properties and advantages.
When developing cellular therapy for neurological diseases the primary concern is the central tenet of medicine – to do no harm. Additional requirements are that the harvesting method should be minimally invasive and the cell count obtained should be predictable, adequate and reproducible. The cells have to survive in the host so must avoid rejection. Ultimately, the treatment must result in an improvement of the patient’s condition. Stem cell transplantation is an attractive potential therapy for neurological diseases and advantages include the potential for replacement of lost or damaged cells, the protection of threatened host cells by immune modulation and the delivery of trophic factors or genes. Stem cells have been shown to migrate and survive in the adult CNS, can be manipulated in vitro, and are susceptible to genetic modification.
 
Potential disadvantages of stem cell therapy include:
·     the possibility that autologous stem cells may have a genetic predisposition to early loss,
·     the unknown risk of introducing genetically modified cells,
·     the theoretical risk of epileptogenesis, especially with transplantation directly into the brain,
·     the risk of oncogenesis, particularly with embryonic cells,
·     the risk of infection, especially with xenografts, allogenic material or any cells modified ex vivo,
·     the media hype associated with the undoubted promise of stem cells which can lead to the generation of unrealistic patient expectation.
 
Bone marrow-derived stem cells are attractive sources of stem cells as they have been clinically used for decades in the treatment of haematological malignancies. The cells are accessible; they are not associated with the ethical issues surrounding the use of the foetal material and bone marrow harvest is a relatively routine clinical procedure. In future, the relevant population(s) of cells could be mobilised using cytokines and collected from peripheral blood thus avoiding the general anaesthetic usually given for bone marrow harvest. The cells have been demonstrated to migrate and survive within the host CNS. Indeed, in animal models the cells appear to have a tropism for sites of disease. Functional benefit has now been demonstrated in animal models of neurological disease and in human cardiological studies.
 
Controversy surrounds some of the research involving adult human bone marrow-derived cells. Concerns include the validity of some research methods (e.g. the specificity of markers and changes in cell morphology) and the fact that the mechanism of action is a matter of on-going research. In addition, the cell population and number likely to be of most benefit are unknown. Another problem is the low frequency of some of the candidate cell populations in normal bone marrow. Although the cells could probably be expanded ex vivo, this would inevitably add an additional layer of complexity and risk. Concern has also been voiced regarding the possible fusion of transplanted cells with host cells. This has been thought to reflect an increased risk of malignant transformation but Dr. Rice would argue that fusion has been recognised in normal tissues and in the physiological response of several organs to stress.  
 
The demonstration that bone marrow-derived stem cells can modulate the immune response and produce beneficial paracrine effects as well as potentially replacing lost or damaged cells means they are likely to exert beneficial effects via more than one mechanism. This, together with their ethical robustness, may be their greatest advantage.
 
 
SESSION III. What is known about stem cell treatment in neurological disease?
 
BAREYRE, Munich, Germany : Neuronal repair and replacement in spinal cord injury.
Spontaneous repair strategies following spinal cord injury could include:
1.       Axonal regeneration: Axonal regeneration does not seem to spontaneously happen in mice.
2.       Axonal reorganization: New collaterals spontaneously emerge above the site of the spinal cord injury that make contact with long interneurons that innervate the hind limb muscles, thereby forming a detour circuitry. After 3 months the electrical signal could pass again to the lower lumbar part of the spinal cord (through this detour circuit) to reach the motoneurons and contribute to functional recovery.
3.       Cell replacement: Spontaneous cell replacement after spinal cord injury has been observed in animals, but often cells differentiated only into oligodendrocytes and astrocytes, not neurons. These new oligodendrocytes could remyelinate spared axons leading to functional recovery.
 
Has stem cell therapy been successful in spinal cord injury? Implanted ES cells give rise only to astrocytes and oligodendrocytes, but not to neurons. Maybe the injured environment prevented this differentiation. Possibilities to circumvent this are 1. to implant undifferentiated cells and aim to at least support the lesioned area with some remyelination or 2. either use oligodendrocytes pre-differentiated progenitors or 3. genetically engineered stem cells. Such pre-differentiated cells were capable of significantly remyelinating damaged axons in acute spinal cord lesions in animals and engineered cells were able to give rise to mature neurons. Several Phase I clinical trials in spinal cord injury that are currently taking place indicate short-term safety. Clinical effects will further be studied in Phase II studies.
 
TRYGGVASON (for ERICSON), Stockholm , Sweden : Stem cell treatment in Parkinson’s Disease.
Parkinson’s Disease (PD) is characterised by stiffness of limbs and slowness to initiate and execute movements caused by loss of dopaminergic (DA) neurons. Since no effective pharmacological treatment is available, PD is a disease eligible for treatment with cell replacement strategies. Over the world more than 300 PD patients have now been treated with fetal ventral-midbrain (VM) grafts with some positive results. The use of fetal transplants raises ethical questions and there is a problem of both the quality and the quantity of the cells. Clearly, alternative sources are needed. Embryonic stem (ES) cells are the most promising material for transplantation in PD. The group of Prof. Ericson and Prof. Perlmann have identified key molecules for the DA neuron development, and ES cells genetically modified to express one of these proteins (Lmx1a) differentiated into DA neurons. In preliminary experiments using rodent models of Parkinson’s disease, the genetically modified ES-transplants produce considerably more fibres that innervate the host striatum compared to non-modified transplants. The major problem of translating these results to the clinic is the occurrence of tumour-like overgrowth associated with transplantation of ES cells. Also, the introduction of genetically modified material in humans may lead to debate in the public domain, which has to be dealt with first.
 
HERMANN, Zürich, Switzerland : Stem cell treatment in stroke.
In stroke, the interruption of blood flow very rapidly leads to brain tissue demise. As such, there is not only a loss of neurons, but of the whole cell matrix, which has implications for treatment. Current pharmacological approaches focus on the recanalisation of brain arteries. So far, neuroprotective agents were not very effective and did not turn out to be efficacious in human patients. So, there is no drug to date that can rescue ischemic tissue after a stroke has occurred.
In the 1990s stem cell research started with transplantation of embryonic tissue, which led to behavioural improvements in rodent stroke models. These positive results were rapidly translated into the clinic. Already in the late 1990s clinical trials were started in patients with ischemic stroke who received cultured human neuronal stem cells directly into the brain (striatum). Notably, there were no adverse effects. Some improvement in motor function was reported, but the absence of a control group hampered the conclusions.
After stroke, the endogenous regenerative response is activated. In the ischemic brain, neural precursor cells (NPCs) come out of the ventricular zone and migrate to the stroke lesion. In rats with experimental stroke, this migration of cells was visualised by tracking labelled precursor cells on MRI. Recent studies showed that transplanted embryonic stem (ES) cells can, in principle, substitute lost cells and exhibit electrophysiological properties similar to host neurons.
Compared with adult NPCs, ES cells have the advantage of a high capacity to divide, differentiate and survive. Potential disadvantages are the ethical/legal concerns, the difficulty of material access and tumour formation.
In Zurich and Milano, the authors are currently conducting studies in mice, analysing the effects of NPCs obtained from adult mice on behavioural recovery following stroke. Three days after experimental stroke one million adult NPCs are administered by i.v. injection. The cells migrate from the periphery to the stroke area, as was visualised by fluorescence imaging. After 3 weeks recovery, there was a significant improvement of motor deficits on several behavioural tests, indicating restitution of neuronal function. Adult NPCs are easier to access than ES cells and raise less ethic and legal debate. This research with i.v. delivery of adult NPCs is very promising for future human application.
   
MAZZINI, Novara, Italy : Stem cell treatment in Amyotrophic Lateral Sclerosis (ALS).
This lecture gave a view on the use of stem cells in ALS. At the moment the mechanism of neurodegeneration of ALS is not entirely clear, and there is no therapy available. Stem cell therapy may therefore be an option. Mesenchymal stem cells (MSCs) from bone marrow have been tried in an animal model of ALS and in patients. Preclinically it has been shown that these cells can migrate, differentiate and display neuroprotective activity and so they might be effective against the neurodegeneration in ALS. The feasibility and safety of BM cells on motoneurons in ALS was assessed in 21 patients. In 9 patients BM stem cells were injected at a high thoracal level of the spinal cord. After 4 years, there were 5 patients still alive that did reasonably well (only one slowly declined). This represented a relatively hopeful result. MSCs may therefore have some use in ALS. The intraspinal administration was found a safe procedure. For the future Prof. Mazzini suggests to increase the number of injections and she hopes that it one day may be possible to define the clinical phenotype of responding patients. Possibly, further developing BM cells into neural stem cells before injection may also help.
 
 
SESSION IV. Stem cell treatment for neurological diseases: Mechanisms
 
CHANDRAN, Cambridge , U.K. : Generation of clinical grade neural cells from human stem cells.
Stem cells have considerable promise for regenerative neurology. Many prerequisites have to be fulfilled for clinical application. This lecture is about standardising all methodological steps necessary for ‘GMP’ stem cell treatment in order to generate defined ‘clinical grade’ cells that may have therapeutic relevance.
The starting material should be ethically and politically acceptable and genetically stable. It is key that cells differentiate in a specific, and targeted direction (regardless of precise clinical application) by controlling all "growing” conditions and that they can be propagated to large numbers with retention of biological function. They must at a minimum, survive, make appropriate neural connections and induce functional recovery in appropriate animal disease models.
Based on research with mice, this group has developed (and validated across two independent hES lines) a method of generating potentially "clinically compatible” neural stem cells from hES lines under totally controlled and humanised defined conditions. Human ES cells become neuralised over 16 days, can be mechanically propagated, and reliably generate functionally active neurons and glia including oligodendrocytes. So the production of clinical grade neural stem cells is feasible. Dr. Chandran advises to exclude genetic modifications or animal products in stem cell therapy for humans. Future hES cell research also enables (human) disease modelling (ex vivo) and, critically, is a unique resource for drug discovery aiming to expand the pharmaceutical armoury for neurological disorders.
 
DUNCAN, Madison , U.S.A. : Replacement of cells.
Do stem cells promote endogenous repair or do they replace lost cells in chronic MS lesions? When stimulating the endogenous repair fails, exogenous cell replacement has to be considered. This has for instance been studied in animals that lack myelin which were transplanted with a mixed glial cell preparation in the ventral column of the spinal cord. After 8 weeks every single axon in the area was remyelinated by the transplant. So yes, in the rodent model there seems to be ‘cell replacement’ by exogenous implants at least on a local level.
But, a mouse is not a man: mouse striatal cells can be grown to a spheric cluster of cells that can be guided to differentiate in a certain direction by adding specific growth factors. A video was shown that demonstrated the migration of stem cells of mice out of the neurosphere. This was not possible with human striatal tissue, so the attention of Prof. Duncan’s research group has shifted to human ES cells.
 
The requirements for the ideal cell for clinical transplantation are:
·     able to migrate
·     capable of many divisions without becoming transformed
·     myelinates many axons
·     should not provoke an immune response
·     should be available in large numbers and can be cryopreserved
 
PLUCHINO, Milan, Italy : Stem cell treatment in MS.
Transplantation of multipotent neural stem/precursor cells (NPCs) significantly ameliorates clinico-pathological features of both chronic as well as relapsing-remitting experimental multiple sclerosis (MS) in mice. In these pre-clinical settings, intravenously-injected NPCs selectively accumulate into central nervous system (CNS) sites of inflammation and/or demyelination. So, after systemic administration these cells know specifically where to go in the CNS. A number of molecules guide the process by which NPCs "home” into CNS sites of injury. Although the whole process allowing this precise migration is not entirely understood, Dr. Pluchino has stressed the importance of the ‘functional immune signature’ of NPCs.
Similarly to blood cells, NPCs express functional cell adhesion molecules and pro-inflammatory (chemokin) receptors that play a major role in regulating the selective accumulation of systemically-injected NPCs within the CNS of mice with experimental MS. Once there, NPCs contribute to the restoration of the microenvironment integrity through (at least) two major mechanisms: i. the direct (rather low) capacity of directly differentiating into myelin forming cells and ii. the (striking) bystander (or chaperone) capacity to support the endogenous reparative potential of the CNS. This latter phenomenon is mainly dependent on the capacity of the injected NPCs to secrete neurotrophic growth factors to rescue endogenous OPCs at the site(s) of lesion and to induce programmed cell death of white blood cells (T lymphocytes).
 
Thus, the message of the recent experience with NPC transplant may be summarized as follows,
·     it works,
·     in rodent as well as monkey models of MS,
·     in chronic as well as relapsing models of MS,
·     through an immunomodulatory ‘bystander’ effect.
 
By showing beneficial effects of NPCs in a monkey model of MS, a possible solution for the human situation has become closer.
 
HOHLFELD, Munich, Germany : Neurotrophic cross-talk between the nervous and immune system.
The edge between immunology and neurology is an interesting area. It is apparent that a lot of cross-talk is going on. Immune cells influence the CNS environment and vice versa CNS-lesions have an effect on the immune system. For instance, brain-derived neurotrophic factor (BDNF) which plays a major role in the CNS (neuronal growth, survival and plasticity) is expressed by immune cells in MS lesions to affect neuronal survival and perhaps functioning. In BDNF-knockout mice it was established that BDNF has an important function in the immune system. Conversely, MS therapeutical agents such as copaxone (glatiramer acetate) might work at least in part by stimulating the production of BDNF by immune cells. Another example of this cross-talk is BAFF, a member of the TNF family that seems to fulfil an important role in the CNS by supporting long-term survival of immune (B) cells. Remarkably, the immunostimulant compound BAFF can be found in the CNS where it exerts a negative action on MS. The development of BAFF-inhibitors might be an option for MS treatment although such agents would not be specific to the CNS. Cross-talk between the nervous and immune system, and the prevention of it, should receive our definite attention.
 
CHOPP, Detroit, U.S.A. : Plasticity and remodelling of the brain.
Do you necessarily need stem cells to replace injured neural tissue for the treatment of neurological diseases, such as MS, or can one employ cell-based, and not necessarily stem cell therapy? Stem cells may be irrelevant for the treatment of MS, says Dr. Chopp. Stem cells as well as other types of cells, e.g. progenitor cells, bone marrow mesenchymal cells, cord blood, all act as mini-factories, providing an array of neurotrophic and other restorative factors that stimulate the endogenous restorative mechanisms inherent in the brain, such as, angiogenesis, synaptogenesis, and neurogenesis. In addition, certain drugs, already in clinical use, also activate endogenous neurorestorative processes. Many types of cell-based and pharmacological therapies to stimulate brain remodelling were investigated to reduce neurological deficits accompanying neural injury (eg. stroke, traumatic brain and spinal cord injury) and neurodegenerative diseases, such as MS. Cell-based therapies primarily activate astrocytes, the workhorses of the brain, to produce a pharmacopoeia of trophic and angiogenic factors.
Preclinical tests for neurological diseases include middle cerebral artery occlusion, i.e. ischemic stroke, and EAE in the rodent. One functional outcome measurement is a somatosensory test in which rats are timed for their capability to remove an adhesive tab from their forelimbs. After stroke, rats have great difficulty in removing adhesive pads from their paws. This was reversed by intravenous cell- based therapy with treatment administered up and possibly beyond a month after onset of injury. The beneficial effects are already observed after 1 week and persist for over 1 year. What is causing this? By applying cells you are in fact stimulating the brain. It doesn’t matter that much of this stimulation is through cells or drugs. The presence of certain exogenously administered cells or drugs (e.g. statins, EPO, PDE5 inhibitors) stimulates astrocytes cells to express neurotrophic factors while reducing apoptosis. When human bone marrow mesenchymal cells are injected intravenously into EAE-mice, there is a significant increase in neurotrophic factors, enhanced functional recovery and remodelling of the white matter (remyelination).
On the question which type of patients to initially treat with bone marrow mesenchymal   cells, Dr. Chopp suggested to treat the more advanced patients since here you may more likely to   obtain approval of the health authorities.
 
BEN-HUR, Jerusalem, Israel : Immunomodulation by neural stem cells.
Another look into the migration of transplanted NPCs. How do they react to a multifocal inflammatory brain process? When NPCs are given intracerebroventricularly (i.c.v.) to EAE-animals (rats and mice) they are attracted by inflammation and migrate to the white matter into the corpus callosum to reduce inflammation and establish remyelination. The reduction of inflammation, and consequent reduction in axonal injury and demyelination, was responsible for a beneficial effect on behaviour.
What would happen if you inject these cells i.v. in EAE-mice? The NPCs reduce again the activity of the immune system and ameliorate neurological damage, but they accumulate in lymph nodes and do not enter the brain. The neurological improvements were in this case caused by a peripheral bystander suppressive effect on the immune system. For clinical use, NPCs should therefore be given intrathecally.
 
 
SESSION V. Requirements for stem cell treatment in trials of multiple sclerosis.
 
EBERS, Oxford, U.K. : Clinical requirements: do we need stem cells for the treatment of MS?
The progression of MS is difficult to predict in individuals. The relation between morphological changes observed with MRI and the actual disability of patients is only weak. Relapse frequency in general does not predict clinical outcome, except for the number of attacks during the first year of illness. It appears that the link between physiological phenomena and actual clinical disability is not always clear.
Do we need stem cell therapy in MS? Prof. Ebers’ personal opinion is that more work is needed to understand the nature of the disease and the effects of stem cells. Of course he wishes to provide new treatments for patients, but only in a well-controlled manner and since this treatment is already coming into uncontrolled use, he stresses to register all cases of stem cell transplantation. This would be helpful for monitoring side effects and to systematically record clinical effects. The founding of ethics and monitoring committees, in which the European Charcot Foundation can play a leading role, is suggested.
 
SCOLDING, Bristol , U.K. : Autological mesenchymal bone marrow stem cells: practical considerations.
Stem cells not only stimulate repair (remyelination) directly, they also re-programme repair processes (neuroprotection). The use of autologous mesenchymal bone marrow stem cells in MS capitalises on what already happens in the body: blood-circulating adult bone marrow stem cells are thought to enter the brain and spinal cord to contribute to the repair of damaged issue. A difficulty is to measure what extra added cells do in the body. Imaging techniques as MRI and PET-scan may help, but clinical improvement (MSIS score) is the bottom-line. In a woman who received male bone marrow cells, a functional integration of transplanted cells into the brain was observed by staining the Y-chromosome (post-mortem). Finally, the potential hazards of stem cells transplantation, such as tumour formation, infection and rejection, are mentioned to monitor by Prof. Scolding. Initiating stem cell therapy requires balancing safety issues against potential benefit. With 11 clinical trials involving more than 350 cardiology patients receiving stem cells for repair of arterial wall damage, the safety database is substantial. Most concerns can be ruled out or overcome, such as infection, purity of cells or GMP regulations. The issue of tumour formation is possibly the most important, but donor mesenchymal stem cell-derived tumours thus far have never been reported in bone marrow transplanted recipients. When using ES cells this risk is higher.
 
SILANI, Milan , Italy : Safety and Ethics.
Autologous haematopoietic (from bone marrow or blood) stem cell therapy (HSCT) is a technically feasible and putative promising therapy for MS patients. HSCT has been applied in haemato-oncology for 20-30 years and is ethically robust, but the risk/benefit ratio of the approach in MS patients is still unclear at this stage of development. Autologous therapy has 3 to 5% mortality rate, versus 15 – 35% with transplantation from allogenic sources. At this phase, pre-clinical evidence in animal models is not quite uniform and the HSCT multi-step procedure in humans is not standardised. Uniformity of procedures, patient selection, and protocols should be part of guidelines for the use of autologous HSCT in MS patients. Prof. Silani calls for caution when obtaining informed consent from patients that need to be fully aware of a complex procedure with potential risk/benefit at each step. Safety is strictly related to the different protocols. Consensus is needed for standardised procedures, including quality of life measures and adequate clinical outcomes supported by biomarkers. Ethics will take great advantage from a synergic action of the different centres involved with this new therapeutical strategy in MS patients.
 
DOUSSET, Bordeaux, France: How to trace stem cells for MRI evaluation?
Stem cell engraftments (derived from bone marrow) can be made visible in the host tissue by attaching radio tracers (PET) or biocompatible contrast agents (MRI) to label the transplanted cells. Cell specific contrast agents are now under development, which allow labelling the transplant cells in vitro, and track the engraftment in vivo in the brain. Most used are ferromagnetics (iron oxide that is incorporated inside the cytoplasma) which make cells visible in MRI or electron microscopy. One technique for labelling is to make the cell membrane receptive for ferromagnetic agents by transfecting DNA-sequences. Via direct injection into the brain, or in the vascular system the labelled cells eventually reach the brain, and stay detectable for 6 to 8 weeks. Several reports have shown the mobility and viability of the grafted cells in host brain tissue in several pathological conditions. One problem is that dead cells or macrophages ‘eating’ the labelled cells will also contain the label, so that the MRI not only visualizes functional cells.
 
BRINCHMANN, Oslo, Norway : Expanding autologous mesenchymal bone marrow stem cells.
Some 100 patients worldwide have been treated over the last 10 years with multipotent autologous mesenchymal stem cells (MSCs) derived from bone marrow.   First patients to be treated had osteogenesis imperfecta, while more recently in this methods were used to treat patients with graft-versus-host disease and neurodegenerative diseases. Although the molecular mechanisms are not entirely clear, it is known that MSCs suppress the immune response (they reduce T-cell proliferation and the response to antigens) and can regenerate tissue. The immunosuppression involves the release of several molecules (cytokines, prostaglandins) that arrest the T-cell cycle. These cells remain inside the host for a while, after which they break down and a next injection can be given. The cells find their own way in the body to reach the site of lesion.
MSCs seem to be immunoprivileged cells, and administering is safe: no short and long term complications are seen.   Dr. Brinchmann presented some practical aspects to maximalise the mesenchymal cell culture. E.g. to use autological serum as growth medium; with serum from 1 blood bag (200 ml) up to 100 million cells can be harvested. Also, it is suggested that fetal bovine serum is not to be used in culture because the risk of zoonoses (transmitting animal viruses to man). Another warning with i.v. administration is that these cells are large and sticky (they belong in a matrix structure) so they can get stuck in lungs or spleen.   One possible solution might be to grow these cells in 3D colonies in alginate beads. This would allow the cells to form their original (round) shape. The alginate may then be removed by simply adding citrate. Direct injection into the damaged tissue would also overcome these ‘sticky’ problems, and the cells develop according to cues provided by the   microenvironment. Finally, the laboratories to culture MSCs should meet extensive GMP regulations to guaranty safety and quality.
 
POSTER WINNER – HUNT , Cambridge , U.K.
Skin-derived precursor cells are an exciting potential source of stem cells for remyelination but are difficult to find in adult skin. The work of Dr. Hunt and colleagues showed that there is a highly enriched niche of these cells at the base of the hair follicle in the adult face, thus allowing these potentially clinically useful cells to be extracted from adult human skin.
 
 
SESSION VI. Structure of a protocol of stem cell treatment in multiple sclerosis
 
SCOLDING, Bristol , U.K. (for HARTUNG, Düsseldorf , Germany ): Report of an expert meeting.
Prof. Scolding reports the results of a consensus meeting held in Düsseldorf, February 2006 and discusses the outline of a possible clinical protocol. From animal studies we have sufficient experimental background to consider clinical trials in MS. There are many different types and sources of stem cells (embryonic-, neural-, bone marrow-, fetal forebrain-, umbilical cord- or skin stem cells), but the autologous mesenchymal bone marrow stem cells seem to be the first choice for clinical stem cell therapy in MS. This choice is prompted by the good safety record of these cells and technical methods. There is, however, room for improvement with regard to containing, storing, delivering and treating. Some regulations are already available. Expansion techniques may, for instance, be improved by using autologous serum.
The mechanism of action is fairly well-known: the stem cells suppress the immune system and generate myelinating oligodendrocytes and neurons that take care of repair. On the question of dosing Prof. Scolding answers it is still an educated guess: 10 to 150 million cells have been used in pilot studies. According to the route of administration (intrathecal or intravenous), dosing could be adjusted: 10 times more is needed for the intravenous route (which is more feasible).
What type of patients should be included in this clinical trial? Treatment-naive patients (with leaking blood-brain barriers) are preferred, as the effect of the treatment can be more clearly evaluated.
Goals of treatment in this trial are to firstly evaluate safety and secondly to repair the brain, as will be monitored with MRI. The call is for a trial in 5-6 centres, with 45-60 patients in a Phase I/II design. The European Charcot Foundation is in a position of direction, guidance and data ownership of such a trial.
   
KARUSSIS, Jerusalem, Israel : Explorative trial with injection of mesenchymal bone marrow cells in multiple sclerosis.
Mesenchymal stem cells (MSCs) may be derived from various sources (bone marrow, adipose tissue, pancreas, blood). It has been demonstrated that these cells migrate into the CNS to exert their regenerative (remyelinating) properties. The role of MSCs on immunomodulation is new. Mouse EAE-studies showed that bone marrow MSCs provided stem cells that could readily enter the brain where they evolved (differentiated) into neuronal and glial cells, and suppressed the local inflammation. They migrated around the ventricles and there was a good correlation between the extent of inflammatory lesions and the degree of migration. The transplant cells expressed NG2 both after intracerebroventricular (i.c.v.) or i.v. delivery. So, transplantation of bone marrow cells strongly suppresses the clinical and histopathological signs of EAE, the model of MS, in mice. Most importantly, this treatment with MSCs induced neuroprotection, since most of the nerve axons in the transplanted animals remained intact in contrast to the extensive damage of the axons that was observed in the untreated mice and which is similar to what happens in patients with MS.
Now a clinical trial is running investigating primarily safety and feasibility MSC infusions in 10 patients with severe MS that fail on current therapy, with clinical performance as secondary endpoint. A similar study is being performed in ALS patients. Bone marrow stem cells were injected (100 million) either i.v. or intrathecal with 1 year follow-up. Protocol of this safety study is run now on 4 patients. Initial improvement has been observed. One spinal injury patient who was a spastic paraplegic showed remarkable improvement upon MSC transplantation: he did not need a cathether anymore and could walk with help. No major adverse effects were observed after either intrathecal or i.v. infusion of MSCs. One case of aseptic meningitis occurred, due to chemical contamination of the cell culture. A large controlled study is needed vs sham or cytotoxic drugs using MRI as measuring tool.
 
HOMMES, Molenhoek, The Netherlands : Can we pass from the experimental to the clinical phase in MS stem cell research?
Prof. Hommes put the question whether we can take the step to move from experimental to clinical studies with stem cell therapy in MS? How do we cope with uncertainties and possible risks of biotechnological therapy? An estimation of overall risk was made using a pharmaceutically-derived check-list. Several aspects of cell therapy were rated based on extensive experiences in preclinical and clinical studies (see Kenter and Cohen, 2006).
 
These items include:
  1. Level of knowledge about mechanism of action:  
    1. Is there a plausible mechanism +
    2. Is there adequate clinical and pathophysiological knowledge about the mechanism  ±
  2. Previous exposure of humans to similar biological mechanism:
    1. Investigation of direct mechanisms +
    2. Assessment of related mechanisms and procedures in analogue disease states +
    3. Investigation of primary and secondary pharmacological and biotechnological processes +
  3. Can the primary or secondary mechanism be induced in animals or in human cell material?
    1. Receptor/tissue homology +
    2. Post receptor/tissue mechanism similar ±
    3. Human ex-vivo tests available -
  4. Selectivity of the mechanism to target tissue in animals:
    1. Receptor/target distribution in tissue +
    2. General pharma/biotechnology studies +
    3. Toxicology +
  5. Analysis of potential harmful effects:
    1. Dose or concentration ±
    2. Intensity, seriousness ±
    3. Vital organ systems affected ±
  6. Pharmacokinetic (PK) - biotechnological (BT) considerations:
    1. Half life in relevant effect compartment +
    2. PK – BT relations ±
    3. Active or toxic metabolites or cells ±
  7. Predictability of effect:
    1. Biomarkers for effect in animal and man +
    2. Precision and accuracy of measurement +
    3. Relation of markers/technology to clinical effect ±
  8. Can effects be managed?
    1. Antidotes or antagonists ±
    2. Other counter measures -
 
Most aspects of the checklist were rated positive or moderate: from the 22 questions there were 10 positive, 10 doubtful and 2 negative.
 
If the step to clinical studies is taken there are a number of issues to be addressed:
·     Only study MS patients, no volunteers
·     Use a sequential protocol (patient after patient)
·     Use standard multiplication and expansion procedures, preferentially at one central laboratory
·     No placebo-control, patient serves as one control (pre- and post treatment)
·     Primary endpoint: MRI, brain atrophy, clinical symptoms
·     Route of administration: i.v. / i.c.v. / intrathecal ?
·     Dosing: how many cells to inject? Repetition of treatment?
·     Number of patients in Phase I-II study: preference 40-80
·     Cooperation between centres? Special organizational structure? Funding?
 
It is expected that stem cell treatment will outperform conventional treatments, with respect to replacement of lost neurons and oligodendrocytes, and that it will stabilize the immune system and exert direct neuroprotection. Prof. Hommes suggests starting clinical trials in MS patients now and invites the panel and the audience to comment.
 
Reference.
Kenter & Cohen: Establishing risk of human experimentation with drugs: lessons from TGN1412. The Lancet 368 14 oct 2006 , 1387-91.
 
PANEL DISCUSSION
Panel members: Blakemore, Hommes, Brinchmann, Ebers, Mazzini, Karussis, Scolding
 
Clinical trial decision
Prof. Hommes asks if a clinical trial to prove the principle of MS stem cell therapy should be started. All panel members agree to initiate such a trial.
 
Clinical protocol
In defining clinical outcome of a stem cell trial in MS, the actual mending of the brain is perhaps too ambitious; stopping further deformation is a more realistic expectation (Blakemore). MSCs are probably a safe option in MS patients since they were well-tolerated in ALS patients (Mazzini). A first MSC trial should try the i.v. route since these cells go easily into the brain (Blakemore). The problem of cells to cross the blood-brain barrier and the correct choice of patients needs to be clarified (Mazzini). Repeated injections of MSCs can be given to enhance their concentration in the brain (Brinchmann). Prof. Karussis suggests to commence with the spinal forms of MS since these lesions more easily reached with intraspinal injection. This would also require less cells (Karussis). Another possibility is to inject these cells in the carotid arteries in order to enhance penetration into the brain (Brinchmann).
 
Cross-talk between the neural and immune system
Prof. Ebers warns to take caution for the possible interference of steroids and stem cells. MSCs rapidly go into lymph nodes, in 2-3 days there are inhibitory effects on the immune system (Ebers). Stem cell therapy should not be used simultaneously with anti-inflammatory therapy. Dr. Brinchmann also stresses that anti-inflammatory drugs will affect MSCs. Be careful with add-on therapy. Dr. Chandran addresses the timing of MSC treatment. In animals there is an active inflammation when they are treated with stem cells, whereas in humans the inflammatory phase will have passed once treatment starts. How will this affect outcome?
 
Patient selection
Prof. Scolding advises not to treat single lesions, but start with well-defined non-MS lesions.
Prof. Ben-Hur pleads for late cell therapy in these illnesses to stimulate surviving brain cells to reorganise. Prof. Karussis: "Yes, we should do a clinical trial to answer this, perhaps in spinal forms of MS”. Prof. Scolding proposes to do a PoP in PPMS patients or in MS patients with a spinal form.
 
Methodological concerns
Dr. Chopp disagrees with some assumptions made and stresses that cell therapy is not a replacement therapy but merely a stimulation of endogenous brain repair mechanism. Both MSCs and NSCs were active in stroke models in his lab. Transplanted cells were highly efficient, also after 1 year. They passed to very few other organs. One hundred million cells were used. Switching from i.v. to intra-carotid injection gained a factor 2 improvement, but occlusion of carotid poses addition risk (unwanted).
 
First, the actions of MSCs have to be compared to normal MS treatments, such as interferons. In clinical studies there has to be a clearly defined lesion (to spinal cord or optic nerve) which can be measured and hence repaired.
 
Practical issues
Prof. Blakemore insists that stem cells need to be cultured in a central growth facility.
 
Other types of cells
Prof. Blakemore suggests to consider using NSCs as a second option for transplantation
Prof. Scolding replies that although there are safety data from NSCs, these are obviously less substantial than with MSCs. For safety reasons we have to start with MSCs.
 
   
LIST OF MEDICAL TERMS
Astrocyte                                 Supporting cell in the central nervous system
Axon                                        Neuron that transmits an electrical signal to an endpoint (eg. organ)
Glia                                           Supportive tissue around the axons
Intrathecal                                In the corticospinal fluid compartment of the spinal cord
Myeline (sheath)                      Insulation tissue around neurons to allow conductance
Neural precursor cells             Cells that develop to neurons
Oligodendrocyte                       Cell that myelinates neurons
Oncohaematology                    Blood/lymph cancer
   
LIST OF ABBREVIATIONS
ALS                                          Amyotrophic lateral sclerosis
BDNF                                        brain derived neurotrophic factor
BM                                            Bone marrow
CNS                                          Central nervous system
DA                                            Dopamine(rgic) (neurotransmitter)
EAE                                          Experimental autoimmune encephalomyelinitis
ES cell                                      Embryonic stem cell
GABA                                       Gamma amino butyric acid (neurotransmitter)
GMP                                          Good manufacturing practice
hES cell                                    Human embryonic stem cell
i.c.v.                                         Intracerebroventricular
i.v.                                            Intravenous
MRI                                           Magnetic resonance imaging
MS                                            Multiple sclerosis
MSC                                          Mesenchymal stem cell
NSC                                          Neural stem cell
OPC                                          Oligodendrocyte progenitor/precursor cell
PET                                           Positron emission tomography
PoP                                           Proof of principle
PPMS                                        Primary progressing MS
   

Prepared by HPRC, Baarn, The Netherlands .

 

   
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